SCHOOL OF NATURAL SCIENCES

General Luna Road, Baguio City Philippines 2600

Telefax No.: (074) 442-3071 Website: www.ubaguio.edu E-mail Address: [email protected]

A Learning Module in

Clinical Chemistry 2
Enzymology, Electrolytes, BGA, Endocrinology,
Toxicology & Drug Testing

Prepared by: Mark Gideon M. Wallis, RMT

Medicalnewstoday.com

Metabolicleader.com

Frontiersin.org
CLSdianostics.com

TABLE OF CONTENTS Page No.

Success is no accident. It is hard work, perseverance, learning, studying, sacrifice, and most of all, love of what you are doing.
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Chapter I Clinical Enzymology ................................................. 7

Introduction to Enzymes ... ............................................. 7

Enzyme Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Enzyme Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Enzymes of Clinical Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Chapter II Major Electrolytes ................................................... 47

The role of Electrolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

The major Electrolyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Chapter III Trace Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Essential Trace Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Ultra-trace Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Chapter IV BGA and Acid Base balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

The Buffer system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Regulation of Acid-base balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Disorders of Acid-base balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Compensation Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Chapter V Clinical Endocrinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Overview of Endocrinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Organs of the Endocrine system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Chapter VI Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Drugs of abuse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Therapeutic drug monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Toxic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Introduction

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I. Course Code and Title: CLNCHM2 (Enzymology, Electrolytes, BGA, Endocrinology, Toxicology & Drug Testing)
II. Course Description and Information: This is a 5-unit course with 3 units lecture and 2 units‘ laboratory. The
course deals with a study of enzymes & enzyme kinetics, together with the quantitative measurement of the
medically important enzymes in blood. In addition, a study of electrolytes, their importance, methods of determination
& clinical correlation is likewise included. Blood gas analysis and tumor markers also form important segments of the
curriculum. An overview of the Endocrine system is given, with elaborate discussions of the hormones from the major
target glands (Adrenal gland, Thyroid gland and the gonads). The unit on Toxicology focuses on drugs of abuse &
techniques for drug analysis, environmental carcinogens, toxins or poisons and the most important therapeutic drugs
which require monitoring. Quality assurance and safety are given due emphasis.
The laboratory part deals with analysis of medically important enzymes and electrolytes as well as the
identification of trace metals and toxins using different chemical and physical methods. Laboratory activities aim to
develop skills in performing routine clinical chemistry procedures as an application of the concepts learned in the
lecture. Other laboratory activities are given as inquiry-based that are intended to develop critical thinking skills as
well as to supplement the topics in the lecture.
III. Requirements of the Course:
1. Regular Attendance to classes: you must attend online classes and live quizzes regularly by logging in to our
scheduled online activities. Online lectures will be done through Google meet and/or facebook live. Assessments
shall be given through Quizziz, Pear Deck, Canvas and/or Google forms. For offline students, your attendance
will be monitored through your responses to text information and through timely correspondence. Offline students
will also be given quizzes and activities through phone calls and text messages.
2. Submission of required activities: All required activities (assignments, research work, and laboratory
illustrations) should be submitted on or before the given deadline. Deadlines will be posted by the teacher in the
google classroom and messenger group chat. It will also be texted to offline students. Learning output for online
students, submit to the teacher‘s email address that will be given during the class orientation; For offline students,
submit via mail or express courier (―padala‖) addressed to: Instructor’s name, School of Natural Sciences,
University of Baguio, Baguio City.
3. Study/Learning Guidelines:
a. Manage your time properly. As students of higher education (College), you are expected to be more
responsible in paying attention to course schedules, requirements, and deadlines. Schedule how you will
accomplish all the requirements in all your enrolled courses (reading the modules, reading on research/
enhancement questions, doing assignments and laboratory illustrations) and focus your attention when doing
your tasks.
b. Observe proper conduct. Despite this online mode of learning, you must still maintain appropriate school
behavior at all times. All standards of student conduct outlined in the University of Baguio Student Handbook
remain in full effect during this time of distance learning. Be honest in answering your quizzes and exams.
Work independently in doing your tasks and assignments.
c. Maintain a performance of high standards. Give your best in accomplishing all the assigned tasks. Do not be
complacent with just a 70% passing cut score. Remember that this is a board subject, and the best preparation
for the board/licensure examination should be during these formative years. The board review is but
supplementary to the knowledge you have already learned during your Med Tech education.
d. Communicate properly. Promptly respond to notifications by regularly visiting our google classroom and
messenger group chat. If you have confusions or queries in any part of this module, I am here to guide you
through. Send your academic concerns using same online platforms. For offline students, text messages and
mobile calls are welcome during scheduled hours of the day and week. Be guided by this schedule when
communicating:
 Respect private hours. I do not always open my laptop/email/messenger 24/7. Send your queries and/or
concerns during regular office hours. For concerns that need immediate attention, send through mobile
text.
e. Show mutual support. Support one another. Let us all be responsible and supportive in making this new
learning process more effective.
f. Live lecture/Video conferencing guidelines:

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f.1 Be punctual. Live lectures/Video conferences will be scheduled during the official class period/time of this
course. Log in to the platform at least 5-10 minutes before the class period. Prepare your learning
materials such as this module, pens, papers, etc. Attendance will be checked during the lecture/video
conference.
f.2 Maintain professionalism.
- Wear appropriate clothing and set your gadget in an appropriate area. You may be asked to turn on
your video/camera at any time during the lecture.
- Log in using your UB gmail account. Unidentified names like nicknames, phone models, etc. will not
be allowed in the video conference.
- Mute your microphone as soon as you log in to the platform to avoid any excess background noise.
Unmute your microphone when instructed to do so.
- Respect privacy. Do not take a screenshot, picture, snapchat, etc. of your teacher or fellow students,
nor make any unnecessary audio or video recordings.
f.3 Remain focused and engaged. Do not be distracted by your gadget. Keep your videoconference

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 Study Schedule
WEEK TOPIC ACTIVITY
1 Chapter I: Enzymology, Section 1: Introduction to Enzymology Lecture: Assessment & Quiz
Laboratory: Activity No.1
2 Section 2: Enzyme Nomenclature Lecture: Assessment & Quiz
Laboratory: Activity No. 2 and Activity No. 3
3 Section 3: Enzyme Classification Lecture: Assessment & Quiz
Laboratory: Activity No. 4, Activity No. 5 &
Activity No. 6
4 Section 4: Enzymes of Clinical Importance Lecture: Assessment & Quiz
5 Laboratory: Activity No. 7, Activity No. 8 &
Activity No. 9
6 FIRST GRADING EXAMINATION Coverage: Chapter I (Lesson 1-4)
7 Chapter II: Section 1: The role of Electrolytes Lecture: Assessment & Quiz
Laboratory: Activity No. 10 & Activity No. 11
8 Section 2: The Major Electrolytes Lecture: Assessment & Quiz
Laboratory: Activity No. 12
9 Lecture: Assessment & Quiz
Laboratory: Activity No. 13
10 Chapter III: Trace Metals, Section 1: Essential Trace metals Lecture: Assessment & Quiz
Laboratory: Activity No. 14
11 Section 2: Ultra trace metals Lecture: Assessment & Quiz
Laboratory: Activity No. 15
12 MIDTERM EXAMINATION Coverage: Chapter II and Chapter III
13 Chapter IV: BGA, Section 1: The Buffer System Lecture: Assessment & Quiz
Laboratory: Activity No. 16
14 Section 2: Regulation of the Acid Base Balance Lecture: Assessment & Quiz
Laboratory: Activity No. 17
15 Section 3: Disorders of the Acid-Base Balance and Section 4: Lecture: Assessment & Quiz
Compensation Mechanism
Laboratory: Activity No. 18 & Activity No. 19
16 Chapter V. Endocrinology, Section 1 Overview of the endocrine Lecture: Assessment & Quiz
system and Section 2: Organs of the Endocrine system
Laboratory: Activity No. 20 & Activity No. 21
17 Chapter VI: Toxicology, Section 1: Drugs of abuse and Section Lecture: Assessment & Quiz
2: TDM
Laboratory: Activity No. 22 & Activity No. 23
18 Section 3: Toxic Agent Lecture: Assessment & Quiz
Laboratory: Activity No. 24
18 FINALS EXAMINATION Coverage: Chapters I to VI

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Rubrics

Rubrics for the evaluation of Essay question activities and Questions for research
CRITERIA HIGHEST Outstanding Satisfactory Poor
SCORE
1. Content and Focus 7 Sharp, distinct & Apparent point made No apparent point and no
substantial controlling about a specific topic to minimal evidence of
-Awareness about the
point about a specific or question with awareness and
specific topic
question or topic with sufficient awareness knowledge. (1 point)
-Presence of relevant ideas evident awareness and knowledge. (4
thru tacts, examples, details, and knowledge. (7 points)
opinions and explanation points)
2. Organization 5 Sophisticated Functional Confused or inconsistent
arrangement of arrangement of arrangement no logical
-Order developed and
content with evident content that sustains order or evidence of
sustained with in the
and/or subtle a logical order w/ transition. (1 point)
paragraph
transition (5 points) some evidence of
transition (3 points)
3. Style and Conventions 3 Good grammar, Sufficient grammar Incorrect grammar and
spelling and sentence and minor spelling major spelling errors
-Choice, use and
formation throughout errors and sentence throughout the paragraph.
arrangement of words
the paragraph. (3 formation (1 point) (0 point)
-Grammar, mechanics, points)
spelling, usage & sentence
formation

Rubrics for the evaluation of Illustrations and Diagrams

CRITERIA HIGHEST Outstanding Satisfactory Poor
SCORE
1. General Appearance 3 Lines or patterns are There are smudges Smudges or stray marks
drawn clearly and not or stray pencil marks obscure details of the
- Neatness and labelling
smudged. Minimal but these do not illustration. Over-all quality
erasure or stray significantly affect the of the drawing shows
pencil marks. over-all appearance minimal effort to complete
Labelling is accurate of the illustration. careful work. Important
and complete. (3 Labels are included labels are missing. (1
points) but there are minor point)
problems in the
accurate identification
of a part. (2 points)
2. Organization and content 5 Provides complete Provides clear Only few important
and well organized illustration however contents are illustrated (1
-Illustrations and sequencing
illustrations (5 points) some details are point)
missing (3-4 points)
3. Attention to details 2 The illustration is Some minor errors Major discrepancies are
accurately drawn (2 are present however very noticeable in the
points) these do not distract illustration. (no point)
the information
conveyed. (1 point)

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 Chapter I. CLINICAL ENZYMOLOGY
Clinical Enzymology is a field of laboratory medicine which focuses on the study of enzymes and their significance
to the diagnosis and treatment of diseases.

Desired learning outcomes:

At the end of this chapter, you should be able to:
a. understand how enzyme kinetics work
b. identify and describe each of the different clinically important enzymes
c. discuss the clinical significance in the assay of the different enzymes
d. describe the principle underlying the different methods of enzyme determination
e. identify factors that may affect test results
f. correlated results with various physiologic and pathologic conditions

Section 1: INTRODUCTION TO ENZYMES

A. What are enzymes?

a. These are substances that catalyzes a given chemical reaction
b. The reaction they catalyze are frequently reversible which means which means that they can synthesize and
decompose molecules
c. They are complicated types of protein in terms of both structure and function
d. They easily denatured with varying molecular weight and mass
e. These enzymes are amphoteric which are capable of ionizing either as acid or base
f. They are synthesized in an inactive state and operates in the presence of a cofactor
g. Enzymes are found in all body tissues, they appear in the serum following cellular injury or they may come from
degraded cells thus changes in enzyme concentration reflects changes in state of health.

B. Definition of terms
a. Coenzymes - non-protein organic biochemical that takes part in the enzyme reaction
 Essential to the catalytic activity as a CO-SUBSTRATE
 Diffusible, heat stable, low molecular weight that when combined tightly to enzymes, the coenzyme will be
called Prosthetic group
 E.g. NAD, Pyridoxal phosphate
b. Activators - Inorganic ionic cofactor
 increase the catalytic activity of an enzyme when it binds to specific site
 Metabolic regulator of enzyme reaction
 Usually metal ions (esp. divalent cations)
 E.g. Mg++, Na+, K+, Zn++
c. Holoenzyme - the combined enzyme & coenzyme
d. Apoenzyme - Enzyme without a cofactor
e. Prosthetic Group - A coenzyme that cannot be removed from its attachment to an enzyme using dialysis
 E.g. Pyridoxal phosphate in transaminase reaction
f. Substrate - Substance acted upon by an enzyme & is converted into a new substance
g. Product - Substance derived from a transformed substrate
h. Active site – Site where substrate interacts with enzymes
i. Allosteric site – Site other than the active site that may lead to either attachment of substrate to the enzyme‘s
active site or inhibition of attachment
j. Isoenzymes – different form of an enzyme with different genetic origins but catalyze the same reaction
k. Isoforms – Results when an enzyme is subject to different post-transitional modification

C. Enzyme Structure
a. Primary Structure - Refers to the sequence of amino acids joined by peptide bonds to form a polypeptide chain
b. Secondary Structure - Conformation of the segments of polypeptide chain
 Made up of alpha helices or beta-pleated sheets which are maintained by hydrogen bonds
c. Tertiary Structure - Arises from the interactions among side chains/groups of the polypeptide chain
 Structure are bent and folded and maintained by covalent disulfide bond
d. Quarternary Structure - Separate bended & folded structures are put together to form a functional unit
 Enzyme variants – LDH, Creatine kinase
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D. The enzyme action model
a. Enzymes act through formation of enzyme substrate complex
b. The substrate must be bound to the active site of the enzyme
c. The enzyme-substrate complex will then break down to give the reaction products and free the enzyme
d. All enzyme reactions are in theory reversible however, in practice, reactions are usually more rapid in one
direction than the other.

Toppr.com

E. Theories Related to Enzyme-Substrate Combination

a. Lock and Key Theory - refers to the active site being complementary in shape & size to the substrate
 First presented by Emil Fisher, the lock represents an enzyme and the key represents a substrate. It is
assumed that both the enzyme and substrate have fixed conformations that lead to an easy fit. Because
the enzyme and the substrate are at a close distance with weak attraction, the substrate must need a
matching shape and fit to join together. At the active sites, the enzyme has a specific geometric shape and
orientation that a complementary substrate fits into perfectly.
b. Induced fit Model - the enzyme changes in shape during binding to accommodate the substrate
 The induced-fit model is generally considered the more correct version. This theory maintains that the
active site and the substrate are, initially, not perfect matches for each other.

Image taken from wikibooks.com

F. Factors that influence the enzymatic reaction

a. Time – The rate of enzymatic reaction
 If the catalytic activity of an enzyme on a substrate is fast, this will mean a shorter reaction time thus
liberating the enzyme to act again on the remaining substrate
b. Molecular compatibility – Commonness between the enzyme and the substrate
c. Space availability – Number of substrate that can be reacted
d. Specificity - capacity of enzymes to recognize and bind only one or few molecules among others
 Absolute specificity – when an enzyme can act and catalyze one unique reaction
 Group specificity – when some enzymes act on different substrates belonging to the same group
 Sterioisomeric – an enzyme acts only on the specific isomer
e. Substrate and Enzyme concentration
 First order Kinetics
o Enzyme conc. is fixed; Substrate conc. is varied
o Rate of reaction is almost directly proportional to substrate conc. at low values
o At low concentration of the substrate, only a fraction of the enzyme is associated with the substrate
o The rate observed reflects the low concentration of the ES complex

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  Zero order Kinetics
o When maximum velocity is reached, the rate of increase in velocity is ―O‖
o Reaction rate is unaffected by increased substrate concentration
o Dependent on enzyme concentration
o In this reaction, the entire enzyme is bound to substrate and a much higher rate of reaction is obtained
o Because the entire enzyme is present in the form of the complex, there is now no further increase in
ES complex conc. No further increment in reaction rate are possible
 Michaelis-Menten Curve - shows the relationship of the reaction velocity to the substrate concentration

f. Temperature
Pharmafacts.com/enzyme-kinetics

 Optimum temperature - °T considered favorable for enzyme activity (30-37°C or 37 – 40°C)

 Q10 value – reaction rate is doubled for every 10°C increase
 50 – 60°C : enzyme undergoes inactivation and denaturation
g. pH – Hydrogen Ion Concentration
 Optimum pH - the point at w/c the reaction rate is greatest
 At pH 7.0 – 8.0, many enzymes show maximum activity
 pH value are seen as low as 1.5 and as high as 10.5
 Optimal pH may be different in forward and reverse reaction
 Maintaining pH is important: it affects the three dimensional confirmation of the enzyme
h. Activators – increased reaction rate
 Bind the substrate to the active site by forming ionic bridges
 Orients the substrate so it is attached to the enzyme in the correct configuration
i. Inhibitors - Decrease the rate of enzyme reaction
 Competitive inhibition - Inhibitors Binds to the active site, blocks access of the S to the E
o A competitive inhibitor is a structural analog of the substrate, but it is not identical thus breakdown to
products do not take place
o If substrate conc. Is significantly higher than the inhibitor, the inhibition may be reversible

Image taken from khanacademy.org/enzymes

 Non-competitive inhibition - Binds elsewhere on the E causing change in shape that interferes w/ S binding
o Attachment of the inhibitor to the enzyme does not alter the affinity but the presence of ESI prohibit the
formation of products

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Image taken from khanacademy.org/enzymes

  Uncompetitive inhibition - Inhibition-inhibitors binds to the ES complex, if substrate will be increased, there
will be increase in ES conc. increasing inhibition, this inhibition does not yield product.
 Reversible inhibition - Inhibitors are possible removed from the system; enzyme is fully restored
o Physical processes that remove inhibitors: dialysis, gel filtration
 Irreversible inhibition - Inhibitors covalently combine w/ the enzyme
o Physical methods are ineffective in separating inhibitors from the enzymes
G. Methods of Enzyme Assay
a. Fixed Timed Assays
 The reactants are combined
 Reaction proceeds for a designated time & is stopped (by inactivating the enzyme)
 reaction is stopped by a weak acid, then measurement is made of the amount of reaction that has
occurred
 The reaction is assumed to be linear over the reaction time, the larger the reaction, the more enzyme is
present
b. Continuous-monitoring or kinetic assay
 Multiple measurements are made at specific time intervals or by a continuous-recording
spectrophotometer
 Advantage: Linearity of the reaction is adequately verified
 This is preferred because any deviation in linearity is readily observable
H. Units of Measurement
a. IU – International Unit
 Proposed by the Commission on Enzymes (IUB)
 Used to standardize the system or reporting of quantitative results
 IU is the amount of enzyme that will catalyze the reaction of 1 µmol of substrate per minute under specified
conditions of temperature, pH, substrate and activators.
 Expressed in terms of U/L or mU/L
b. Katal
 unit of enzyme activity w/c converts 1 mol of substrate per second
 conforms w/ the Systemè International (SI) scheme of units
 Mole is the unit for substrate concentration while the unit of time is second
 Enzyme concentration is then expressed as katals per liter
I. Factors that influence rate of entry
a. Leakage of enzymes from cells
 Impaired energy production: promote deterioration of cell membrane
 Direct attack on the cell membranes (viruses or organic chemicals)
 Reduction in the supply of oxygenated blood perfusing any tissue (e.g. MI)
b. Altered enzyme production decrease
 Genetic deficiency of enzyme production
 Enzyme production is depressed as a result of disease
J. Clearance of Enzymes
a. Urinary excretion – NOT a major route for elimination
 Except: Amylase = ↑blood levels (e.g. Acute pancreatitis)
b. Enzyme Inactivation in Plasma
 Inactivated enzymes are removed by the RES
 Half – life in plasma: 24 – 48 hours

Reference:
Ashwood E., D. Bruns and C. Burtis (2012). Tietz’s Textbook of Clinical Chemistry and Molecular Diagnostics 5th ed.
Philadelphia: W.B. Saunders Co.
th
Bishop, Michael (2013) Clinical Chemistry Procedures, Correlation. (7 ed.). Holland
th
Crook, Martin (2006). Clinical Chemistry and Metabolic Medicine (7 ed.). USA: Hodder Arnold Publication.

McPherson, Richard. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Elsevier
Saunders, 2011.
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 Laboratory Activity No. 1: PRINCIPLES OF ENZYME REACTIONS

Objectives: At the end of the activity, the students are expected:

1. To describe the biochemical nature and structure of enzymes.
2. To illustrate the Michaelis-Menten and Lineweaver-Burke plots; describe how the MM and LB equations relate
to enzyme kinetics.
3. To briefly explain the factors that affect the enzyme reactions.
4. To list the physiologic causes of abnormal blood enzyme levels.
5. To describe methods employed for enzyme assays.
6. To relate the rate of enzyme catalyzed-reactions to the amount of enzyme activity present in a system.

Materials:
1. Visual aids
2. Chart illustrations
3. Reference books

Procedure:
The instructor discusses the various aspects of enzyme and enzyme-catalyzed-reactions through a video
presentation. The clinical relevance of enzyme assays is also pointed out. For the drawing and illustration, it will be
placed on a short bond paper which will contain a margin of .5‖ in all sides with the name and section of the student. The
students can scan or take pictures of their drawing, then submit it online. For offline students, they can submit it through
courier.

Drawing:

Illustrate and label the following:

1. Enzyme-substrate complex formation in a reaction.
2. Michaelis-Menten plot
3. Lineweaver-Burke plot
4. Effects of pH, temperature and inhibitors on enzyme reaction.

Questions for research: The instructor will create an online forum in which the answers for the QFR will be written. For
offline students, they may send via courier or through text message.

1. Explain the basis of specificity of enzyme reaction.

2. Describe the Michaelis-Menten kinetics of enzyme reaction.
3. What is the Lineweaver-Burke equation?
4. Enumerate and briefly describe the various factors that affect enzyme assays.
5. Describe the factors that contribute to abnormal levels of enzymes in blood?
6. Describe briefly:
a. Fixed-time reaction
b. Continuous monitoring method
c. Multi-enzyme methods
d. International unit of enzyme activity and katal unit
7. Describe some techniques to measure/differentiate isoenzymes.

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 Assessment No. 1:

True or False: Please answer the following questions; write your answer on the space provided.

1. A zero-order reaction rate is independent of substrate concentration

because there is sufficient substrate to saturate the enzyme.

2. Enzymes alter the energy of activation by forming a metastable

intermediate, the enzyme substrate complex.

3. An enzyme will accelerate the rate of a reaction, reducing the time

required to reach equilibrium.

4. A constant change in absorbance per unit of time occurs only when the
rate of the reaction is first order.

5. The IU is a rate expressed in micromoles per minute. Activity is reported

as IUs per liter (IU/L) or mIU/mL.

6. Uncompetitive inhibitors bind to the active site where the enzyme binds
substrate and are overcome by increasing the substrate concentration.

7. The concentration of reactants and products at equilibrium will be the

same with or without the enzyme.

8. A coenzyme is an organic molecule required for full enzyme activity.

9. A prosthetic group is a coenzyme that is tightly bound to the apoenzyme

and is required for activity.

10. The SI unit for enzyme activity is the katal (1 katal converts 1 mol of
substrate to product in 1 second).

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 SECTION 2: ENZYME NOMENCLATURE AND CLASSIFICATION

A. Classifications of Enzymes - Based on catalytic activity of an enzyme

The International Union of Biochemistry (IUB) Enzyme Commission categorized enzymes into six (6) classes
a. Oxidureductase
 Older names: Dehydrogenases & Oxidases
 Catalyze oxidation-reduction reactions
 Oxidoreductases catalyze the transfer of electrons from one molecule (the oxidant) to another molecule
(the reductant).
 Oxidoreductases catalyze reactions similar to the following: A– + B → A + B– where A is the oxidant and B
is the reductant
 Assayed for investigation of cardiac & liver disorders
b. Transferases
 Move an intact group of atoms (NH2 or PO4) from one molecule to another
 catalyze reactions similar to the following A + BX = AX + B
 Gives information about liver damage
c. Hydrolases
 Splits molecules with water as part of the reaction process
 catalyze reactions similar to the following: L A + H20 = B + C
 Three (3) Groups:
o Esterases: ACP, ALP, Lipase
o Peptidases: Leucine aminopeptidase, Pepsin
o Glycosidases: Amylase, Amylo-1,6-glucosidase
d. Lyases
 Responsible for splitting molecules or breaking of bonds (C to C; C to O; C to N, etc.)
 catalyze reactions similar to the following: A = B + C
 Assayed in disorders of skeletal muscles
e. Isomerases
 Responsible for the conversion of one isomer to another
 catalyze reactions similar to the following: A > B
 All reactions are reversible
f. Ligases
 Enzymes causing bond formation between two molecules to form a larger molecule
 catalyze reactions similar to the following: A + B = AB

B. Enzyme Nomenclature
Enzymes are classified according to the type of reaction they catalyze. In naming an enzyme, the suffix ―ase‖
is added to the name of the substrate
a. Systematic name
 Describes the nature of the reaction catalyzed
 Numerical code designation prefixed w/ the letters E.C.
 Example: E.C. 3.1.3.1 = ALP and E.C. 3.1.3.2 = ACP
1st digit = denotes the class of the enzyme
2nd digit = sub-class of the enzyme
3rd digit = sub sub-class
4th digit = specific serial number
b. Trivial name
 a.k.a Non- specific, Practical name, Working name
 Uses acronyms and abbreviations
 Examples: SGOT, SGPT

WordPress.com/Biochem-enzyme-nomenclature

Page | 13
 Assessment No. 2

Write the systematic name and the trivial, practical or other name of the following enzymes

Enzyme Systematic name Other name/Working name

1. Lactate dehydrogenase

2. Acid phosphatase

3. Gamma glutamyl transferase

4. Lipase

5. Amylase

6. Creatine kinase

7. Glucose-6-phosphate
dehydrogenase

8. 5‘-Nucleotidase

9. Alkaline phosphatase

10. Angiotensin converting enzyme

Reference:

Ashwood E., D. Bruns and C. Burtis (2012). Tietz’s Textbook of Clinical Chemistry and Molecular Diagnostics 5th ed.
Philadelphia: W.B. Saunders Co.
th
Bishop, Michael (2013) Clinical Chemistry Procedures, Correlation. (7 ed.). Holland
th
Crook, Martin (2006). Clinical Chemistry and Metabolic Medicine (7 ed.). USA: Hodder Arnold Publication.

McPherson, Richard. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Elsevier
Saunders, 2011.

Page | 14
 Section 3: ENZYMES OF CLINICAL IMPORTANCE

A. AMINOTRANSFERASES

 Catalyze interconversions of the amino acids & alpha-ketoacids by transfer of amino groups
 Pyridoxal phosphate as obligate coenzyme
 Pyrodoxal-5‘-phosphate will be bound to the apoenzyme and serves as a true prothetic group
 it will accept the amino group from the first substrates (aspartate/alanine) to form pyridoxamine-5-phosphate and
the first product of the reaction – oxaloacetate and pyruvate
 the coenzyme in amino form will then transfer the amino group to the acceptor/second substrate (oxoglutarate)-to
form the second products of the reaction-p5p is regenerated
 Function: Amino acid metabolism
 Ketoacids formed are ultimately oxidized by the TCA Cycle

A1. ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1

 Formerly SGOT (Serum glutamic-oxalocacetic transaminase)
 involved in the transfer of an amino group between aspartate and a-keto acids
 Reaction catalyzed:

 Tissue Sources:
o widely distributed in human tissue
o Highest concentration: cardiac tissue, liver & skeletal muscle
o Smaller amounts: kidney, pancreas & RBCs
o Isoenzymes of AST in the cytoplasm & the mitochondria
o Both mitochondria and cytoplasmic forms of AST are found in cells.
o About 5-10% of the AST activity in serum from healthy individuals is of mitochondrial origin
 Diagnostic Significance
o Evaluation of hepatocellular disorders & skeletal muscle involvement
 Liver disease-most important cause of elevated transaminase activity in serum
 In most liver disease, ALT is higher than AST (Except:Alcoholic hepatitis, Hepatic Cirrhosis and liver
neoplasia)
o Mild degree of liver tissue injury: cytoplasmic isoenzyme is predominant
o Severe tissue damage: release of mitochondrial isoenzyme
 mitochondrial AST activity in serum shows marked increase in patients with extensive liver cell
degeneration and damage
o AST Elevations
 Pulmonary embolism
 Following congestive heart failure
 following congestive heart failure, AST levels also may be increased, probably reflecting liver
involvement as a result of inadequate blood supply to the organ
 Viral hepatitis: up to 100x ULN
 In viral hepatitis and other forms of liver disease associated with acute hepatic necrosis,
serum AST and ALT activities are elevated before the clinical signs of jaundice-although ALT
activity persists longer
 Cirrhosis: ~4x ULN
 Skeletal muscle disorders: Muscular dystrophies & inflammatory conditions ~ 4 – 8x ULN
 In AMI: AST levels begin to rise w/in 6 – 8 hrs., peak at 24 hrs. & return to normal w/in 5 days
 AST levels are NOT useful in the diagnosis of AMI because of the wide tissue distribution

Page | 15
 Laboratory Activity No. 2: AMINOTRANSFERASE
ASPARTATE AMINOTRANFERASE (AST)
Continuous monitoring Assay

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure the activity of Aspartate aminotransferase in serum using
a continuous monitoring or kinetic assay by viewing simulation video which will be recorded by the laboratory
instructor.
2. To be able to understand the precautions in processing the specimens for such enzyme assays and
recognize the factors that affect the reaction.
3. To be able to describe the basic principle of reaction involved in the assay.
4. To be able to associate an elevated AST activity with pathological conditions.

Materials and Equipment:

1. Venipuncture set 8. Water bath (37 C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
3. Beaker 10. Labeling tapes
4. Stat fax tubes 11. Parafilm
5. Pasteur pipets 12. AST/SGOT Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

Principle of Reaction:
The enzyme aspartate aminotransferase (E.C. 2.6.1.1) catalyzes the transaminase reaction between L-aspartate
and 2-oxoglutarate. The 2-oxaloacetate formed is reduced to malate in the presence of malate dehydrogenase. As the
reactions proceed, NADH is oxidized to NAD. The disappearance of NADH per unit time is followed by measuring the
decrease in absorbance at 340 nm.
The present method has been made according to IFCC (2002).

Precautions:
Reagent may contain some non-reactive and preservative components. It is suggested to handle it carefully,
avoiding skin contact and ingestion.
Specimen: Serum, plasma; Collect blood with a minimum of venous stasis. AST is stable up to 4 days at 2 - 8C or 1
month at -20C.

Procedure: The instructor will make a recorded video of the actual laboratory performance of the procedure and
explanation of the principle of the test. The video will be uploaded for the students to be able to familiarize themselves
with the procedure. The absorbance reading will then be shown to the students for them to be able to compute for the
results.

BLANK SAMPLE
Working reagent 1.0mL 1.0mL
Pre-incubate at 37C for 5 minutes
water 100 μL
Standard reagent - -
serum - 100 μL
Mix; incubate at 37C and obtain the first absorbance reading after 90 seconds against reagent blank.
Perform three (3) other readings at 60 second intervals. Calculate the A/min.

Calculations:
AST (U/L) = A/min x 1746
Activity in ukat/L = U/L x 0.0167

Page | 16
Expected Values:
Males: <35 U/L (<0.58 ukat/L)
Females: <31 U/L (<0.52 ukat/L)

Linearity: The method is linear up to 440 U/L

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for research: The instructor will make an online forum where the students can type and post their QFRs. For
offline students, they can submit it via mail, express couriers or through text message.

1. Characterize aspartate aminotransferase as to activity and source.

2. What is the clinical significance of AST determination?
3. Aside from the method employed in the laboratory, describe current methods for AST determination in routine
laboratories.

Page | 17
A2. ALANINE AMINOTRANSFERASE (ALT); E.C. 2.6.1.2
 Formerly SGPT (Serum glutamic pyruvic transaminase)
 specifically, catalyze the transfer of an amino group from alanine to a-ketoglutarate with the formation of
glutamate and pyruvate
 Reaction catalyzed:

 Tissue sources:
o Distributed in many tissues
o High concentrations in the liver (more liver-specific enzyme of the transferases)
o low levels in the heart and skeletal muscle
o Isoenzyme: exclusively cytoplasmic form
o RBC contains 5-8X as much ALT activity as does the serum
 Diagnostic Significance:
o Evaluation of hepatic disorders (hepatocellular)
o Progressive inflammatory liver conditions: higher ALT elevations than AST
o Higher elevations are found in hepatocellular disorders than extrahepatic ot intrahepatic obstructive disorders

 Important Note on De Ritis Ratio (AST:ALT ratio)

 diagnostic marker of alcoholic liver disease
 implication: most causes of liver cell injury, associated w/ greater ALT than AST, however, there are cases where
in AST to ALT ratio is 2:1 or greater
o Normally <1.0 (also in viral hepatitis)
o If >1.0 but <2.0: associated with cirrhosis
o If >2.0: associated with alcoholic hepatitis or hepatocellular carcinoma
o Acute hepatocellular injury: AST>ALT initially; w/in 24-48 hrs., ALT>AST
 Higher AST activity in hepatocytes
 In acute inflammatory conditions of the liver, ALT elevations are frequently higher than those of AST
and tend to remain elevated longer as a result of the longer half-life of ALT in serum (16 and 24
hours, respectfully).
 Methods for the Measurement
a. Continuous Monitoring Method
o Transaminase reactions coupled to specific dehydrogenase reactions
o multiple measurement of absorbance change during the reaction is observed
o The oxo-acids formed in the reaction are measured indirectly by enzymatic reduction to the corresponding
hydroxyl acids
o The accompanying change in NADH concentration is being monitored spectrophotometrically
 Assay reaction for AST Activity
o Karmen Method
 coupled enzymatic reaction w/ MDH (indicator reaction)
 Monitors change in absorbance at 340 nm (NADH to NAD)
 Optimal pH: 7.3 – 7.8
o Sources of errors
 Hemolysis: increase serum AST
 AST activity in RBC is 15x higher than in serum
 AST activity is stable in serum for 3 – 4 days at ref °T
 Reference Range: 5 – 30 U/L (37°C)

 Assay reaction for ALT Activity

o Pyruvate formed is converted to lactate by LDH
o LDH as the indicator enzyme
o NADH formed is oxidized to NAD
o The accompanying change in NADH concentration is being
monitored spectrophotometrically
o the disappearance of NADH is followed by measuring he
decrease in absorbance
o Change in absorbance is proportional to the micromoles of
NADH oxidized that reflects the number of substrate transformed
Page | 18
 o Sources of Errors:
 Relatively unaffected by hemolysis (ALT activity in RBCs is 7x higher than in normal serum)
 Stable for 3 – 4 days at 4°C
 Reference Range: 6 – 37 U/L (37°C)
b. Colorimetric Method
o Coupling w/ 2,4- DNPH (Reitman-Frankel)
o Still feasible
o Phenylhydrazones of oxaloacetate & pyruvate are more chromogenic
o Simple; limited but acceptable accuracy
o Derivative formed will give a strong blue color measured as 505 nm

Page | 19
 Laboratory Activity No. 3: AMINOTRANSFERASE
ALANIN AMINOTRANSFERASE (ALT)
Continuous monitoring Assay

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure the activity of Alanine aminotransferase in serum using a
continuous monitoring or kinetic assay by viewing simulation video which will be recorded by the laboratory
instructor.
2. To be able to understand the precautions in processing the specimens for such enzyme assays and
recognize the factors that affect the reaction.
3. To be able to describe the basic principle of reaction involved in the assay.
4. To be able to associate an elevated ALT activity with pathological conditions.

Materials and Equipment:

1. Venipuncture set 8. Water bath (37C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
3. Stat fax tubes 10. Labeling tapes
4. Beaker 11. Parafilm
5. Pasteur pipets 12. ALT/SGPT Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

Principle of the Reaction:

The enzyme alanine aminotransferase (E.C. 2.6.1.2) catalyzes the transaminase reaction between L-alanine and
2-oxoglutarate. The pyruvate formed is reduced to lactate in the presence of lactate dehydrogenase. As the reactions
proceed, NADH is oxidized to NAD. The disappearance of NADH per unit time is followed by measuring the decrease in
absorbance at 340 nm.
The present method has been made according to IFCC (2002).

Precautions:
Reagent may contain some non-reactive and preservative components. It is suggested to handle it
carefully, avoiding skin contact and ingestion.
Specimen: Serum, plasma; Collect blood with a minimum of venous stasis. AST is stable up to 4 days at 2 - 8C or 1
month at -20C.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students be able to familiarize themselves with the procedure. The
absorbance reading will then be shown to the students for them to be able to compute for the results.
BLANK SAMPLE
Working reagent 1.0mL 1.0mL
Pre-incubate at 37C for 5 minutes
water 100 μL
Standard reagent - -
serum - 100 μL
Mix; incubate at 37C and obtain the first absorbance reading after 90 seconds against reagent blank. Perform
three (3) other readings at 60 second intervals. Calculate the A/min.

Calculations:
AST (U/L) = A/min x 1746
Activity in ukat/L = U/L x 0.0167

Linearity: The method is linear up to 440 U/L

Expected Values:
Males: <45 U/L (<0.74 ukat/L)
Females: <34 U/L (<0.56 ukat/L)

Page | 20
Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail or express couriers.

Characterize alanine aminotransferase as to activity and source.

1. What is the clinical significance of ALT determination?
2. What is the De Ritis ratio? Associate variations in the ratio to different pathological conditions.
3. Aside from the method employed in the laboratory, describe current methods for ALT determination in routine
laboratories.

Page | 21
B. Creatine Kinase (E.C. 2.7.3.2)
 An enzyme w/ MW of approximately 82,000
 Catalyzes reversible phosphorylation of Creatine by ATP
 it is generally associated w/ ATP regeneration in transport system
 Its dominant physiologic function is in muscle cells where it is involved in storage of high-energy creatine
phosphate (phosphocreatine - major phosphorylated compound in muscle)
 Reaction catalyzed:

 When muscle contracts, ATP is consumed (to form ADP) & CK catalyzes the rephosphorylation of ADP to form
ATP, using Phosphocreatine
 Mg-forms complexes with ATP and ADP with narrow concentration because excess will be inhibitory
 Tissue Sources
o Greatest in striated muscle, brain tissue & heart tissue
o Smaller quantities: bladder, placenta, GIT, Thyroid, uterus, kidney, lung, prostate, spleen & pancreas
o liver and erythrocyte is devoid of the activity of CK
 Structure
o Dimer w/ 2 sub-units: B (brain) & M (muscle)
o Each sub-unit w/ a MW of ~ 40,000
o Three major isoenzymes:
 CK – BB (brain type) CK1
 Found in the brain, prostate, gut, lung, bladder, uterus, placenta & thyroid
 CK – MB (hybrid type) CK2
 present in varying degrees in heart muscle (25 – 46% of CK activity) and minor degree in
skeletal muscle
 CK – MM (muscle type) CK3
 predominates in skeletal & cardiac muscle
 All three isoenzymes are found in the cell cytosol
o Unusual CK isoenzymes
 CK-Mt : 4th isoenzyme
 migrates cathodic of CK-MM
 Located b/w the inner & outer membranes of the mitochondria
 Differs immunologically & in electrophoretic mobility
 Constitutes up to 15% CK activity in the heart
 Its presence does not correlate with any specific disease however it correlates with severe
illness and cases of malignant tumor and cardiac abnormality
 Macromolecular forms of CK
 Macro CK Type 1
 CK1 associated with IgG or CK3 w/ IgA
 Macro CK Type 2
 Oligomeric CK-Mt
 Diagnostic Significance
o Disease of the Skeletal Muscle
 All types of muscular dystrophy, esp. Duchenne type sex-linked progressive muscular dystrophy)
 which may show up to 50x the ULN
 Normal Serum CK activity is seen in Neurogenic muscle disease
 CKMM major CK in serum of healthy people where Skeletal muscle contains almost exclusively of
CKMM and heart muscle activity is attributed to CKMM
 Injury on both heart and skeletal will mean elevation of CKMM
o Disease of the Heart
 CK level: sensitive indicator of AMI (Total CK & CK-2)
 CK-MB levels begin to rise w/in 4 – 8 hrs. Peak at 12 – 24 hrs. & return to normal levels w/in 48 – 72
hrs.
 Elevation of Total CK: Cardiac trauma ff. heart surgery (including transplantation)
 CKMB activity has been observed in other cardiac conditions, not entirely specific for AMI although
specificity can increase if tested in conjunction with LDH
Page | 22
  The time course of CK is unique in AMI
o Disease of the CNS
 CKBB isoenzyme will be elevated in conditions such as cerebral ischemia and cerebrovascular
ischemia where blood flow to the brain is insufficient
 Elevations are also noted during head injury and acute cerebrovascular disease
 It is also observe that the activity may increase in conditions that affects children such as Reye‘s
syndrome where the liver and the brain is swollen as a side effect of viral infections
o Disease of the Thyroid
 60% of hypothyroid subjects
 Major isoenzyme is CK-3
 Hypothyroidism results in CK-MM elevations because of the involvement
 of muscle tissue (increased membrane permeability), the effect of thyroid hormone on enzyme
activity, and, possibly, the slower clearance of CK as a result of slower metabolism.
o CK activity in Malignancy
 CKBB is rarely seen in the serum bec. Of its molecular size
 Extensive damage to the brain may lead to the leakage of it in the serum
 CKBB is associated also with patients with carcinoma of various organs such as adenocarcinoma,
lung tumors, tumors of the prostate, kidney breasts and ovary the CKBB can be a useful tumor
marker
 Methods of Determination
o Makes use of coupled enzymatic reaction, either the forward or reverse reaction
 The forward reaction is set at a pH of 9.0 and the reduction of NADH to NAD is monitored
spectrophotometrically
 The change in absorbance is proportional to the activity of CK

 The method by Oliver-Rosalki utilizes the reverse reaction

 The reverse reaction is preferred in the laboratory because it is about six times faster than the
forward reaction.
 The rate of NADPH formation is a measure of the CK activity

o Sources of errors
 Adenylate kinase (AK) Effect – an enzyme released from erythrocytes in hemolyzed samples and
appearing as a band cathodal to CK-MM. AK may interfere with chemical or immunoinhibition
methods, causing a falsely elevated CK or CK-MB value.

 reacts w/ ADP to produce ATP
 causes falsely elevated CK activity
 Hemolysis – RBCs are devoid of CK but are rich in AK, thus hemolysis should be avoided
o Reference range
 TOTAL CK:
 Male : 15 – 160 U/L (37°C)
 Female : 15 – 130 U/L (37°C)
 CK – MB : < 6% Total CK
 Separation Techniques
Page | 23
o Electrophoresis – reference method and the most useful method
 bands are visualized by incubating the support with a concentrated CK assay using the reverse reaction
 NADPH formed is observed with the bluish-white fluorescence after excitation by ultra violet light
 Allows visualization of AK
o Ion Exchange Chromatography
 Potential for being more sensitive & precise than electrophoresis
 On unsatisfactory column:
 CK-MM may merge into CK-MB
 CK-BB may be eluted w/ CK-MB
 Macro-CK may elute w/ CK-MB
o Immunoassays
 Measure the conc. of Enzyme protein rather than Enzyme activity
 detects enzymatically inactive CK-2
o Immunoinhibition
 an anti-CK-M subunit antiserum is used to inhibit both M subunits of CK-MM and the single M subunit
og CK-MB and allows determination of the enzyme activity of the B subunit of CK-MB and the B
subunit of CK-BB
 The residual activity after inhibition is multiplied by 2 to account for MB activity (50% inhibited). The
major disadvantage of this method is that it detects BB activity, which, although not normally
detectable, will cause falsely elevated MB results when BB is present.
 In addition, the atypical forms of CK-Mi and macro-CK are not inhibited by anti-M antibodies and also
may cause erroneous results for MB activity.

Page | 24
 Laboratory Activity No. 4: CREATINE KINASE
CK-MB Immunologic Method

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure the activity of Creatine kinase in serum using
immunologic method by viewing simulation video which is recorded by the laboratory instructor.
2. To be able to understand the precautions in processing the specimens for such enzyme assays and
recognize the factors that affect the reaction.
3. To be able to describe the basic principle of reaction involved in the assay.
4. To be able to associate an elevated CK activity with pathological conditions.

Materials and Equipment:

1. Venipuncture set 8. Water bath (37C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
3. Stat fax tubes 10. Labeling tapes
4. Beaker 11. Parafilm
5. Pasteur pipets 12. CK Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

Principle of the Reaction:

CK-MB consists of the subunits CK-M and CK-B. Specific antibodies against CK-M inhibit completely CK-MM
activity (main part of the total CK activity) and CK-M subunit of CK-MB. Therefore, only CK-B activity is measured, which
is half of the CK-MB activity.

Reagent Components:
Buffer (pH 6.7) ADP
Creatine phosphate NADP
Glucose Diadenosine-5‘-pentaphosphate
N-acetyl-L-cysteine Glucose-6-phosphate dehydrogenase
Magnesium acetate Hexokinase
EDTA Anti-CK-M monoclonal antibodies

Precautions:
Reagent may contain some non-reactive and preservative components. It is suggested to handle it carefully,
avoiding skin contact and ingestion.

Specimen:
Serum is the preferred specimen. Plasma containing heparin, EDTA, citrate or fluoride may produce
unpredictable reaction rates. CK activity in serum is unstable and is rapidly lost during storage. CK is inactivated both by
bright daylight and by increasing specimen pH owing to loss of carbon dioxide; accordingly, specimens should be stored
in the dark in tightly closed tubes. CK is susceptible to thermal denaturation; the degree of inactivation corresponds to the
degree of temperature increase. Therefore, the serum specimen should be chilled at 4C as rapidly as possible after
collection. A slight degree of hemolysis can be tolerated because erythrocytes contain NO CK activity. However,
moderately or severely hemolyzed specimens are unsatisfactory because enzymes and intermediates liberated from the
erythrocytes may affect the lag phase and the side reactions may occur in the assay system.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The absorbance reading will then be shown to the students for them to be able to compute for the results.

BLANK SAMPLE
Working reagent 1.0mL 1.0mL
Pre-incubate at 37C for 5 minutes
water 40 μL
Standard reagent - -
serum - 40 μL
Mix; incubate at 37C and obtain the first absorbance reading after 1 minute against reagent blank. Perform
five (5) other readings at 60 second intervals. Calculate the A/min.

Page | 25
Calculations:
CK (U/L) = A/min x 8254
Activity in ukat/L = U/L x 0.0167

Linearity: The method is linear up to 2000 U/L

Expected Values: Serum= <24 U/L (<0.40 ukat/L)

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text message.

1. Describe the importance of Creatine kinase activity in muscle metabolism.

2. Describe the tissue distribution of the isoenzymes of Creatine kinase and their clinical significance.
3. What are the atypical forms of Creatine kinase? Associate these to pathological conditions.
4. Enumerate & describe the factors which may affect CK determination in serum.

Page | 26
C. LACTATE DEHYDROGENASE – LDH (E.C. 1.1.1.27)
 catalyzes the interconversion of lactic acid and pyruvic acids
 NAD - a hydrogen transfer enzyme that uses the coenzyme NAD as a hydrogen acceptor
 Reaction catalyzed:

 Tissue Sources:
o Present in all cells of the body
o Widely distributed in the body
o High activities: heart, liver, skeletal muscle, kidney & RBCs
o Lesser amounts: Lungs, smooth muscle & brain
 Structure:
o Has a molecular weight of 128,000 daltons with 5 isoenzymes fractions, each with 4 sub-units
o The structure has two different polypeptide chains (H-heart and M-muscle)
 LDH Isoenzymes
Isoenzymes Distribution Localization Associated conditions
in %
LDH1 (HHHH) 14 – 26 Heart and RBC Myocardial infarct and
hemolytic anemia
LDH2 (HHHM) 29 – 39 Heart and RBC Megaloblastic anemia, acute
renal infarct and hemolyzed
sample
LDH3 (HHMM) 20 – 26 Lungs, lymphocytes, spleen and pancreas P. embolism, lymphocytosis
and acute pancreatitis
LDH4 (HMMM) 8 – 16 Liver Hepatic injury
LDH5 (MMMM) 6 – 16 Skeletal muscle S. muscle injury
*Note:
-in the sera of healthy individuals, the major isoenzyme fraction is LDH2 followed by LDH1, LDH3, LDH4 and LDH5.
-LDH1 migrates fastest towards the anode followed in sequence by the other fractions with LDH5 migrating slowest
-LDH1 & LDH2 are present in the same extent in tissues however, cardiac tissue and RBC contains a higher conc. Of
LDH1 and for this reason, in conditions involving cardiac necrosis (AMI) and intravascular hemolysis, LDH1 will
increase to a point at w/c it is presented in greater conc. Than LDH2 (LDH flipped Pattern) LDH1>LDH2
- LDH1 is not specific to cardiac tissue and not a preferred marker for AMI
LDH6 (Alcohol Migrates LDH5 is elevated concurrently with the Patients w/ arteriosclerotic
dehydrogenase) cathodic to appearance of LDH6, this may be caused by CV failure which signifies
LDH5 hepatic congestion due to cardiovascular grave prognosis &
disease.Therefore it is suggested that LDH6 may impending death
reflect liver injury secondary to severe circulatory
insufficiency
 Assay for LDH Activity
o Either a forward or reverse reaction has been used in clinical assays
o Lactate is a more specific substrate compared to pyruvate
o LD-1 prefers the forward reaction whereas LD-5 prefers the reverse reaction
1. Wacker Method (forward rxn) – pH 8.8
 Most commonly used method; produces NADH; not affected by product inhibition
2. Wrobleuski La Due (reverse rxn) – pH7.2
 About 2x faster than the forward reaction and is preferred method for dry slide technology
 Smaller specimen volume requirement
3. Electrophoresis
 widely used historically
 Isoenzymes can be detected either fluorometrically or colorimetrically
 α-hydroxybutyrate: greater affinity w/ H-subunits
 used to measure LDH-1 activity
 Sources of errors
o HEMOLYSIS – LDH in RBCs is 100 – 150x than that in serum; any degree is unacceptable
o Unstable in serum at any storage °T
 Stored at 25°C & analyzed w/in 48 hrs.
o LDH-5: most labile enzyme
 loss of activity at 4°C than at 25°C
o Reference Range: 100 – 225 U/L (37°C)
Page | 27
 Laboratory Activity No. 5: LACTATE DEHYDROGENASE
Continuous monitoring Assay

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure the activity of Lactate dehydrogenase in serum using a
continuous monitoring or kinetic assay by viewing simulation video which is recorded by the laboratory
instructor.
2. To be able to understand the precautions in processing the specimens for such enzyme assays and
recognize the factors that affect the reaction.
3. To be able to describe the basic principle of reaction involved in the assay.
4. To be able to associate elevated LDH activity with pathological conditions.

Materials and Equipment:

1. Venipuncture set 8. Water bath (37C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
3. Stat fax tubes 10. Labeling tapes
4. Beaker 11. Parafilm
5. Pasteur pipets 12. LDH Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

Principle of the Reaction:

Lactate dehydrogenase (E.C. 1.1.1.27) catalyzes the conversion of pyruvate to L-lactate in the presence of
+ +
NADH, which is converted to NAD . The rate of conversion of NADH to NAD is monitored at 340 nm and is proportional
to LDH activity.

Reagent Components:
Phosphate buffer (pH 7.5)
Sodium pyruvate
NADH

Precautions:
Reagent may contain some non-reactive and preservative components. It is suggested to handle it carefully,
avoiding skin contact and ingestion.
Specimen: Serum, plasma (heparinized or EDTA). Avoid hemolysis. LDH activity is stable for 3 days in samples stored
at 2 - 8C

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The absorbance reading will then be shown to the students for them to be able to compute for the results.
BLANK SAMPLE
Working reagent 1.0mL 1.0mL
Pre-incubate at 37C for 5 minutes
water 10 μL
Standard reagent - -
serum - 10 μL
Mix; incubate at 37C and obtain the first absorbance reading after 1 minute against reagent blank. Perform
three (3) other readings at 60 second intervals. Calculate the A/min.

Calculations:
LDH (U/L) = A/min x 16030
Activity in ukat/L = U/L x 0.0167

Linearity: The method is linear up to 4000 U/L

Expected Values: 225 – 450 U/L (3.75 – 7.51 ukat/L)

Page | 28
Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or text
message.

1. Distinguish the isoenzymes of Lactate dehydrogenase in terms of polypeptide components, tissue distribution and
clinical significance.
2. What are the atypical forms of Lactate dehydrogenase? Associate these to pathological conditions.
3. In relation to electrophoretic migration pattern of LD isoenzymes, what is the so-called ―flipped pattern‖?
4. Enumerate and describe the factors which may affect LDH determination in serum.

Page | 29
D. ACID PHOSPHATASE (E.C. 3.1.3.2)
 ACP belongs to the same group of phosphatase enzymes as ALP and is a hydrolase that catalyze the same
reactions
 Major difference between ACP and ALP is the pH where ACP activity takes place at a pH of 5.0
 Isoenzymes
o Prostatic ACP (band 1) - inhibited by tartrate
o Granulocytes (bands 2 & 4)
o Platelets, RBCs & Monocytes (band 3) - major form in plasma
o Bone - osteoclasts (band 5) - Resistant to tartrate inhibition
 ACP Elevation:
o Prostatic isoenzyme
 Historically, ACP measurement aid in detection of prostatic carcinoma & metastatic carcinoma of the
prostate
 Although today newer markers such as PSA is more specific screening and diagnostic tool
 Elevations may also be found in prostatic hyperplasia and prostatic infarction
 elevations however may return to normal within 24 hours
 Elevations is also found in urinary tract obstruction, carcinoid tumors of rectum & prostatic massage
 Medico legal cases
 proved to be useful in forensic chemistry and suspected rape cases
 Vaginal washings are examined for seminal fluid where ACP activity can be observed from first
12 hours up to 4 days.
 Elevations of ACP activity is presumptive evidence of rape in such cases
o Bone isoenzyme
 Elevations of the bone isoenzyme is seen in active osteoclast-mediated bone resorption, Gaucher‘s
cells and Hairy cell leukemia
 Method of measuring ACP Activity
METHODS SUBSTRATE END-PRODUCTS
1. Gutman and gutman Phenylphosphate Inorganic PO4
2. Shinowara PNPP p-nitrophenol
3. Babson, Read and Philips Alpha-naphthyl PO4 Alpha-naphthol
4. Roy and Hillman Thymolphthalein monophosphate Free thymolphthalein
Note: Assays for total ACP use the same techniques as in ALP assay but are performed in acidic pH
- Reaction products are colorless in reaction pH, and alkali solution is used to stop reaction and transform
products into chromogens w/x is measure spectrophotometrically
- Thymolphthalein monophosphate is the substrate of choice for quantitative endpoint reaction
Chemical Inhibition • Reaction is measured before addition of Tartrate (Total ACP)
• After addition: Residual activity
• Total ACP – Residual activity = Prostatic ACP
Prostatic ACP • Thymolphthalein monophosphate – preferred substrate for
quantitative endpoint reactions (Modified by Roy)
• α-naphthyl phosphate – continuous monitoring methods (Hillman
method)

Page | 30
 Laboratory Activity No. 6: ACID PHOSPHATASE
Bessy, Lowrey and Brock Method

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure the activity of Acid Phosphatase in serum by viewing a
simulation video which is recorded by the laboratory instructor.
2. To be able to understand the different factors that may influence the assay
3. To be able to describe the different methods employed for laboratory determination of ACP activity.
4. To be able to associate elevated ACP activity with different pathological conditions.

Materials and Equipment:

1. Venipuncture set 8. Water bath (37 C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
3. Stat fax tubes 10. Labeling tapes
4. Beaker 11. Parafilm
5. Pasteur pipets 12. ACP Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

Principle:
The enzyme acid phosphatase (E.C. 3.1.3.2) catalyzes the hydrolysis of 4-nitrophenylphosphate (p-NPP) to
release p-nitrophenol at an acid pH. The p-nitrophenol formed is determined spectrophotometrically at 450 nm to give a
measurement of acid phosphatase activity in the sample.
Reagent Composition:
p-nitrophenylphosphate (PNPP)
Acid phosphatase diluent
p-nitrophenol standard (15.6 U/L)

Specimen collection and Storage

1. Use only clear, unhemolyzed serum.
2. Serum must be separated from the clot within two hours after collection.
3. ACP activity is extremely labile at room temperature. Stabilization of the enzyme can only be achieved by
acidifying with the acetate buffer provided. Add 20 μL of buffer per 1.0 ml of serum then mix. Treated serum will
remain stable for seven days when kept refrigerated at 2-8 °C.
4. Do not use plasma. Some anticoagulants inhibit ACP or cause turbidity.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The absorbance reading will then be shown to the students for them to be able to compute for the results.
STANDARD SAMPLE
ACP Substrate (PNPP) 250 μL
Standard reagent 50 μL -
serum - 50 μL
Mix & allow to stand for 10 minutes at room temperature or 5 minutes at 37C
ACP Diluent 1.5mL 1.5mL
Mix and read the absorbance at 450 nm against water blank

Calculation:
ACP (U/L) = Abs of sample x value of standard (15.6 U/L)
Abs of standard
Normal Values:
Males: 4.7 – 13.6 U/L
Females: 5.0 – 11.0 U/L

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.
Page | 31
Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or txt
message.

1. Describe the different isoenzymes of Acid Phosphatase in terms of electrophoretic mobility and inhibition by
various agents.
2. What are the sources of interferences in the ACP assay?
3. Discuss the clinical significance associated with elevated activity ACP in serum.
4. Describe how ACP activity can be used for forensic studies in medico-legal cases.

Page | 32
E. ALKALINE PHOSPHATASE (E.C. 3.1.3.1)
 Belongs to a group of enzymes that catalyzes the hydrolysis of various phosphomonoesters in alkaline pH
 Considered a non-specific enzyme, able to react with many different substrate
 It liberates inorganic phosphate from an organic phosphate ester w/ the concomitant production of an alcohol
 Mg is required as an activator
 Major Isoenzymes
o Placental isoenzyme
o Intestinal isoenzyme
o Liver isoenzyme
o Bone isoenzyme
 Clinical significance
o ALP elevations is found in the following conditions
 Obstructive hepatic disorder (Liver isoenzyme)
 Paget‘s disease, Osteitis deformans and increased osteoblastic activity
 (Bone isoenzyme)
 DM, renal failure and cirrhosis (Intestinal isoenzyme)
o ALP Isoenzymes in cancer
 in addition to the major isoenzymes, these are fractions associated with neoplasm
 They are referred to as carcinoplacental alkaline phosphatase because of their similarities to the
placental Isoenzyme
 Regan isoenzyme – patients w/ a particular type of lung CA
 Nagao isoenzyme - Adenocarcinoma
 Kasahara isoenzyme – hepatoma
 Measurement of ALP Activity
o Bowers and McComb Method
 Most specific method recommended by IFCC
 Kinetic or continuous monitoring method method (pH 10.2) which is based on the molar absorptivity
of para-nitrophenol
 p-nitrophenylphosphate (colorless cmpd) is hydrolyzed p-nitrophenol (yellow) and subsequent
liberation of phosphate ions
 increase in absorbance is directly proportional to ALP activity
o Separation of isoenzymes
 Inhibition method
 Phenylalanine - reduces activity of intestinal & placental isoenzymes
 Levamisole - inhibits bone & liver isoenzymes
 Heat fractionation (measurement before and after heating)
 Shows stability of isoenzyme at 56 degree Celsius
o Placental isoenzyme is the most heal stable followed by intestinal, liver then bone
isoenzyme is the most heat labile fraction
 Electrophoresis
 Makes use of agarose gel, polyacrylamide gel and cellulose acetate
 Liver fraction migrates the fastest, followed by bone, placental then the intestinal is the
slowest isoenzyme.
 Use of enzymes or lectins
 Because of the similarity between bone and liver forms, enzymes or lectins maybe used to
further separate the two.
Summary of specificity of enzymes
High specificity Moderate specificity Low specificity
-ACP: RBC & Prostate -AST: Liver, Heart & Skeletal muscle -LDH: All tissues
-ALT: Liver -CK: Heart, Skeletal muscle & Brain
-AMS: Pancreas & Salivary Glands -ALP: Liver, Bone, Kidney & Pancreas
-Lipase: Pancreas

Page | 33
 Laboratory Activity No. 7: ALKALINE PHOSPHATASE
Continuous monitoring Assay

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure the activity of alkaline phosphatase in serum by employing
a continuous monitoring or kinetic method by viewing a simulation video which is recorded by the laboratory
instructor.
2. To be able to describe different methods employed for laboratory determination of ALP activity.
3. To be able to correlate ALP activity with other liver profile test results.
4. To be able to associate elevated ALP activity with different pathological conditions.

Materials and Equipment:

1. Venipuncture set 8. Water bath (37 C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
3. Stat fax tubes 10. Labeling tapes
4. Beaker 11. Parafilm
5. Pasteur pipets 12. ALP Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

Principle of reaction:
The enzyme alkaline phosphatase (E.C. 3.1.3.1) catalyzes the hydrolysis of 4-nitrophenylphosphate (4-NPP) to
release 4-nitrophenol under alkaline conditions. The 4-nitrophenol formed is determined spectrophotometrically at 405 nm
to give a measurement of alkaline phosphatase activity in the sample. The present method has been made according to
IFCC.

Reagent Composition:
2-amino-2-methyl-1-propanol buffer (pH 10.4)
Magnesium acetate
Zinc sulfate
HEDTA
4-nitrophenylphosphate
Specimen / Stability: serum or heparinized plasma
Sera kept at room temperature usually show a slight but real increase in activity, which varies from 1% over a 6-
hour period to 3 – 6% over 1-4 day period. Even in sera stored at refrigerator temperature, activity increases slowly. In
frozen sera, activity decreases but slowly recovers after thawing the serum.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The absorbance reading will then be shown to the students for them to be able to compute for the results.
BLANK SAMPLE
Working reagent 1.0mL 1.0mL
Pre-incubate at 37C for 5 minutes
water 20 μL
Standard reagent - -
serum - 20 μL
Mix; incubate at 37C and obtain the first absorbance reading after 1 minute against reagent blank. Perform
three (3) other readings at 60 second intervals. Calculate the A/min.

Calculation:
ALP (U/L) = A/min x 2757
Activity in ukat/L = U/L x 0.0167

Expected Values:
Males: 35 – 104 U/L (0.58 – 1.74 ukat/L)
Females: 40 – 129 U/L (0.67 – 2.15 ukat/L)

Linearity:
The method is linear up to 3000 IU/L.
Page | 34
Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or text
message.

1. Characterize the various ALP isoenzymes in terms of electrophoretic mobility, stability towards heat and inhibition
with various agents.
2. Discuss the clinical significance of ALP.
3. Describe the atypical forms of ALP, particularly those which are detected in the presence of malignancy.
4. Aside from the method employed in the laboratory, describe current methods for the determination of ALP activity
in routine laboratories.

Page | 35
F. GAMMA-GLUTAMYL TRANSFERASE (E.C. 2.3.2.1)
 Catalyzes the transfer of γ-glutamyl residue from γ-glutamyl peptides to amino acids, other peptides, or H2O
 in most biological systems, glutathione serves as the gamma glutamyl donor
 Tissue sources:
o found primarily in tissue of the kidney, brain, prostate, pancreaas and liver
o they are cell membrane bound with increased secretory and absorptive properties
o Half-life: 7-10 days but may persist up to 28 days in cases of alcoholic liver disease.
 Diagnostic significance:
o Liver damage – major source
 In the liver, GGT is located in the canaliculi of the hepatic cell and the epithelial cell lining of the biliary
ducts. Because of its location, it is elevated in virtually all hepatobiliary disorders
o Smoking – moderate = 10%
 smoking increase the risk of liver cancer and liver cirhosis, promotes production of cytokines and
chemicals that cause inflammation to the liver
o Medication – heavy = 20%
o Ethanol abuse
 Alcohol affects GGT activity-Elevations may indicate alcoholism, particularly chronic alcoholism/heavy
drinkers – 2-3X ULN
 GGT assay are useful in monitoring abstinence from alcohol and used by treatment centers
o GGT Decrease:
 Pregnancy: 1st trimester = dec. 25%
 Oral contraceptives: dec. 20%
 Estrogens and oral contraceptives are both associated with several liver related
complications including intrahepatic cholestasis, sinusoidal dilatation, hepatic adenomas,
hepatocellular carcinoma, hepatic venous thrombosis and an increase risk of gallstones thus
may affect GGT levels
 Assay for GGT
Note:
- Nitroaniline-a chromogenic product w/ strong
absorbance at 405-420nm
- Activity is stable at least 1 month at 4dc and 1
year at -20 degree Celsius
- Non hemolyzed sample is preffered but EDTA
plasma has also been used
- Heparin-produce turbidity in the reaction mixture
- Citrate, oxalate and fluoride-depress GGT
activity by 10 to 15 %
- Normal Value: 6-45 U/L men, 3-30 U/L women
G. Pancreatic Enzymes

G1. AMYLASE (AMS): E.C. 3.2.1.1

 Chemical Name: α-1,4-glucan-4-glucanohydrolase
o Hydrolases are enzymes that catalyzes the breakdown of starch and glycogen
o Starch is made up of both Amylose and Amylopectin
o Amylose-long unbranched chain of glucose linked by a,1-4 glycosidic bonds (glucose molecule)
o Amylopectin-branched chain polisaccharide with a,1-6 linkage (polysaccharide)
o a-AMS attacks only the a,1-4 glycosidic bonds to produce the degradation products: Glucose, Maltose and an
intermediate chain (Dextrin)
o Characteristics
 MW: 45,000 daltons – considered the smallest enzyme and can pass through glomerular filter
 Metalloenzyme: Calcium
 Optimum pH: 6.9 – 7.0 in serum
 Tissue Sources:
o The greatest source is pancreas but present also in the salivary gland
 Isoenzymes of Amylase
S-type Isoamylase P-type Isoamylase
- Its activity starts in the salivary gland where it initiates the - Synthesized by the pancreatic acinar cells and secreted
hydrolysis of starch while food is in the mouth and into the intestinal tract via the pancreatic duct system
Page | 36
esophagus - Its action is favored by the mildly alkaline condition of the
-Its action is terminated by the acidity of the stomach duodenum
- Inhibited by monoclonal antibody - Inhibited by monoclonal antibody
- Inhibited by protein isolated from wheat - Not inhibited by protein isolated from wheat
Note: The isoenzymes of salivary origin migrates most quickly (S1,S2,S3) , P type are slower (P1,P2,P3)
- The most commonly observed fractions: P2, S1 and S2
- In acute pancreatitis: P3 predominates
- S type represent 2/3 of AMS activity of normal serum
- P type predominates in the normal urine
Macroamylases
- Abnormal amylase(usually the S-type) in combination with Immunoglobulins (IgA or IgG) or other high MW proteins
- Not found in urine and concentrated in serum/plasma
- No clinical significance
 Diagnostic significance
o The primary reason for an elevated amylase is Acute pancreatitis
Henry Tietz AMS Activity
2 – 12 H 5 – 8 hours Rise upon onset of symptoms
48 Hours 12 - 72 hours Peak activity (fourfold to sixfold)
3rd – 5th day 3rd – 4th day Returns to normal
Note: The degree of elevation of AMS is helpful in differentiating diagnosis of acute pancreatitis
o Other conditions
 Mumps, perforated peptic ulcer, appendicitis, ruptured ectopic pregnancy, dissecting aortic aneurysm
and biliary tract disease.
 Methods of AMS Determination
o Continuous Monitoring Method
 Improved the reaction stoichiometry; more controlled and consistent hydrolysis conditions
 makes use of coupling of several enzyme systems to monitor amylase activity
o Historical methods
 Saccharogenic: (Nelson Somogyi mod.by Henry & Chiamon)
 Uses starch substrate hydrolyzed to carbohydrate molecule w/ reducing properties
 The amount of reducing sugars are then measured where the concentration is directly
proportional to AMS activity
 Amyloclastic (Iodometric method)
 Hydrolysis of starch by amylase causes a decrease in color intensity
 Starch substrate-attached to iodine, AMS hydrolyze starch and iodine is released-decrease in
the intensity of the initial dark blue complex-the decrease is proportional to AMS conc.
 Chromogenic (Klein, Foreman, Searcy)
 Use a starch substrate to which a chromogenic dye has been attached, forming an insoluble
dye–substrate complex.
 The increase in color intensity of soluble dye-substrate solution is proportional to AMS
activity, measure increase in color from production of product couples with a chromogenic
dye
 Turbidimetry and Nephelometry (Peralta & Reinhart)
 Measures the change in turbidity of starch solution over a short reaction period
Notes
o Normal serum contains both salivary and pancreatic AMS
o Normal amylase / creatinine ratio = 1.0% – 4.0% (0.01 – 0.04)
o A:C ratio (Acute Pancreatitis) = >4.0% (up to 15%)
o An elevated amylase-creatinine clearance ratio has been established as being highly specific for the
diagnosis of acute pancreatitis

Page | 37
 Laboratory Activity No. 8: AMYLASE
Continuous monitoring Assay

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure the activity of Amylase in serum using continuous
monitoring or kinetic method by viewing a simulation video which is recorded by the laboratory instructor.
2. To be able to compare this method used with other methods, noting its advantage over them.
3. To be able to discuss the clinical significance of amylase determination, particularly for the diagnosis of acute
pancreatitis

Materials and Equipment:

1. Venipuncture set 8. Water bath (37 C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
4. Stat fax tubes 10. Labeling tapes
5. Beaker 11. Parafilm
6. Pasteur pipets 12. Amylase Reagent
7. Serological pipets 13. Stat fax machine
8. Centrifuge 14. Applicator sticks

Principle of Reaction:
The enzyme α-amylase (E.C. 3.2.1.1) catalyzes the hydrolysis of 2-chloro-4-nitrophenyl-α-D-maltotrioside
(CNPG3) to release 2-chloro-4-nitrophenol and form 2-chloro-4-nitrophenyl-α-D-maltoside (CNPG2), maltotriose (G3) and
glucose (G). The rate of formation of 2-chloro-4-nitrophenol can be detected spectrophotometrically at 405 nm to give a
measurement of α-amylase activity in the sample.

Reagent Composition:
CNP-G3
NaCl
Calcium acetate
Potassium thiocyanate
Good‘s buffer (pH 6.9)
Stabilizers

Specimen / Stability:
1. After blood collection, promptly separate serum from clot.
2. Non-hemolyzed specimen is the ideal specimen.
3. Heparinized plasma may be used.
4. Other anticoagulants such as citrate and EDTA bind calcium, an ion needed for amylase activity.
5. Amylase in serum is stable for one week at room temperature and up to 2 months when stored refrigerated at 2-8
C and protected against evaporation and bacterial contamination.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The absorbance reading will then be shown to the students for them to be able to compute for the results.
BLANK SAMPLE
Working reagent 1.0mL 1.0mL
Pre-incubate at 37C for 5 minutes
water 25 μL
Standard reagent - -
serum - 25 μL
Mix; incubate at 37C and obtain the first absorbance reading after 1 minute against reagent blank. Perform
three (3) other readings at 60 second intervals. Calculate the A/min.

Calculations:
Amylase (U/L) = A/min x 3178
Activity in ukat/L = U/L x 0.0167

Expected values: Serum / Plasma: <96 U/L (<1.60 ukat/L)

Page | 38
Linearity: The method is linear up to 3000 U/L

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or text
message.

1. What is the importance of determining amylase activity in urine?

2. Describe the clinical significance of determining amylase/ creatinine clearance.
3. What is macroamylasemia?
4. Discuss the pathogenesis of acute pancreatitis.
5. Describe the reference method employed for amylase determination in serum.

Page | 39
 G2. LIPASE (LPS)
 Chemical name: Triacylglycerol Acylhydrolase
 Systematic name: E.C. 3.1.1.3
 Reaction catalyzed:
o Hydrolyzes glycerol esters of long chain fatty acids
o Acts only on emulsified substrate
o Small molecule and filtered in the glomeruli but not normally seen in urine bec. It is totally reabsorbed by the
renal tubules
 Importance: Responsible for triglyceride metabolism
 Tissue sources: Pancreas (1° source), GIT, leukocytes, adipose cells, colostrum
 Methods of Determination
o Specimen: serum (stable at RT), pleural fluid, ascitic fluid
o Inhibitors: heavy metals, quinine & some esterase inhibitors
o Not inhibited by fluoride or arsenilate
o Bacterium increases concentration of LPS
 Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia
o For Long-Chain Triglycerol Substrate:
 Titrimetric Methods: fatty acid is titrated w/ alkali solution
 Turbidimetric Methods: emulsion of fats produces milky appearance
 Spectrophotometric Method: Myrtle and Zell method: fatty acids are extracted using Petroleum ether
 Fluorometric Method: Fatty acid + chemical reagent: Fluorescein (4-methyl bellifuzone)
o For Short-Chain TG Substrate:
 Advantages: Analytical, greater solubility in aqueous medium
 Disadvantage: unphysiologic substrate
 Erlanson & Bergstrom – Tributyrin
o Other methods
 Radio-immunoassay : makes use of 1251-labeled lipase
 Latex Agglutination : antibodies to pancreatic lipase are bound to latex particles
 Coupled enzymatic method
 Diagnostic significance
Henry Tietz AMS Activity
4-8H 4 – 8 hours Rise upon onset of symptoms
24 Hours 24 hours Peak activity
8 to 14 days 7 to 14 days Returns to normal
Note: diagnostic marker for acute pancreatitis
- The increase in serum LPS in not necessarily proportional to the severity of the attack
o Other conditions:
 Elevations of lipase are also observed in perforated peptic ulcer, duodenal ulcer, intestinal
obstruction and mesenteric vascular obstruction.

Page | 40
 Laboratory Activity No. 9: LIPASE
Continuous monitoring Assay

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure the activity of Lipase in serum using continuous
monitoring or kinetic method by viewing a simulation video which is recorded by the laboratory instructor.
2. To be able to compare this method used with other methods, noting its advantage over them.
3. To be able to discuss the clinical significance of Lipase determination, particularly for the diagnosis of
acute pancreatitis

Materials and Equipment:

1. Venipuncture set 8. Water bath (37 C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
3. Stat fax tubes 10. Labeling tapes
4. Beaker 11. Parafilm
5. Pasteur pipets 12. Lipase Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

Principle of Reaction:
The colorimetric substrate 1,2-o-Dilauryl-rac-glycero-3-glutaric acid-(6‘ methyl-resorufin) ester is cleaved by
pancreatic lipase and the resulting dicarboxylic acid ester is hydrolyzed under alkaline conditions to yield the chromophore
methylresorufin. The kinetic of color formation at 580 nm is monitored and it is proportional to lipase activity in the sample.

Reagent Composition:
Reagent A: Good‘s buffer (pH 8.0)
Co-lipase
Desoxycholate
Taurodesoxycholate
Calcium ions
Detergent
Preservative
Reagent B: Tartrate buffer (pH 4.0)
Lipase substrate
Stabilizer
Preservative

Specimen / Stability: Serum or heparinized plasma. Samples are stable for 7 days at 2 – 8 C.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The absorbance reading will then be shown to the students for them to be able to compute for the results.
BLANK CALIBRATOR SAMPLE
Reagent A 1.0mL 1.0mL 1.0mL
water 20 μL
calibrator - 20 μL -
serum - - 20 μL
Mix carefully (do not shake); incubate at 37C for 5 minutes.
Reagent B 250 μL 250 μL 250 μL
Mix; incubate at 37C and obtain the first absorbance reading after 2 minutes against reagent blank. Perform
two (2) other readings at 60 second intervals. Calculate the A/min.

Calculations:
A/min = A/min(calibrator or sample) - A/min(blank)
U/L (methylresorufin at 37C) = A/min (sample) x calibrator value
A/min (calibrator)

Expected values: Serum / Plasma: ≤60 U/L

Page | 41
Linearity: The method is linear up to 300 U/L

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text massage.

1. Compare the measured activities of Amylase & Lipase in acute pancreatitis.

2. Enumerate other conditions which will cause an elevation in lipase activity.
3. Completely describe Trypsin.
4. Discuss the importance of determining Trypsin in serum for differential diagnosis of Pancreatitis.

Page | 42
G3. Trypsin
 Pancreas-specific serine protease
 Solely produced by pancreatic acinar cells
 Cleaves peptide bonds formed between the –COOH group of lysine or arginine with other amino acids
 Zymogen: trypsinogen-1 & trypsinogen-2
o Zymogen-an inactive substance that is converted into an enzyme when activated by another enzyme.
 Activator: Enterokinase
 Inactivated in plasma by: α-1-antitrypsin & α-2-macroglobulin
 Diagnostic significance
o TRYPSIN-1 (Cationic)
o Elevated in:
 Acute pancreatitis – TRY-1 rises in parallel with serum AMY activity
 Chronic renal failure
 Cystic fibrosis - fibrosis-a common inherited genetic disease in pediatric patients, newborns has
severe pancreatic insufficiency, accumulation of thick muscous secretion in the pancreatic ducts that
inhibit secretion of pancreatic digestive enzymes
o TRYPSIN-2 (Anionic)
 Acute pancreatitis – Serum trypsinogen-2 increases more than trypsinogen-1 (10-fold greater)
 larger amounts are excreted into urine
 Method for determination
o Commercial IMMUNOASSAYS detect: Trypsinogen-1, Try-1 and Try-1-a1-antitrypsin complex
o Urinary trypsinogen-2 test strip: based on the use of immunochromatography with monoclonal antibodies

H. Miscellaneous Enzymes
H1. Angiotensin-Converting Enzyme (ACE) - E.C. 3.4.15.1
 Other names: A.k.a peptidyl dipeptidase A / Kininase II
 Conversion of angiotensin I to angiotensin II
o Takes part in the RAAS - System that control blood pressure by regulating the volume of fluid in the body by
regulating aldosterone (sodium balance)
 if there is perceived volume depletion or low sodium filtered, Renin is produced juxtaglomerular
apparatus (proteolytic enzyme), initiate cleavage of angiotensinogen
 Converts Angiotensin I to the vasoconstrictor angiotensin II
 It increased blood pressure and stimulate aldosterone release
 Inactivation of bradykinin, encephalin, tachykinin
 Most activity: lungs & endothelial cells
 Diagnostic significance:
o Diagnosis and monitoring sarcoidosis (decrease in ACE)
 an inflammatory disease that affects multiple organs in the body, but mostly the lungs and lymph
glands. In people with sarcoidosis, abnormal masses or nodules (called granulomas) consisting of
inflamed tissues form in certain organs of the body are found
o Elevations are more likely in pulmonary involvement

H2. True Cholinesterase (E.C. 3.1.1.7)

 an enzyme that splits acetylcholine (a neurotransmitter) into acetic acid and choline
 Major source: CNS, RBCs, Lung and Spleen
 Measurement of activity: True Cholinesterase uses acetylcholine
 Diagnostic significance: Diagnosis of organophosphate insecticide poisoning (cholinesterase activity is inhibited)

H3. Pseudocholinesterase (E.C. 3.1.1.8)

 Other name: Acylcholine Acyl hydrolase
 Cleaves succinylcholine (muscle relaxant used during surgery)
 Major source: Liver, myocardium & pancreas
 Measurement of activity: Pseudocholinesterase uses butyrylthiocholine
o Released thiocholine reacts with Ellman‘s reagent
o Product: measured photometrically
 Diagnostic significance:
o Liver function test: Pseudocholinesterase prod‘n (decreased activity in malnutrition)
o For diagnosis of genetic variants - Prolonged apnea after using succinylcholine during anesthesia

Page | 43
H4. Leucine aminopeptidase (E.C. 3.4.11.1)
 Hydrolyzes amino acids from the N-terminal end of the peptides
 An exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of polypeptide
chains
 LAPs are widely distributed, ubiquitous in nature, and are of critical biological importance because of their role in
protein degradation
 LAP isoenzymes:
o Liver isoenzyme
 major isoenzyme found in the canalicular membrane with similar activities to GGT and ALP
 Diagnostic significance
 Increased in obstructive liver diseases (NOT in bone disease)
o Sensitive than ALP & 5‘-NT
o Less sensitive & specific than GGT
 Elevated in SLE, breast, endometrial and ovarian carcinomas, germ cell tumors of the ovary
and testis
o Placental isoenzyme
 Important in hydrolysis of Oxytocin & Angiotensin II
 Increased during 3rd trimester of pregnancy
 Measurement of activity: Starch gel electrophoresis
 Normal Values:
o MALES: 19.2 – 48.0 IU/L
o FEMALES: 18.0 – 44.4 IU/L

H5. 5’-Nucleotidase (E.C.3.1.3.5) or 5’-NT

 Other name: 5‘-ribonucleoside phosphohydrolase
 Acts on nucleosides w/ PO4: ATP & GTP – Guanosine triphosphate
 Tissue source: Widely distributed but predominates in the liver
 Measurement of activity: Assay uses large amounts of other non-nucleoside substrates
o Chelating agents interfere
 Diagnostic significance:
o may reflect hepatobillary disease with considerable specificity seen in acute hepatitis and also in ovarian
carcinoma and rheumatoid arthritis
o together with other enzymes such as ALP, LDH it can also serve as a tumor marker

Page | 44
 Assessment No. 3

True or False: Please answer the following questions; write your answer on the space provided.

1. Most enzymes are measured by monitoring the rate of absorbance change at 340 nm
as NADH is produced or consumed.
2. The amount of enzyme in the serum can be increased by necrosis, altered
permeability, secretion, or synthesis.
3. No enzyme is truly tissue specific and diagnostic accuracy depends upon recognizing
changes in plasma levels that characterize different diseases.
4. Most enzymes require metals as activators or cofactors.
5. LD is increased moderately to high levels in most causes of liver disease.
6. Liver disease produces an elevated LD-3, LD-4 and LD-5.
7. RBCs are rich in LD-1 and LD-2, and even slight hemolysis will falsely elevate results.
8. Hemolytic, megaloblastic, and pernicious anemias are associated with LD levels of
10–50 times the URL.
9. The Oliver–Rosalki method for CK is based upon the formation of ATP from creatine
phosphate.
10. Positive interference in the Oliver–Rosalki method can occur when adenylate kinase
is present in the serum from hemolysis or damaged tissue.
11. Total CK is neither sensitive nor specific for AMI.
12. CK activity is lost with excessive storage, the most labile isoenzyme being CK-3.
13. In some noncardiac causes of elevated plasma CK-MB such as muscular dystrophy,
there is a persistent elevation of both total CK and CK-MB
14. ALT catalyzes the transfer of an amino group from alanine, a three-carbon amino
acid, to α–ketoglutarate (2–oxoglutarate), forming pyruvate.
15. AST forms oxaloacetate and glutamate from aspartate and α–ketoglutarate (2–
oxoglutarate).
16. Because glutamate is a common product for transaminases, pyruvate (a three-
carbon ketoacid) and glutamate would be generated from the transamination reaction
between aspartate and α–ketoglutarate.
17. Patients with liver disease often have high levels of pyruvate and LD.
18. SGOT refers to the products measured in the in vitro reaction, and is more correctly
named ASTfor the four-carbon amino acid substrate aspartate.
19. ALT may be slightly elevated after an AMI.
20. Although AST and ALT are elevated in alcoholic hepatitis, GGT is a more sensitive
indicator of alcoholic liver disease.
21. GGT and 5´ nucleotidase are markedly elevated in intra- but not in posthepatic
obstruction
22. The method of Bowers–McComb (Szasz modification) is the IFCC-recommended
method for ACP.
23. Amylase is commonly measured using synthetic substrates.
24. Serum amylase usually peaks 2–12 hours following acute abdominal pain resulting
from pancreatitis.
25. Both salivary and pancreatic amylases designated S-type and P-type, respectively,
are present in normal serum and urine.
26. Lipase elevation is of greater magnitude (2-50 × N) and duration than amylase in
acute pancreatitis.
27. 5‗-Nucleotidase is increased primarily in obstructive liver disease and liver cancer.
28. Amylase in humans is a hydrolase that splits the second α 1-6 glycosidic bonds of
polyglucans forming maltose.
29. Urinary amylase peaks concurrently with serum but rises higher and remains
elevated for up to 1 week in cases of acute pancreatitis.
30. Leukemia and Hodgkin‘s disease may cause an elevated leukocyte or bone-derived
ALP.

Page | 45
Reference:

McPherson, Richard. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Elsevier
Saunders, 2011.

Ashwood E., D. Bruns and C. Burtis (2012). Tietz’s Textbook of Clinical Chemistry and Molecular Diagnostics 5th ed.
Philadelphia: W.B. Saunders Co.
th
Bishop, Michael (2013) Clinical Chemistry Procedures, Correlation. (7 ed.). Holland
th
Crook, Martin (2006). Clinical Chemistry and Metabolic Medicine (7 ed.). USA: Hodder Arnold Publication.

Suba, Sally C. (2008). Laboratory Guide in Clinical Blood Chemistry. Rex Book Store, Inc.
th
Wu, Allan (2006). Tietz’s Clinical Guide to Laboratory Tests (4 ed.). Philadelphia: W.B. Saunders.

Clinical Chemistry Links. Institute for Quality in Laboratory Medicine retrieved from http://www.dgrhoads.com/links.shtml

Clinical Chemistry Links. Clinical Laboratory Management Association retrieved from http://www.dgrhoads.com/links.shtml

Clinical Chemistry Links. Clinical and Laboratory Standards Institute retrieved from http://www.dgrhoads.com/links.shtml

Page | 46
 Chapter II. MAJOR ELECTROLYTES

Electrolytes are anions and cations, meaning they are capable of carrying electric charge, which essential
components of all living matter.

Desired learning outcomes:

At the end of this chapter, you should:
a. Recognize the importance of the electrolytes in homeostasis
b. Describe the laboratory methods for electrolyte determination
c. Correlate the plasma level of electrolytes with various metabolic and pathologic processes

Section 1. THE ROLE OF ELECTROLYTES

Electrolytes are essential component in numerous processes. They are responsible in the maintenance of
osmotic pressure & water distribution in the various body fluid compartments. They help in the maintenance of the proper
pH/acid-base balance. Electrolytes also have regulatory properties where some are important in the proper function of the
heart and other muscles. Some electrolytes are involved in oxidation-reduction reactions or electron transfer reaction and
many are cofactors important for enzymes activity. Some electrolytes are even involved in blood coagulation. The major
electrolytes occur as free ions whose properties are unaffected by other ions or molecules.

Assessment No. 4

Question: In your own words, what is the role of electrolytes in patients with severe illness like severe coronavirus
infection? Please place your answer on the space provided.

Reference:

Ashwood E., D. Bruns and C. Burtis (2012). Tietz’s Textbook of Clinical Chemistry and Molecular Diagnostics 5th ed.
Philadelphia: W.B. Saunders Co.

Page | 47
 Section 2. THE MAJOR ELECTROLYTES
+
A. Sodium (NA )
 Sodium is the major cation of ECF and it represents about 90% of extracellular cations
 Sodium plays a central role in maintaining the normal H2O distribution and the osmolality of plasma.

Regulation of Plasma Sodium

1. Intake of Water
 As excess intake of H2O (polydipsia) begins to lower plasma osmolality (measure of hydration status), AVP and
thirst are suppressed, in the absence of AVP, water is not reabsorbed causing large volume of diluted urine to be
excreted
2. Excretion of H2O (affected by AVP release)
 Deficit in water increase plasma osmolality (Both AVP & thirst are activated), AVP contributes by minimizing renal
water loss although thirst is major defense against hyper osmolality and hypernatremia
+
3. Blood volume status, which affects Na excretion
 Adequate blood volume is important to maintain pressure and ensure good perfusion to all tissues and organs.
 The RAAS responds primarily to a decreased blood volume. Renin is secreted in response to decrease blood
flow. Renin converts angiotensinogen to angiotensin I w/c will become angiotensin II that causes vasoconstriction
which increased blood pressure and secretion of aldosterone w/c will increase retention of Na and water
 ANP(Atrial natriuretic peptide)-peptide released form the myocardial atria in response to volume expansion,
promotes Na excretion in the kidney

Renal handling of Sodium

Sodium is initially filtered by the glomeruli. About 60 to 70% of sodium filtered is reabsorbed in the proximal
convoluted tubules along with bicarbonate and water. About 25-30% is reabsorbed in the loop of Henle with chloride and
more water. While reabsorption in the distal convoluted tubules is controlled by aldosterone, a hormone that conserves
sodium.

Image taken from

studylib.net/doc/formation-of-
urine

Important points to remember

The renal threshold of sodium is between 110–130 mmol/L and the kidneys are the ultimate regulators of the
+ +
amount of Na or K in the body. The following are the normal value for sodium:
 Serum/Plasma :136 – 145 mmol/L
 Urine (24 hr) : 40 – 220 mmol/L (varies w/ diet)
 CSF : 136 – 150 mmol/L

Diagnostic Significance
1. Hyponatremia
This is one of the most common electrolyte disorders which happen when plasma sodium levels go down to <135
mmol/L. Hyponatremia is caused by the following conditions:

DEPLETIONAL loss caused by an DILUTIONAL loss due to an ARTIFACTUAL loss or also known
absolute losses of total body increase in water volume as pseudo hyponatremia caused by
sodium analytical errors
Renal losses caused by diminished SIADH or syndrome of inappropriate Pseudo hyponatremia occur when
tubular reabsorption and renal tubular AVP secretion can cause an sodium is measured using ion
acidosis in which tubular transport of uncontrolled increase in water selective electrodes in patients who
electrolytes are impaired. retention hence dilution of salt will have hyperproteinemia and
Non-renal losses are caused by GIT occur. hyperlipidemia
Page | 48
loss through diarrhea and vomiting. In nephrotic syndrome or hepatic
Salt losing enteropathies or salt cirrhosis, plasma proteins are
wasting is when the kidneys are decreased resulting in decreased
normal but conservation of salt is colloidal osmotic pressure which will
impaired. result to migration of water to tissues
Excessive sweating may also lead to causing edema.
hyponatremia. In hyperglycemia, high solutes causes
shift of water from cells to blood
causing dilutional effects.

2. Hypernatremia
This is a less common abnormality which may result because of the following conditions:
Excess water loss Increased sodium intake and Decreased water intake
retention
Hypernatremia because of water loss Diabetes insipidus whether This usually happens in patients or
can be a consequence of GIT losses hypothalamic or nephrogenic may individuals who are not able to tell their
like vomiting and diarrhea. This may cause excess production of need like for example infants, older
also be due to excessive sweating, aldosterone which may lead to over people and those who are mentally
fever and exercise. conservation of sodium. challenged.
This also occurs in people with adipsia
who has impaired thirst sensation.

Page | 49
 Activity No. 10: SODIUM DETERMINATION
Colorimetric Method Based on the Modification of Maruna and Trinder

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure serum sodium level by viewing a simulation video which is
recorded by the laboratory instructor.
2. To be able to describe its principle of reaction.
3. To be able to describe the basic principles of Flame Photometry and Ion Selective
Spectrophotometry used in the determination of sodium.
4. To be able to associate hyponatremia and hypernatremia with various clinical conditions

Materials and Equipment:

1. Venipuncture set 8. Water bath (37C)
2. 13 x 100 mm test tubes for blood 9. Test tube rack
3. Stat fax tubes 10. Labeling tapes
4. Beaker 11. Parafilm
5. Pasteur pipets 12. Sodium Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

Principle of Reaction:
Sodium is precipitated as the triple salt, sodium magnesium uranyl acetate, with the excess uranium then being reacted
with ferrocyanide, producing a chromophore whose absorbance varies inversely as the concentration of sodium in the test
specimen.

Reagent Deterioration:
Turbidity may be a sign of contamination.

Storage and Stability

Store all reagent set at room temperature.

Specimen:
Freshly drawn serum is the serum of choice. Plasma from non-sodium containing anticoagulant (e.g., lithium,
calcium, magnesium or heparin) is an acceptable alternative. Sodium is stable for at least 24 hours at room temperature
and for 2 weeks when refrigerated.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The absorbance reading will then be shown to the students for them to be able to compute for the results.

Filtrate Preparation:
1. Label test tubes: Blank, standard, control, patient, etc.
2. Pipette 1.0 ml of filtrate Reagent to all tubes.
3. Add 50 uL of sample to all tubes and distilled water to the blank.
4. Shake all tubes vigorously.
5. Centrifuge tubes at high speed (1500g) for 10 minutes and test the supernatant fluids as described below, taking
care not to disturb the protein precipitate.
Color development
1. Label test tubes corresponding to the above filtrate tubes.
2. Pipette 1.0 ml Acid Reagent to all tubes.
3. Add 50 uL of supernatant to respective tubes and mix.
4. Add 50 uL of color reagent to all tubes and mix.
5. Zero Spectrophotometer with distilled water at 550 nm.
6. Read and record absorbance of all tubes.

NOTE: The Chemistry reaction of this procedure involves a reduction in absorbance, as opposed to the usual absorbance
increase. The absorbance of blank should be higher than the test samples.

Page | 50
Calculation
(Abs. of blank – Abs. of Sample) x Conc. Of standard = Sodium Concentration
(Abs. of Blank – Abs. of Standard) (150 mEq/L)

Expected Values
135 – 155 mEq/L

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text message.

1. Characterize sodium in the blood. Give its clinical significance.

2. What are the causes of pseudohyponatremia?
3. Describe the underlying principles of Flame Emission Spectrophotometry (FES) and Ion-Selective Electrode
(ISE)

Page | 51
 +
B. Potassium (K )
 This is the major intracellular cation which is found in tissue cells (average of 150mmol/L) and in the RBC (105
mmo/L)

Regulation of Potassium
Like sodium, kidney is also the most important organ in the regulation and handling of potassium. Potassium once
filtered by the glomerulus if almost completely reabsorbed in the proximal convoluted tubules. Under the influence of
aldosterone, potassium is secreted in the distal tubules and collecting duct hence the distal nephron is the principal
determinant of urinary potassium excretion.

Factors that influence potassium distribution

+ + +
1. K loss: Na -K -ATPase pump inhibited by hypoxia, hypomagnesemia or digoxin overdose
 Hypoxia inhibits Na,K-ATPase activity by decreasing the number of active Na pump molecules at the plasma
+

membrane
 The pump is activated by magnesium. Under magnesium deficiency the pump function is impaired, because the
membrane ATpase, the enzyme responsible, now shows reduced activity, The energy substrate for the transport
activity of the sodium/potassium pump is represented by ATP in form of its magnesium complex
 Digoxin serves as a potent inhibitor of pump
+
2. Insulin promotes acute entry of K into skeletal muscle & liver
 insulin promotes acute entry of K into skeletal muscle and liver by increasing Na, K-ATPase activity
3. Catecholamines
 Epinephrine (β2-stimulator) – promote cellular entry of K
+

 Propranolol (β-blocker) – impairs cellular entry of K

+

Diagnostic significance:
1. Hypokalemia – Decreased amount of potassium which is caused by the following:
Increased Cellular uptake Renal losses Excessive GIT losses
Alkalemia promotes intracellular loss RTA may lead to tubular excretion of H GI loss occurs when GI fluid is lost
of H to minimize elevation of Hyperaldosteronism-aldosteron through vomiting, diarrhea, gastric
intracellular pH-both K and Na enters promotes retention of Na and loss of K suction or discharge from an intestinal
cell to promote electronutrality. Cushing's syndrome can also lead fistula
Insulin increase promotes cellular to hypokalemia due to excess cortisol Increased K loss in the stool also
+ +
uptake of K binding the Na /K pump and acting occurs with certain tumors,
like aldosterone. malabsorption, cancer therapy and
Magnesium deficiency deminishes large doses of laxative
activity of Na-K ATPase pump and
enhance secretion of aldosterone
Renal K loss also occur in acute
myelogenous leukemia and acute
lymphocytic leukemia

2. Hyperkalemia – elevated concentration of potassium caused by the following:

Increased intake Cellular shift Decreased renal excretion Artifactual causes
The most common cause of In metabolic acidosis, as Acute or chronic renal Seen often in hemolyzed
hyperkalemia is due to excess H moves failure and samples, thrombocytosis
therapeutic K administration intracellularly to be buffered, hypoaldosteronism and prolonged tourniquet
and the risk is greatest with K leaves the cell to maintain use and excessive fist
IV K replacement seen in electroneutrality. clenching that leads to
dialysis patient Hyperkalemia may result hemoconcentration
when K is released into the
ECF during enhanced tissue
breakdown or catabolism
cytotoxic agents, hemolysis
or tumors)

Reference Range:
 Serum of adults :3.5 – 5.0 mmol/L
 Plasma :3.5 – 4.5 mmol/L
 Newborn :3.7 – 5.9 mmol/L
Page | 52
  CSF :~70% of values in serum
 Urine :25 – 125 mmol/L (varies w/ dietary intake)

Determination of Sodium and Potassium

Specimen for Sodium Specimen for Potassium
+
SPECIMEN for Na : Serum, heparinized plasma, sweat, Serum & plasma – avoid hemolysis
+ +
24-hour urine, liquid feces or GIT fluids (timed collection).
Plasma K < serum (K released from platelets during
For delayed analysis: serum, plasma or urine stored at ref coagulation)
T or frozen Plasma - specimen of choice
Whole blood samples for K determinations should be stored
at room temperature (never ice) and analyze promptly or
centrifuged to remove the cells
Ice cold temperature drive K out of the cell
Methods: Colorimeteric method, FES, AAS and Ion selective electrode
Note: ISE is the most routinely used method which makes use of reference electrodes and measuring electrodes. Sodium
uses glass ion exchange membrane while potassium used liquid ion exchange membrane with valinomycin.

Page | 53
 Activity No. 11: POTASSIUM DETERMINATION
Turbidimetric Method

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure Potassium level in serum by viewing a simulation video
which is recorded by the laboratory instructor.
2. To be able to describe its principle of reaction.
3. To recognize the different factors that can adversely affect potassium assay.
4. To associate hypokalemia and hyperkalemia with various clinical conditions.

Materials and Equipment:

1. Venipuncture set 8. Test tube rack
2. 13 x 100 mm test tubes for blood 9. Labeling tapes
3. Stat fax tubes 10. Parafilm
4. Beaker 11. Potassium Reagent
5. Pasteur pipets 12. Stat fax machine
6. Serological pipets 13. Applicator sticks
7. Centrifuge

Principle of Reaction:
Potassium ions react with Sodium tetraphenyl boron to produce a colloidal suspension. The turbidity of which is
proportional to the potassium concentration in the range of 2-7 mmol/L.
Storage and Stability:
Store all reagents at room temperature. All reagents are stable up to expiration date on the individual bottle label.

Reagent Deterioration:
Do not use if:
1. The reagent is very cloudy
2. The reagent fails to achieve assigned values on fresh control serum.

Specimen:
1. Serum or Lithium-heparin plasma is recommended.
2. Plasma from anticoagulants not containing potassium is also recommended.
3. Potassium in serum is stable for at least 2 weeks at 2-8 °C.
4. Specimens for serum potassium analysis should be free from hemolysis.
5. Serum should be separated from the red cells shortly after collection.
6. Turbid or icteric samples produce falsely elevated results. Bilirubin above 40 mg/dL and Urea Nitrogen above 80
mg/dL will produce elevated results. Sera containing high levels of ammonia should be avoided.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the test. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The absorbance reading will then be shown to the students for them to be able to compute for the results.

1. Label test tubes: Standard, Blank, test, controls etc.

2. Pipet into appropriate tubes:
BLANK STANDARD TEST
Potassium reagent 1.0 ml 1.0 ml 1.0 ml
water 10 μL - -
Potassium Standard - 10 μL -
serum/plasma - - 10 μL
Mix and allow to stand for 3 minutes and measure absorbance of the standard and the test against
reagent blank at 500 nm.

Calculation:
Abs(Test) x conc. of Std (4.0 mmol/L) = Concentration of Test (mmol/L)
Abs (Std)
Notes:
1. Contaminated glassware is the greatest source of error.
2. Sample with values above 7 mmol/L should be diluted 1:1 with NSS, reassayed and the results to be multiplied by
2.
Page | 54
Normal Values:
3.4 – 5.3 mmol/L or 3.5 – 5.3 mEq/L

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text message.

1. Characterize potassium in blood.

2. Discuss the clinical significance of potassium determination in blood.
3. What are the sources of errors that can adversely affect the potassium assay?
4. Aside from the method performed in the laboratory, describe current methods for potassium determination in
routine laboratories.

Page | 55
 -
C. Chloride (Cl )
 Chloride is major extracellular anion and it represents the largest fraction of the plasma total inorganic anion
concentration. In RBC chloride is approximately about 45 to 54 mmol/L while in tissue cell about 1 mmol/L.
Chloride has main importance in maintaining osmolality and water distribution, maintaining osmotic pressure and
has an important role in anion-cation balance in the ECF in achieving electrical neutrality. Chloride shifts
secondary to the movement of sodium or bicarbonate.
Chloride’s role in electrical neutrality
+ -
1. Na is reabsorbed along w/ Cl in the PCT & LH
 In effect, chloride acts as the rate limiting component, in that, Na reabsorption is limited by the amount of Cl
available
 The result is sodium chloride (NaCl), composed of one positively charged sodium ion (Na ) and one negatively
+
−
charged chloride ion (Cl ).
2. Chloride shift
 CO2 generated by cellular metabolism w/in the tissue diffuses out into both the plasma & the RBC
 In RBC CO2 forms carbonic acid(H2CO3)
 This then splits into H and HCO3-(Bicarbonate)
 Deoxyhemoglobin buffers H whereas HCO3 diffuses out into plasma and Cl diffuses into the red cell to maintain
the electric balance of the cell

Image taken from

Regulation of Chloride researchgate.net/figure/shift-of-chloride
Chloride is filtered by the glomerulus. It is passively absorbed along with sodium in the proximal convoluted
tubules and active reabsorption through the chloride pump happens in the ascending loop of Henle. Excessive sweating
+
triggers release of Aldosterone (causes the sweat glands to reabsorb more Na and Chloride)

Diagnostic significance
Hypochloremia - Decrease plasma concentration of Cl Hyperchloremia - Increase plasma concentration of Cl
Cl disorders are often a result of the same cause that Hyperchloremia occur when there is an excess loss of
disturb Na Levels because Cl passively follows Na HCO3 as a result of GI losses, RTA or metabolic acidosis
Hypochloremia-occur with excessive loss of Cl from Acidosis is characterized by a lowered bicarbonate
prolonged vomiting, diabetic acidosis, aldosterone concentration, which is counterbalanced by an equivalent
deficiency of salt losing renal disease such as increase in plasma chloride concentration. For this reason,
pyelonephritis it is also known as hyperchloremic metabolic acidosis
A low serum level of Cl may also be observed in conditions
associated with high serum HCO3 concentration
Addisonian crisis is also known as adrenal insufficiency-
condition occurs when the body cannot produce enough
homones (aldosterone)

Two methods have been widely used for chloride determination

 Mercumetric Titration or the Schales and Schaled method which used diphenylcarbazone as the indicator and
HgCl2 as the end product of the reaction
 Whitehorn Titration Method is done using spectrophotometric reading which uses diphenylcarbazone as the
reagent with reddish complex end point product which is read spectrophotometrically.

Page | 56
 Activity No. 12: DETERMINATION OF CHLORIDE
Colorimetric Method Based on the Modification of Skeggs and Hochestrasser

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure Chloride level in serum by viewing a simulation video
which is recorded by the laboratory instructor.
2. To be able to describe its principle of reaction.
3. To be able to describe the basic principle of sweat chloride determination.
4. To be able associate hypochloremia and hyperchloremia with various conditions.

Materials and Equipment:

1. Venipuncture set 8. Test tube rack
2. 13 x 100 mm test tubes for blood 9. Labeling tapes
3. Stat fax tubes 10. Parafilm
4. Beaker 11. Chloride Reagent
5. Pasteur pipets 12. Stat fax machine
6. Serological pipets 13. Applicator sticks
7. Centrifuge

Principle:
Chloride ions form a soluble, non-ionized compound with mercuric ions and will displace thiocyanate ions from
non-ionized mercuric thiocyanate. The released thiocyanate ions react will ferric ions to form a color complex that absorb
light at 480 nm. The intensity of the color produced is directly proportional to the chloride concentration.
- ____________ -
Hg(SCN)2 + 2Cl > HgCl2 + 2SCN
- 3+ ___________
3SCN + Fe > 4 Fe(SCN)2 red complex

Reagent Composition:
1. Chloride reagent(Active ingredients):
Mercuric Nitrate 0.058 mM
Mercuric thiocyanate 1.75 mM
Mercuric chloride 0.74 mM
Ferric Nitrate 22.3 mM
Non-reactive ingredients and stabilizers in dilute acid and methanol
Precautions: Chloride reagent is POISONOUS. It contains mercury and methanol. Maybe harmful or
fatal if swallowed. DO NOT PIPET BY MOUTH. Call physician if taken internally.
2. Chloride calibrator:
Sodium chloride 100 mEq/L

Specimen Collection and Storage:

1. Use serum that has been separated from the clot soon after drawing.
2. Grossly hemolyzed serum should not be used as it may create falsely decreased values.
3. Avoid contamination of blood with tissue fluid.
4. Store serum in tightly stoppered tubes.
5. Chloride is stable in serum for 1 day at room temperature and for 3 moths frozen when stored tightly capped.

Procedure: The instructor will make a recorded video of the actual laboratory performance of the procedure and
explanation of the principle of the test. The video will be uploaded for the students to be able to familiarize themselves
with the procedure. The results will then be showed and will be used by the students for the computation.
BLANK STANDARD SAMPLE
Reagent 1.0mL 1.0mL 1.0mL
water 5 μL
Standard reagent - 5 μL -
serum - - 5 μL
Mix; incubate at 37C for 5 minutes. Read absorbances of standard (As) and sample (Ax) against reagent
blank.

Calculation:

Page | 57
 Abs. of sample x Conc. of Standard (100 mEq/L) = Chloride (mEq/L)
Abs. of standard

Reference Values:
98 – 110 mEq/L

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text message.

1. Characterize Chloride in terms of its metabolism.

2. What is the clinical significance associated with chloride?
3. What are the sources of interferences that can adversely affect the chloride assay?
4. Briefly describe the performance of sweat chloride determination.
5. What is the clinical importance of sweat chloride?

Page | 58
 -
D. Bicarbonate (HCO3 )
 Bicarbonate is the 2 most abundant anion in the ECF which accounts for >90% of the total CO2
nd

 Because HCO3 composes the largest fraction of total CO2, total CO2 measurement is indicative of HCO3
- -

measurement
Regulation of Bicarbonate
About 85% of filtered bicarbonate is reabsorbed in the proximal convoluted tubules and the rest in the distal
convoluted tubules. Tubules are known to be only slightly permeable to bicarbonate because bicarbonate after being
filtered into the tubules combines with hydrogen to form carbonic acid. Carbonic acid then dissociates into molecules of
water and carbon dioxide where carbon dioxide readily diffuses back into the ECF.

Diagnostic significance
Alterations of HCO3 & CO2 in plasma are characteristic of acid-base imbalance. Evaluation of blood gases & pH:
-
provide a definitive picture of the over-all pattern of imbalances. A decreased HCO3 may occur from metabolic acidosis as
-
HCO3 combines with H to produce CO2, which is exhaled by the lungs.

Determination of Bicarbonate
 Specimen: Serum or Lithium heparinized plasma (from venous or capillary blood)
o Specimen drawn in evacuated tube: unopened & centrifuged ASAP
o Analyzed promptly after the tube is opened
o Note: Exposure to air = CO2 loss; decrease by 6 mmol/L within an hour
Anion Gap
This is the mathematical formula used to demonstrate electro neutrality of body fluids. The anion gap is the
+ +
difference between primary measured cations (sodium Na and potassium K ) and the primary measured anions (chloride
- -
Cl and bicarbonate HCO3 ) in serum. This test is most commonly performed in patients who present with altered mental
status, unknown exposures, acute renal failure, and acute illnesses. Anion gap is increased in uremia, lactic acidosis,
ketoacidosis, hypernatremia and ingestion of alcohol while decreased in hypoalbuminemia and hypercalcemia.

Two calculations:
+ - -
1. Na - (Cl + HCO3 ) = anion gap (Expected anion gap: 7 – 16 mmol/L)
+ + - -
2. (Na + K ) - (Cl + HCO3 ) = anion gap (Expected anion gap: 10 – 20 mmol/L)

E. Magnesium
 Magnesium is he 4 most abundant cation in the body and the second most abundant intracellular ion.
th

 It is distributed in primarily in the bones (53%), intracellularly (46%) and extracellularly (<1%).
 The serum magnesium is further divided into protein bound (30%), ionized or free (55%) and complexed with
other molecules such as phosphate and citrate (15%)
 Magnesium serves as a cofactor for >300 enzymes
Regulation of Magnesium
About 20 to 65% of dietary magnesium is absorbed in the small intestines while the overall regulation like the
other electrolytes is a responsibility of the kidney. Non-protein bound magnesium is readily filtered by the glomerulus and
about 25 to 30% is reabsorbed in the proximal convoluted tubules while the majority about 50 to 60% in the ascending
loop of Henle. Only 2 to 5% is reabsorbed in the distal convoluted tubules. The renal threshold of magnesium is 0.60 to
0.85 mmol/L. Approximately 6% of filtered magnesium is excreted in the urine per day. Parathyroid hormone increases
the renal reabsorption of magnesium and enhances absorption of magnesium in the small intestines while aldosterone
and thyroxine provides the opposite effects.

Diagnostic significance
1. Hypomagnesemia is most frequently observed in hospitalized and in intensive care unit patients may be due to overall
depletion due to severe loss. Hypomagnesemia may cause by reduced intake because of starvation and poor diet. It may
also be caused by decreased absorption in malabsorption syndrome, laxative abuse and pancreatitis. Increased renal
excretion due to tubular disorders and glomerulonephritis may also be associated with this.

2. Hypermagnesemia is less frequently observed than hypomagnesemia. The most common cause is renal failure and
the most severe elevations are usually a result of the combined effects of decreased renal function and increased intake
of commonly prescribed medications such as antacids. Dehydration may cause pseudo hypermagnesemia which can be
Page | 59
corrected with hydration and mild serum magnesium can be observed in increased bone loss such as in the case of
multiple myeloma and metastases.

Determination of Magnesium
 Specimen: non-hemolyzed serum or Li heparinized plasma
++
o Oxalate, Citrate & EDTA bind w/ Mg
o 24–hour urine (acidified w/ HCl) to avoid precipitation
 The methods that can be employed are the following: Colorimetric method that uses either of these dyes,
Calgamite, Formazen and methylthymol blue. Dye lake method has also been used while AAS is considered the
reference method.

Page | 60
 Activity No. 13: MAGNESIUM DETERMINATION
Dye-binding Method (Xylidyl blue)

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure Magnesium level in serum by viewing a simulation video
which is recorded by the laboratory instructor.
2. To be able to describe other methods employed for magnesium determination
3. To be able to associate hypomagnesemia and hypermagnesemia to various clinical conditions.

Materials and Equipment:

1. Venipuncture set 8. Test tube rack
2. 13 x 100 mm test tubes for blood 9. Labeling tapes
3. Stat fax tubes 10. Parafilm
4. Beaker 11. Magnesium Reagent
5. Pasteur pipets 12. Stat fax machine
6. Serological pipets 13. Applicator sticks
7. Centrifuge

Principle:
Xylidyl blue combines with magnesium at alkaline pH to form a purple complex, the absorbance of which is
measured at 546 nm. Interference from other cations are avoided by specific chelating agents.

Reagent Composition:
Xylidyl blue
NaCl
EGTA
Triethanolamine
Good‘s buffer (pH 11.0)
Surfactant
Preservative

Specimen Collection and Storage:

Serum (preferred) or heparinized plasma; do NOT use citrate, oxalate and EDTA as anticoagulant. The specimen
should be drawn without venous stasis if possible. Do not use samples from patients in therapy with EDTA. Remove
serum from clot without delay.
Serum or plasma samples are stable at 2 - 8C for one week.

Procedure: The instructor will make a recorded video of the actual laboratory performance of the procedure and
explanation of the principle of the test. The video will be uploaded for the students to be able to familiarize themselves
with the procedure. The results will then be showed and will be used by the students for the computation.
BLANK STANDARD SAMPLE
Reagent 1.0mL 1.0mL 1.0mL
water 10 μL
Standard reagent - 10 μL -
serum - - 10 μL
Mix; incubate at 37C for 2 minutes. Read absorbances of standard (As) and sample (Ax) against reagent
blank.

Calculation:
Abs. of sample x Conc. of Standard (2 mEq/L) = Magnesium (mEq/L)
Abs. of standard
Expected Values:
Newborn (2-4 days) 1.20 – 1.80 mEq/L (0.60 – 0.90 mmol/L)
5 months – 6 yrs. 1.42 – 1.88 mEq/L (0.71 – 0.94 mmol/L)
6 – 12 years 1.38 – 1.74 mEq/L (0.69 – 0.87 mmol/L)
12 – 20 years 1.35 – 1.77 mEq/L (0.67 – 0.88 mmol/L)
Adult 1.30 – 2.10 mEq/L (0.65 – 1.05 mmol/L)

Page | 61
Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text message.

1. Characterize magnesium in terms of its distribution and function in the body.

2. What is the clinical significance associated with magnesium?
3. What are the sources of interferences that can adversely affect the magnesium assay?
4. Aside from xylidyl blue, identify other agents used for magnesium in dye-binding methods.
5. What is EGTA? Indicate the purpose of this agent in the Xylidyl blue method for magnesium.

Page | 62
F. Calcium


th
This is the 5 most common element and a major component of bones and teeth (99%) while a small
concentration is found in the soft tissues and the ECF (1%)
 In the serum 45% is protein bound in which 80% is bound to albumin and rest are bound to globulins.
 10% of serum calcium is complexed with other molecules such as citrate and phosphate
 About 45% of the serum calcium are ionized or in their free form
 Calcium has regulatory function, they act as inorganic messenger
 They participate also in several enzyme activities as cofactors
 Calcium is also important in muscle contraction where it binds to troponins.
 Calcium also regulates cell membrane permeability in it stabilizes the cell membrane.
 It also controls the secretion of some endocrine glands such as parathyroid gland and C cells
 It is important in blood coagulation and in providing rigidity to the bones and teeth

Diagnostic Significance
Hypercalcemia Hypocalcemia
Primary hyperparathyroidism – adenoma or glandular Primary hypoparathyroidism
hyperplasia Hypomagnesemia
Hyperthyroidism Hypermagnesemia
Malignancy Acute pancreatitis
Multiple myeloma Vit. D deficiency
Increased Vit. D

Page | 63
 Activity No. 14: CALCIUM DETERMINATION
Dye-binding Method (Arsenazo III dye)

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure calcium level in serum by viewing a simulation video which
is recorded by the laboratory instructor.
2. To be able to describe other methods employed for calcium determination
3. To be able to associate hypocalcemia and hypercalcemia with various conditions.

Materials and Equipment:

1. Venipuncture set 8. Test tube rack
2. 13 x 100 mm test tubes for blood 9. Labeling tapes
3. Stat fax tubes 10. Parafilm
4. Beaker 11. Calcium Reagent
5. Pasteur pipets 12. Stat fax machine
6. Serological pipets 13. Applicator sticks
7. Centrifuge

Principle:
Arsenazo III combines with calcium at slight acidic pH to form a blue complex, the absorbance of which is
measured at 660 nm. The reaction has high specificity and interference from magnesium is avoided due to pH.
Reagent Composition:
Arsenazo III
Good‘s buffer (pH 6.8)
Stabilizers

Specimen Collection and Storage:

Serum (preferred) heparinized plasma. Do not use citrate, oxalate and EDTA as anticoagulant. Total calcium is
stable for 7 days at 2 - 8C and for several months when frozen at -20C.

Procedure: The instructor will make a recorded video of the actual laboratory performance of the procedure and
explanation of the principle of the test. The video will be uploaded for the students to be able to familiarize themselves
with the procedure. The results will then be showed and will be used by the students for the computation.
BLANK STANDARD SAMPLE
Reagent 1.0mL 1.0mL 1.0mL
water 10 μL
Standard reagent - 10 μL -
serum - - 10 μL
Mix; incubate at 37C for 2 minutes. Read absorbances of standard (As) and sample (Ax) against reagent
blank.
Calculation:
Abs. of sample x Conc. of Standard (10 mg/dL) = Calcium (mg/dL)
Abs. of standard

Expected Values: 8.6 – 10.3 mg/dL (2.15 – 2.57 mmol/L)

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text message.
1. Characterize calcium in terms of its distribution and function in the body.
2. What is the clinical significance associated with calcium?
3. What are the sources of interferences that can adversely affect the calcium assay?
4. Describe current methods employed for the determination of calcium in blood.
Page | 64
G. Phosphate
 The body contains about 20 mol of phosphorus in the form of phosphate
 Intracellular phosphate can be either organic which are of macromolecules or inorganic which participates in high
energy transfer reactions.
 The majority of the extracellular Phosphate (85%) is inorganic form and acts as part of the hydroxyapatite.
 Phosphate has many important functions; it is an essential component of normal bones and teeth.
 It is associated with high energy nucleotides such as ATP
 It is a component of nucleic acids which serves as its backbone and cell membrane
 Phosphorus are required in glycogenolysis, in particular, it plays a role during fight or flight responses in which
glycogen degradation serves to provide an immediate source of glucose-6-phosphate for glycolysis, to provide
energy for muscle contraction.
 It is also an important determinant of bone mineral turnover
 2,3-diphosphoglycerate (2,3-DPG) in red blood cells increases in response to anemia/hypoxia and causes a shift
of the oxygen dissociation curve, allowing a more effective oxygen delivery
Diagnostic Significance
Hyperphosphatemia Hypophosphatemia
Acute or chronic renal failure CAUSES:
- Increased intake of PO4: among neonates w/ cow‘s milk or DKA
laxatives COPD
- Increased release of cellular PO4 Malignancy
Severe infections Increased renal excretion: hyperparathyroidism
Intensive exercise Decreased intestinal absorption: Vit. D deficiency or
Neoplastic disorders antacid use
Intravascular hemolysis
Lymphoblastic leukemia
Hormones that regulate Calcium and Phosphate
Parathyroid Hormone Calcitonin Vit. D and its metabolites
-Chief cells of PT glands: production & - Produced by C cells of the thyroid 1. Vitamin D3 (Cholecalciferol)
secretion of PTH gland - Natural form produced in the skin
-PTH most important factor in the renal - Secreted when the concentration of from the action of sunlight
regulation of PO4-lowers blood Ca in blood increase 2. Vitamin D2 (Ergocalciferol)
concentration and increase renal - Secretion is regulated by the level of - Manufactured commercially from
excretion Calcium in the circulation precursors of plant origin
++
-Regulation of the ECF Ca - Hypercalcemia = stimulates CT -To be physiologically active, (2)
concentration release hydroxylations are required (by
-Hypocalcemia: stimulates PTH - Hypocalcemia = suppresses CT specific hydroxylases)
secretion secretion 1,25 (OH)2 D – most potent; it
++
-Hypercalcemia: suppresses secretion stimulates: Ca absorption by the SI,
++
of PTH intestinal absorption of PO4, Ca
++
resorption from bone, Ca
reabsorption by the distal tubules

Page | 65
 Activity No. 15: PHOSPHATE DETERMINATION
Fiske and Subbarow Method

Objectives: At the end of the activity, the students are expected:

1. To be able to appreciate how to accurately measure inorganic phosphate level in serum by viewing a
simulation video which is recorded by the laboratory instructor.
2. To be able to describe other methods employed for inorganic phosphate determination
3. To be able to associate hypophosphatemia and hyperphosphatemia with various clinical conditions.

Materials and Equipment:

1. Venipuncture set 8. Test tube rack
2. 13 x 100 mm test tubes for blood 9. Labeling tapes
3. Stat fax tubes 10. Parafilm
4. Beaker 11. Phosphate Reagent
5. Pasteur pipets 12. Stat fax machine
6. Serological pipets 13. Applicator sticks
7. Centrifuge

Principle:
The phosphate ions react with ammonium molybdate to form a phosphomolybdate complex. The colorless
phosphomolybdate complex can be measured directly by ultraviolet (UV) absorption at 340 nm. An acid pH is necessary
for the formation of complexes.
Reagent Composition:
Ammonium molybdate
Sulfuric acid
Surfactant

Specimen Collection and Storage:

Serum is the preferred specimen. Although heparinized plasma is acceptable, levels of inorganic phosphate are
about 0.2 – 0.3 mg/dL lower than in serum. Anticoagulants such as citrate, oxalate and EDTA interfere with formation of
the phosphomolybdate complex and should not be used.
Inorganic phosphate in whole blood specimens may either decrease or increase with time, depending on the type
of specimen, temperature, and duration of storage. Levels in plasma or serum are increased by prolonged storage with
cells at room temperature or 37C. It is important to promptly separate serum or plasma from erythrocytes. Hemolyzed
specimens are unacceptable because erythrocytes contain high concentrations of organic phosphate esters which can be
hydrolyzed to inorganic phosphate during storage. Inorganic phosphate increases by 4 – 5 mg/dL per day in hemolyzed
specimens stored at 4C. Glucose phosphate, creatine phosphate and other organic phosphates may also be hydrolyzed
by assay conditions, resulting in over-estimation of inorganic phosphate levels.
Phosphate is considered to be stable in serum that has been separated from the clot for days at 4C and months
when frozen.

Procedure: The instructor will make a recorded video of the actual laboratory performance of the procedure and
explanation of the principle of the test. The video will be uploaded for the students to be able to familiarize themselves
with the procedure. The results will then be showed and will be used by the students for the computation.
BLANK STANDARD SAMPLE
Reagent 1.0mL 1.0mL 1.0mL
water 10 μL
Standard reagent - 10 μL -
serum - - 10 μL
Mix; incubate at 37C for 5 minutes. Read absorbances of standard (As) and sample (Ax) against reagent
blank.

Calculation:
Abs. of sample x Conc. of Standard (5 mg/dL) = Phosphate (mg/dL)
Abs. of standard

Expected Values:
Adults 2.5 – 4.5 mg/dL (0.81 – 1.45 mmol/L)
Children 4.0 – 7.0 mg/dL (1.29 – 2.26 mmol/L)

Page | 66
Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
compute for the values and determine whether the values computed are normal or outside the reference range. For offline
students, the absorbance readings will be given through a text message. Calculations will be written on a separate bond
paper. Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text message.

1. Characterize phosphate in terms of its distribution and function in the body.

2. What is the clinical significance associated with phosphate?
3. What are the sources of interferences that can adversely affect the phosphate assay?
4. Describe the enzymatic method for the determination of phosphate in blood.

Page | 67
 Assessment No. 5

Please answer the following questions; write your brief answers to the following questions.

1. What is the relationship of magnesium to the K/NA ATPase pump?

2. Briefly explain the importance of bicarbonate in the acid base balance.

3. What is the importance of potassium in the body?

4. Why is chloride test done in sweat?

5. Explain why hypokalemia is both a cause and result of alkalosis.

Page | 68
Reference:

Anderson, Shauna and Susan Cockyane (2007). Clinical Chemistry: Concepts and Applications. USA: Waveland
Press Inc.

Arneson, W. and J. Brickell (2007). Clinical Chemistry: A Laboratory Perspective. USA: F.A. Davis Co.

Ashwood E., D. Bruns and C. Burtis (2012). Tietz’s Textbook of Clinical Chemistry and Molecular Diagnostics 5th ed.
Philadelphia: W.B. Saunders Co.
th
Bishop, Michael (2013) Clinical Chemistry Procedures, Correlation. (7 ed.). Holland
th
Crook, Martin (2006). Clinical Chemistry and Metabolic Medicine (7 ed.). USA: Hodder Arnold Publication.

McPherson, Richard. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Elsevier
Saunders, 2011.

Page | 69
 Chapter III. TRACE METALS

Almost half of the elements found in the periodic table have been found in the human body. The essential trace
elements are usually associated with enzymes and serves as cofactor in enzymatic reactions. An element is to be
considered essential if the deficiency impairs a biochemical or functional process and replacement of the impairment
corrects this impairment. Nonessential trace elements are also of medical importance because many of them toxic.

Desired learning outcomes:

At the end of this chapter, you should be able to:
a. Describe the importance of the trace elements in metabolism
b. Explain the various methods for the laboratory determination of trace elements
c. Discuss the clinical significance of trace metals

Section 1. PROVEN ESSENTIAL TRACE METALS

A. Iron
 Most abundant trace element in the body wherein about 40 to 50 mg of iron is present per kilogram body weight
 Iron containing proteins are important in the metabolism such as collagen, tyrosine and catecholamines
 Iron majorly found in the following:
o Hemoglobin in RBC
o Ferritin and hemosiderin as iron stores
o Moderate amount is found in body tissue such as myoglobin and non heme enzymes
o Iron bound to transferrin which is the first to become diminished in iron deficiency conditions

Metabolism and Regulation

To be absorbed by intestinal cells, iron must be in the Fe(II) (ferrous) oxidation state and bound to protein.
Because Fe(III) is the predominant form of iron in foods, it must first be reduced to Fe(II) by agents such as vitamin C,
ascorbic acid or the acidity of the stomach before it can be absorbed. However, if gastric acid production is impaired then
iron absorption will be reduced substantially. The flow of Fe(II) is sped up by several other mechanisms until it is picked
up by transferrin, which delivers it into tissues.

Diagnostic significance
Iron Deficiency Iron Overload
-Iron deficiency affects about 15% of the -Common causes: Hereditary hemochromatosis - Hereditary
worldwide population. Those with a higher hemochromatosis is a disorder that causes the body to absorb too much
than average risk of iron deficiency anemia iron from the diet. Sideroblastic anemia, Chronic ingestion of medicinal iron,
include pregnant women, young children Chronic hepatitis
- Increased blood loss, decreased dietary -The following shows symptoms of hemochromatosis
iron intake, or decreased release from
ferritin may result in iron deficiency
- Reduction in iron stores usually precedes
both a reduction in circulating iron and
anemia, as demonstrated by a decreased
red blood cell count, mean corpuscular
hemoglobin concentration, and the
presence microcytic RBCs.
- Common causes: Blood loss due to GIT
bleeding, Chronic drug ingestion, Parasitic,
Image taken from
Impaired absorption of iron and Renal msdmanuals.com/professional/hematology/H
failure H

Analytical methods for Iron

Direct measurements Indirect methods
-quantitative, specific, & sensitive -Serum iron is measure by: colorimetric method and AAS
-involve invasive procedures
*Liver biopsy is digested and analyzed for iron
* quantitative phlebotomy is used as a substitute for biopsy

Page | 70
in monitoring iron overload
*Bone marrow biopsy
Note:
 Serum transferrin is measured using Direct immunoassay and TIBC or the maximum amount of iron that can bind
to serum transferrin
 Serum ferritin is measured by RIA, ELISA, IFA and Chemiluminiscence assay
 Serum transferrin receptor is a useful marker for body iron stores and sensitive measurement of tissue iron
deficiency
Reference Interval:
++
Total Fe 60 – 150 µg/dL Ferritin:
TIBC 250 – 400 µg/dL Male: 15 – 200 µg/L
Iron Sat‘n 20 – 55% Female: 12 – 200 µg/L

B. Zinc
 Zinc is the second most abundant trace metal in the body and is known to be a cofactor for almost 300 enzymes
 It occurs in enzyme that is important in the synthesis and metabolism of RNA and DNA
 Zn plays a role in the synthesis, storage and secretion of insulin as well as in conformational integrity of insulin,
the decrease in Zn will affect the ability of the islet cell to produce and secrete insulin, that may contribute to the
development of Type 2 diabetes

Metabolism and Regulation

• Zinc is mainly absorbed from the duodenum. In blood, bound to albumin and α-2 macroglobulin while the bones
and the muscle contain most of the body‘s Zn stores. In the liver, zinc is bound to metallothionein.

Diagnostic significance
Zinc deficiency is common in patients with the following disorders: Diabetes mellitus, alcohol abuse,
malabsorption syndrome, liver and kidney disease and Acrodermatitis enteropathica which is rare genetic autosomal
recessive disorder, characterized by periorificial dermatitis, alopecia, and diarrhea. It is caused by mutations in the gene
that encodes for the membrane protein that binds zinc.

Analytical methods
Diurnal variation should be considered when testing for zinc because Zinc is highest in the morning. Postprandial
variation should also be considered because serum values are 10% greater than plasma values. The RBC has 10 times
more zinc than the plasma hence hemolysis should be avoided. The reference method for zinc measurement is AAS.

C. Copper
 Copper is the third most abundant trace metal in the body
 It participates in several body mechanisms such as cellular respiration, DNA and RNA reproduction, Maintenance
of cell membrane integrity and sequestration of free radicals.
 About half of the dietary copper is excreted in feces per day and approximately 3% of it is lost in urine.

Metabolism and Distribution

The liver has the highest copper concentration while in the blood copper is contained in proteins such as
ceruloplasmic, transcuperin and metallothionein.

Diagnostic significance
Copper deficiency Copper toxicity
- Copper deficiency is related to malnutrition, malabsorption, - An increased tissue and serum levels of copper
and chronic diarrhea and prolonged feeding with low-copper, - An example is in acute copper poisoning through
total-milk diets. fungicides
- Osteoporosis/bone and joint abnormality refelects copper -Wilson‘s disease is a genetically determined copper
dependent cross linking of collagen and connective tissue accumulation disease that causes copper deposits in
- Subclinical copper depletion contributes to an increased tissues (liver, brain & cornea)
risk of coronary heart disease. -Main manifestations of Wilson‘s disease includes:
- An extreme form of copper deficiency is seen in Menkes Neurologic disorders, liver dysfunction and Kayser-
disease. X-linked recessive disorder caused by mutations in Fleischer rings in the cornea.

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genes coding for the copper-transport protein, leading to
copper deficiency. This is a fatal, progressive brain disease
in children characterized by peculiar hair, called kinky or
steely, and retardation of growth.

Image taken from pinterest.ph

Image taken from
forgottendisease.org/assets/menkes
Analytical methods
Serum or urine copper can measure using AAS which is the method of choice or colorimetric method.
Ceruloplasmin is measured by photometric method and immunochemical methods.

Reference Intervals
Serum Copper Ceruloplasmin: 23 – 50 mg/dL
Male: 70 – 140 µg/dL (11 – 22µmol/L)
Female: 80 – 155 µg/dL (13 – 24µmol/L)

Section 2. ULTRA-TRACE ELEMENTS

Cobalt Chromium Fluoride Manganese Molybdenum Selenium
- Naturally -Naturally -Readily -Activator for -Cofactor for -Cofactor in
occurring occurring absorbed by the several enzyme oxidase enzymes glutathione
-Constituent of -Industrial waste Gut -Transported by -Absorbed in the peroxidase and
Vit. B12 -Insulin cofactor -Distributed plasma protein stomach and iodothyronine
-Toxic at high in glucose almost totally in -Deficiency: small intestines deiodinase
doses metabolism bones and teeth seizure and -Absorption of -Antioxidant
-Prevents dental epilepsy iron & copper properties
carries -Excess: Locura -Excess: Gout -involved in
-Excess: causes manganica thyroid hormone
mottling of teeth metabolism

Reference:

Anderson, Shauna and Susan Cockyane (2007). Clinical Chemistry: Concepts and Applications. USA: Waveland
Press Inc.
th
Bishop, Michael (2013) Clinical Chemistry Procedures, Correlation. (7 ed.). Holland
th
Crook, Martin (2006). Clinical Chemistry and Metabolic Medicine (7 ed.). USA: Hodder Arnold Publication.

Page | 72
 Assessment No. 6
Please answer the following questions; write your brief answers to the following questions.

1. Explain the Pathogenesis of Hemochromatosis.

2. Explain the Pathogenesis of Wilson’s disease

3. Explain the Pathogenesis of Acrodermatitis enteropathica.

4. Explain the Pathogenesis of Lucura manganica.

5. Explain how fluoride can be result to toxicity.

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 Chapter IV. ARTERIAL BLOOD GAS AND ACID BASE BALANCE

Proper physiological functioning depends on a very tight balance between the concentrations of acids and bases
in the blood. A variety of buffering systems permits blood and other bodily fluids to maintain a narrow pH range, even in
the face of perturbations. A buffer is a chemical system that prevents a radical change in fluid pH by dampening the
change in hydrogen ion concentrations in the case of excess acid or base. Most commonly, the substance that absorbs
the ions is either a weak acid, which takes up hydroxyl ions, or a weak base, which takes up hydrogen ions.

At the end of this chapter, you should be able to:

a. Describe the role of blood gases in the maintenance of body fluid equilibrium.
b. Distinguish the different metabolic and respiratory disturbances in the body.
c. Explain the methods of blood gas determination
d. Correlate blood gas results with various physiologic and pathologic processes.

Section 1. THE BUFFER SYSTEM

A. Buffer system in the body

The buffer systems in the human body are extremely efficient, and different systems work at different rates. It
takes only seconds for the chemical buffers in the blood to make adjustments to pH. The respiratory tract can adjust the
blood pH upward in minutes by exhaling CO2 from the body. The renal system can also adjust blood pH through the
+
excretion of hydrogen ions (H ) and the conservation of bicarbonate, but this process takes hours to days to have an
effect.
The buffer systems functioning in blood plasma include plasma proteins, phosphate, and bicarbonate and
carbonic acid buffers. The kidneys help control acid-base balance by excreting hydrogen ions and generating bicarbonate
that helps maintain blood plasma pH within a normal range. Protein buffer systems work predominantly inside cells.

B. Protein buffer in blood plasma

Nearly all proteins can function as buffers. Proteins are made up of amino acids, which contain positively charged
amino groups and negatively charged carboxyl groups. The charged regions of these molecules can bind hydrogen and
hydroxyl ions, and thus function as buffers. Buffering by proteins accounts for two-thirds of the buffering power of the
blood and most of the buffering within cells.

C. Hemoglobin as buffer
Hemoglobin is the principal protein inside of red blood cells and accounts for one-third of the mass of the cell.
During the conversion of CO2 into bicarbonate, hydrogen ions liberated in the reaction are buffered by hemoglobin, which
is reduced by the dissociation of oxygen. This buffering helps maintain normal pH. The process is reversed in the
pulmonary capillaries to re-form CO2, which then can diffuse into the air sacs to be exhaled into the atmosphere. This
process is discussed in detail in the chapter on the respiratory system.

D. Bicarbonate-Carbonic Acid Buffer

The bicarbonate is regulated in the blood by sodium, as are the phosphate ions. When sodium bicarbonate
(NaHCO3), comes into contact with a strong acid, such as HCl, carbonic acid (H 2CO3), which is a weak acid, and NaCl are
formed. When carbonic acid comes into contact with a strong base, such as NaOH, bicarbonate and water are formed.
As with the phosphate buffer, a weak acid or weak base captures the free ions, and a significant change in pH is
prevented. Bicarbonate ions and carbonic acid are present in the blood in a 20:1 ratio if the blood pH is within the normal
range. With 20 times more bicarbonate than carbonic acid, this capture system is most efficient at buffering changes that
would make the blood more acidic. This is useful because most of the body‘s metabolic wastes, such as lactic acid and
ketones, are acids. Carbonic acid levels in the blood are controlled by the expiration of CO 2 through the lungs. In red
blood cells, carbonic anhydrase forces the dissociation of the acid, rendering the blood less acidic. Because of this acid
dissociation, CO2 is exhaled (see equations above). The level of bicarbonate in the blood is controlled through the renal
system, where bicarbonate ions in the renal filtrate are conserved and passed back into the blood. However, the
bicarbonate buffer is the primary buffering system of the IF surrounding the cells in tissues throughout the body.

Respiratory Regulation of Acid-Base Balance

The respiratory system contributes to the balance of acids and bases in the body by regulating the blood levels of
carbonic acid. CO2 in the blood readily reacts with water to form carbonic acid, and the levels of CO 2 and carbonic acid in
the blood are in equilibrium. When the CO2 level in the blood rises (as it does when you hold your breath), the excess
CO2 reacts with water to form additional carbonic acid, lowering blood pH. Increasing the rate and/or depth of respiration
(which you might feel the ―urge‖ to do after holding your breath) allows you to exhale more CO 2. The loss of CO2 from the
Page | 74
body reduces blood levels of carbonic acid and thereby adjusts the pH upward, toward normal levels. As you might have
surmised, this process also works in the opposite direction. Excessive deep and rapid breathing (as in hyperventilation)
rids the blood of CO2 and reduces the level of carbonic acid, making the blood too alkaline. This brief alkalosis can be
remedied by rebreathing air that has been exhaled into a paper bag. Rebreathing exhaled air will rapidly bring blood pH
down toward normal.
The chemical reactions that regulate the levels of CO2 and carbonic acid occur in the lungs when blood travels
through the lung‘s pulmonary capillaries. Minor adjustments in breathing are usually sufficient to adjust the pH of the blood
by changing how much CO2 is exhaled. In fact, doubling the respiratory rate for less than 1 minute, removing ―extra‖ CO 2,
would increase the blood pH by 0.2. This situation is common if you are exercising strenuously over a period of time. To
keep up the necessary energy production, you would produce excess CO2 (and lactic acid if exercising beyond your
aerobic threshold). In order to balance the increased acid production, the respiration rate goes up to remove the CO 2. This
helps to keep you from developing acidosis.
The body regulates the respiratory rate by the use of chemoreceptors, which primarily use CO 2 as a signal.
Peripheral blood sensors are found in the walls of the aorta and carotid arteries. These sensors signal the brain to provide
immediate adjustments to the respiratory rate if CO2 levels rise or fall. Yet other sensors are found in the brain itself.
Changes in the pH of CSF affect the respiratory center in the medulla oblongata, which can directly modulate breathing
rate to bring the pH back into the normal range.
Hypercapnia, or abnormally elevated blood levels of CO2, occurs in any situation that impairs respiratory
functions, including pneumonia and congestive heart failure. Reduced breathing (hypoventilation) due to drugs such as
morphine, barbiturates, or ethanol (or even just holding one‘s breath) can also result in hypercapnia. Hypocapnia, or
abnormally low blood levels of CO2, occurs with any cause of hyperventilation that drives off the CO 2, such as salicylate
toxicity, elevated room temperatures, fever, or hysteria.

Image taken from chem.libretexts.org

Section 2.Renal Regulation of Acid-Base balance

The renal regulation of the body‘s acid-base balance addresses the metabolic component of the buffering system.
Whereas the respiratory system (together with breathing centers in the brain) controls the blood levels of carbonic acid by
controlling the exhalation of CO2, the renal system controls the blood levels of bicarbonate. A decrease of blood
bicarbonate can result from the inhibition of carbonic anhydrase by certain diuretics or from excessive bicarbonate loss
due to diarrhea. Blood bicarbonate levels are also typically lower in people who have Addison‘s disease (chronic adrenal
insufficiency), in which aldosterone levels are reduced, and in people who have renal damage, such as chronic nephritis.
Finally, low bicarbonate blood levels can result from elevated levels of ketones (common in unmanaged diabetes
mellitus), which bind bicarbonate in the filtrate and prevent its conservation.
−
Bicarbonate ions, HCO3 , found in the filtrate, are essential to the bicarbonate buffer system, yet the cells of the
tubule are not permeable to bicarbonate ions. The steps involved in supplying bicarbonate ions to the system are seen in
previous diagram and are summarized below:

+
Step 1: Sodium ions are reabsorbed from the filtrate in exchange for H by an antiport mechanism in the apical
membranes of cells lining the renal tubule.
 Step 2: The cells produce bicarbonate ions that can be shunted to peritubular capillaries.

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  Step 3: When CO2 is available, the reaction is driven to the formation of carbonic acid, which dissociates to form a
bicarbonate ion and a hydrogen ion.
 Step 4: The bicarbonate ion passes into the peritubular capillaries and returns to the blood. The hydrogen ion is
secreted into the filtrate, where it can become part of new water molecules and be reabsorbed as such, or
removed in the urine.

Image taken from lumenlearning.com

It is also possible that salts in the filtrate, such as sulfates, phosphates, or ammonia, will capture hydrogen ions. If
this occurs, the hydrogen ions will not be available to combine with bicarbonate ions and produce CO 2. In such cases,
bicarbonate ions are not conserved from the filtrate to the blood, which will also contribute to a pH imbalance and
acidosis.
The hydrogen ions also compete with potassium to exchange with sodium in the renal tubules. If more potassium
is present than normal, potassium, rather than the hydrogen ions, will be exchanged, and increased potassium enters the
filtrate. When this occurs, fewer hydrogen ions in the filtrate participate in the conversion of bicarbonate into CO 2 and less
bicarbonate is conserved. If there is less potassium, more hydrogen ions enter the filtrate to be exchanged with sodium
and more bicarbonate is conserved.
Chloride ions are important in neutralizing positive ion charges in the body. If chloride is lost, the body uses
bicarbonate ions in place of the lost chloride ions. Thus, lost chloride results in an increased reabsorption of bicarbonate
by the renal system.

Section 3. Disorders of Acid-Base balance

Normal arterial blood pH is restricted to a very narrow range of 7.35 to 7.45. A person who has a blood pH below
7.35 is considered to be in acidosis (actually, ―physiological acidosis,‖ because blood is not truly acidic until its pH drops
below 7), and a continuous blood pH below 7.0 can be fatal. Acidosis has several symptoms, including headache and
confusion, and the individual can become lethargic and easily fatigued. A person who has a blood pH above 7.45 is
considered to be in alkalosis, and a pH above 7.8 is fatal. Some symptoms of alkalosis include cognitive impairment
(which can progress to unconsciousness), tingling or numbness in the extremities, muscle twitching and spasm, and
nausea and vomiting. Both acidosis and alkalosis can be caused by either metabolic or respiratory disorders.
As discussed earlier in this chapter, the concentration of carbonic acid in the blood is dependent on the level of
CO2 in the body and the amount of CO2 gas exhaled through the lungs. Thus, the respiratory contribution to acid-base
balance is usually discussed in terms of CO 2 (rather than of carbonic acid). Remember that a molecule of carbonic acid is
lost for every molecule of CO2 exhaled, and a molecule of carbonic acid is formed for every molecule of CO 2 retained.

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 Normal
Values:
pH: 7.35 –
7.45
CO2: 35 – 45
mmHg
Bicarbonate:
22 – 26%
Image taken from lumenlearning.com

Metabolic Acidosis: Metabolic Alkalosis: Respiratory Acidosis: Respiratory Alkalosis:

Primary Bicarbonate Primary Bicarbonate Primary Carbonic acid/ Primary Carbonic
Deficiency Excess CO2 Excess acid/CO2 Deficiency
Metabolic Acidosis Metabolic Alkalosis Respiratory Acidosis Respiratory Alkalosis
Occurs when the blood is Is the opposite of metabolic Occurs when the blood is Occurs when the blood is
too acidic (pH below 7.35) acidosis. It occurs when the overly acidic due to an overly alkaline due to a
due to too little bicarbonate, blood is too alkaline (pH excess of carbonic acid, deficiency in carbonic acid
a condition called primary above 7.45) due to too resulting from too much and CO2 levels in the blood.
bicarbonate deficiency. At much bicarbonate (called CO2 in the blood. This condition usually
the normal pH of 7.40, the primary bicarbonate Respiratory acidosis can occurs when too much
ratio of bicarbonate to excess). A transient excess result from anything that CO2 is exhaled from the
carbonic acid buffer is 20:1. of bicarbonate in the blood interferes with respiration, lungs, as occurs in
If a person‘s blood pH drops can follow ingestion of such as pneumonia, hyperventilation, which is
below 7.35, then he or she excessive amounts of emphysema, or congestive breathing that is deeper or
is in metabolic acidosis. The bicarbonate, citrate, or heart failure. more frequent than normal.
most common cause of antacids for conditions such An elevated respiratory rate
metabolic acidosis is the as stomach acid reflux— leading to hyperventilation
presence of organic acids or known as heartburn. can be due to extreme
excessive ketones in the Cushing‘s disease, which is emotional upset or fear,
blood. Table 1 lists some the chronic hypersecretion fever, infections, hypoxia, or
other causes of metabolic of adrenocorticotrophic abnormally high levels of
acidosis. hormone (ACTH) by the catecholamines, such as
anterior pituitary gland, can epinephrine and
cause chronic metabolic norepinephrine.
alkalosis. The oversecretion Surprisingly, aspirin
of ACTH results in elevated overdose—salicylate
aldosterone levels and an toxicity—can result in
increased loss of potassium respiratory alkalosis as the
by urinary excretion. Other body tries to compensate for
causes of metabolic initial acidosis.
alkalosis include the loss of
hydrochloric acid from the
stomach through vomiting,

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 potassium depletion due to
the use of diuretics for
hypertension, and the
excessive use of laxatives.

Section 4. Compensation Mechanism

Various compensatory mechanisms exist to maintain blood pH within a narrow range, including buffers,
respiration, and renal mechanisms. Although compensatory mechanisms usually work very well, when one of these
mechanisms is not working properly (like kidney failure or respiratory disease), they have their limits. If the pH and
bicarbonate to carbonic acid ratio are changed too drastically, the body may not be able to compensate. Moreover,
extreme changes in pH can denature proteins. Extensive damage to proteins in this way can result in disruption of normal
metabolic processes, serious tissue damage, and ultimately death.

Respiratory Compensation Metabolic Compensation

Respiratory compensation for metabolic acidosis increases Metabolic and renal compensation for respiratory diseases
the respiratory rate to drive off CO2 and readjust the that can create acidosis revolves around the conservation
bicarbonate to carbonic acid ratio to the 20:1 level. This of bicarbonate ions. In cases of respiratory acidosis, the
adjustment can occur within minutes. Respiratory kidney increases the conservation of bicarbonate and
+
compensation for metabolic alkalosis is not as adept as its secretion of H through the exchange mechanism
compensation for acidosis. The normal response of the discussed earlier. These processes increase the
respiratory system to elevated pH is to increase the amount concentration of bicarbonate in the blood, reestablishing
of CO2 in the blood by decreasing the respiratory rate to the proper relative concentrations of bicarbonate and
conserve CO2. There is a limit to the decrease in carbonic acid. In cases of respiratory alkalosis, the kidneys
respiration, however, that the body can tolerate. Hence, the decrease the production of bicarbonate and reabsorb
+
respiratory route is less efficient at compensating for H from the tubular fluid. These processes can be limited by
metabolic alkalosis than for acidosis. the exchange of potassium by the renal cells, which use a
+ +
K -H exchange mechanism (antiporter).

Reference:
th
Crook, Martin (2006). Clinical Chemistry and Metabolic Medicine (7 ed.). USA: Hodder Arnold Publication.

Libretexts. (2020, July 14). 25.4B: Chemical Buffer Systems. Retrieved August 07, 2020, from
https://med.libretexts.org/Bookshelves/Anatomy_and_Physiology/Book:_Anatomy_and_Physiology_(Boundless)/
25:_Body_Fluids_and_Acid-Base_Balance/25.4:_Acid-Base_Balance/25.4B:_Chemical_Buffer_Systems

OpenStax, L. (n.d.). Anatomy and Physiology II. Retrieved August 07, 2020, from
https://courses.lumenlearning.com/suny-ap2/chapter/disorders-of-acid-base-balance/

Page | 78
 Activity No. 16: ELECTROCHEMISTRY PRINCIPLES OF BLOOD GAS ANALYSIS

Objectives: At the end of the activity, the students are expected:

1. To be able to describe the basic principles involved in the measurement of pH, pO 2 and pCO2.
2. To be able to discuss the different precautions in collecting and handling samples for pH and blood gas analysis.
3. To be able to calculate partial pressures for pO2 and pCO2 for various percentages of CO2 and O2.
-
4. To be able to estimate/quantitate CO2, HCO3 and H2CO3 given 2 of the 3 parameters.

Apparatus and Materials:

1. Chart illustrations
2. Reference books

Procedure:
1. The instructor discusses the principles of pH, pO2 and pCO2 measurements through a recorded video.
2. Different case studies will be presented to the students for analysis and the students discuss each in group.

Drawings: Drawings must be placed on a short bond paper with .5‖ margins in all sides bearing the name and section of
the student. Students will scan or will take a picture of their drawings to be submitted online. For offline students, submit
via mail or express courier (―padala‖) addressed to: Instructor’s name, School of Natural Sciences, University of Baguio,
Baguio City.

1. Illustrate the pH electrode

2. Illustrate the Clarke electrode
3. Illustrate the Severinghaus electrode

Questions for Research: The instructor will make an online forum where the students can type and post their answers to
the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or
through text message.

1. Define the following:

a. Electrochemical cell b. Electrode/half-cell c. electromotive force
d. Electrode potential e. Membrane potential
2. Describe the electrochemical principles of pH, pO2 and pCO2 electrodes.
3. Describe how arterial pO2 and pCO2 can be estimated by transcutaneous measurement.
4. What specimens can be used for blood gas analysis? Briefly describe the collection process of each sample and
the different precautions in handling such samples.
5. If the sample is not maintained on ice until analysis, what effect would this have on the results?
6. How are the pH, pCO2 and pO2 measurement systems calibrated?

Page | 79
 Assessment No. 7

Please answer the following questions; write your answers on the space provided.

_____1. A patient‘s blood gas results are as follows: pH = 7.26, dCO2 = 2.0 mmol/L, HCO3– = 29 mmol/L
a. Respiratory alkalosis c. Metabolic alkalosis
b. Respiratory acidosis d. Metabolic acidosis

_____2. A patient‘s blood gas results are: pH = 7.50, PCO2 = 55 mm Hg, HCO3– = 40 mmol/L
a. Respiratory alkalosis c. Metabolic alkalosis
b. Respiratory acidosis d. Metabolic acidosis

_____3. Which set of results is consistent with uncompensated respiratory alkalosis?

a. pH 7.36 HCO3 22 mmol/L PCO2 38 mm Hg
b. pH 7.46 HCO3 38 mmol/L PCO2 55 mm Hg
c. pH 7.66 HCO3 22 mmol/L PCO2 20 mm Hg
d. pH 7.70 HCO3 30 mmol/L PCO2 25 mm Hg

_____4. Which condition results in metabolic acidosis with severe hypokalemia and chronic alkaline urine?
a. Diabetic ketoacidosis c. Renal tubular acidosis
b. Phenformin-induced acidosis d. Acidosis caused by starvation

_____5. Which of the following is the primary mechanism of compensation for metabolic acidosis?
a. Hyperventilation c. Aldosterone release
b. Release of epinephrine d. Bicarbonate excretion

_____6. Which PCO2 value would be seen in maximally compensated metabolic acidosis?
a. 60 mm Hg c. 30 mm Hg
b. 40 mm H d. 15 mm Hg

_____7. Which of the following contributes the most to the serum total CO2?
–
a. PCO2 c. HCO3
b. dCO2 d. Carbonium ion

_____8. Which of the following best represents the reference (normal) range for arterial pH?
a. 7.35–7.45 c. 7.38–7.68
b. 7.42–7.52 d. 6.85–7.56

_____9. In addition to sodium bicarbonate, what other substance contributes most to the amount of base in the blood?
a. Hemoglobin concentration c. Inorganic phosphorus
b. Dissolved O2 concentration d. Organic phosphate

_____10. Which of the following effects results from exposure of a normal arterial blood sample to room air?
a. PO2 increased PCO2 decreased pH increased
b. PO2 decreased PCO2 increased pH decreased
c. PO2 increased PCO2 decreased pH decreased
d. PO2 decreased PCO2 decreased pH decreased

Page | 80
 Chapter V. CLINICAL ENDOCRINOLOGY

Endocrinology is a field of medicine which studies hormones and their actions. These hormones are produced
from endocrine glands. The endocrine system is a finely integrated system whereby the hypothalamus, pituitary & target
glands continually communicate through feedback inhibition & stimulation, to control all aspects of metabolism, growth
and reproduction (Henry) and by understanding this interplay, and carefully manipulating these systems via provocative
and suppressive stimuli, it is possible to characterize an underlying abnormality and provide directed treatment

At the end of this chapter, you should be able to:

a. Describe the mechanisms which regulate hormone secretion.
b. Describe methods that are employed for hormone assays.
c. Correlate results of hormonal assays with various physiologic and pathologic conditions
d. Accurately perform thyroid function tests

Section 1. AN OVERVIEW OF THE ENDOCRINE SYSTEM

What are Hormones?

Secretory products transported in the bloodstream from their place of synthesis to a distant location where they
exert their action. These are chemical substances that have a specific regulatory effect on the activity of a certain organ or
organs or cell types. Hormones have the following important functions:
 Control rates of certain chemical reactions
 Transport substances across cell membranes
 Help regulate water and electrolyte balance
 Play a role in reproduction
Three General classes of Hormones
Steroid Hormones Polypeptides and Proteins Hormones derived from amino
acids
generally hydrophobic water-soluble  Half-lives vary
 Many circulate in plasma bound  Circulate unbound in plasma  Examples:
to high-affinity proteins  Half-life: vary from 5 to 60 mins.  Thyroxine – protein-bound
 Small % are free for biologic  Initiate their response by (Half-life: almost 1 week)
activity binding to cell membrane  Epinephrine – not protein-
 Half-life: 60 – 100 mins. receptor bound (Half-life: <1 min.)

Image taken from slideserve.com Image taken from slideserve.com

Types of Hormone Action according to Tietz

1. Endocrine hormone synthesized in one location & released into plasma
Binds to specific receptors in cells at a distant site to elicit characteristic response
2. Neuroendocrine hormone synthesized in nerve ending & released into EC space
Interacts w/ receptors of cells at distant site
3. Neurocrine hormone synthesized in neurons & released into EC space
Binds to receptor in nearby cell & affects its function
4. Neurotransmission hormone synthesized in neurons & released from nerve endings
Crosses synapse & binds to specific receptors in another neuron
5. Paracrine hormone synthesized in endocrine cells & released into EC space
Binds to specific receptor of nearby cell & affects its function
6. Exocrine hormone synthesized in endocrine cells & released into lumen of gut
Page | 81
Hormone-Receptor Interaction
The biological response to a hormone is initiated by the binding of the hormone to target cell receptors. Receptors
provide the target cell with a mechanism for recognizing & concentrating the hormone. The hormone-receptor complex
activates the target cell to begin the chain of events that constitutes the biological effect(s) of that hormone.

3 ways in which control of Hormonal Secretion occurs

1. Releasing (trophic) hormones from the HYPOTHALAMUS control secretions of the anterior pituitary.
2. The NERVOUS SYSTEM influences certain endocrine glands directly.
3. FREE-STANDING GLANDS: respond directly to changes in fluid composition.

Mechanism of Hormone Secretion

Positive Feedback Mechanism Negative Feedback Mechanism
An increase in one hormone would result in the increase of An increase in one hormone causes a decrease in the
a second hormone second hormone

Page | 82
 Activity No. 17: THE ENDOCRINE SYSTEM
Hypothalamus-Anterior Pituitary-Target Organ (HPO Axis)

OBJECTIVE: At the end of the activity, the students are expected:

 To understand the association & communication that occurs among the glands within the endocrine system
 To learn the mode of action and clinical significance of the hormones produced by the endocrine glands

MATERIALS:
 Charts, Reference Materials

PROCEDURE: The instructor will explain the Endocrine system through a recorded video.
 In a tabulated form, enumerate the hormones produced by the Pituitary (Anterior & Posterior) and Thyroid glands,
their mode of action, target organ/s, and clinical significance.

DRAW AND LABEL: Drawings must be placed on a short bond paper with .5‖ margins in all sides bearing the name and
section of the student. Students will scan or will take a picture of their drawings to be submitted online. For offline
students, submit via mail or express courier (―padala‖) addressed to: Instructor’s name, School of Natural Sciences,
University of Baguio, Baguio City.

 Hypothalamus
 Anterior pituitary
 Posterior pituitary

GUIDE QUESTIONS: The instructor will make an online forum where the students can type and post their answers to the
QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or through
text mesage.

1. Describe the three (3) major classes of hormones and give examples of each class.
2. Discuss the three (3) mechanisms that control hormonal secretions from endocrine glands.
3. How does the feedback mechanism occur in the endocrine system?
4. In tabular form, enumerate the different hypothalamic hormones (Column A) which will regulate the release of
Anterior Pituitary hormones (Column B). In the last column (Column C), identify the target gland & the effector hormone/s
produced.

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 Section 2. ORGANS OF THE ENDOCRINE SYSTEM

A. Hypothalamus
Located behind the frontal lobe and below the thalamus. It controls a number of bodily functions including the
release of hormone from pituitary gland. It secretes 7 hormones, 3 regulatory hormones and 2 pairs of regulatory and
inhibitory hormones. Known as the master organ since it affects the secretion of hormones from the other organs of the
endocrine system.
HYPOTHALAMIC HORMONE ANT. PITUITARY HORMONE
TRH - Thyrotropin releasing hormones TSH
CRH - Corticotropin-releasing hormone ACTH
GnRH – Gonadotropin releasing hormone LH and FSH
GHRH – Growth hormone releasing hormone GH
Somatostatin Suppress GH
PRF – Prolactin releasing factor PRL
PIF (Dopamine) – Prolactin inhibitory factor Suppress PRL

Image taken from boliclinic.com Image taken from yumpu.com Image taken from geekymedics.com

B. Pituitary Gland
Also known as hypophysis located within the stella turcica which is connected wo the median eminence of the
hypothalamus by the infundibular stalk. The pituitary gland is made up of anterior and posterior part.

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Anterior pituitary gland Posterior pituitary gland
-Hormone synthesizing and secreting cells: - Does not synthesize hormones rather stores hormones
a. Somatotrophs – secretes Growth Hormone produced by the hypothalamus
b. Lactotrophs – secretes Prolactin - Secretes Arginine vasopressin and Oxytocin which are
c. Thyrotrophs – secretes thyroid stimulating hormone hormones synthesized by the hypothalamus and released
d. Gonadotrophs – secretes alpha and beta subunits of into the circulatory system via neural signaling from the
FSH and LH hypothalamus.
e. Corticotrophs – secretes pro-opiomelanocortin
-Main target organs:
a. Thyroid gland
b. Adrenal Cortex
c. Gonads

Clinically important hormones of the Anterior Pituitary gland

1. Human Growth Hormone
 The primary target of GH is the liver- liver receptors occupied by GH causes the liver to secrete a group
polypeptide hormones insulin like growth factors
 Promote growth of cartilage, bone & many soft tissues by stimulating protein synthesis
 IGF is structurally similar to insulin however it is more potent mediator of linear growth and metabolism of infants
 insulin like growth factor I and II-polypeptides synthesized and release in response to GH-functions similarly to
insulin
 IGFs circulate the blood complexed to specific plasma-binding protein

Image taken from sematicscholar.org

Image taken from transitional-medicine-
Diagnostic significance biomedicentral.com
Acromegaly
 A pathologic or autonomous conditions caused by a pituitary gland tumor.
 Results in increased width of bone rather than length and usually affects the hand, feet and jaw.
 It may also result to generalized organomegaly and coarsening of the facial features with hyperglycemia

Image taken from Medscape.com Image taken from ultimate.altervista.org Image taken from pinterest.com
Diagnostic method for Acromegaly
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  The screening test measure randomly collected IGF-1 and the confirmatory test is the oral glucose tolerance test

Dwarfism or Growth hormone deficiency

 Congenital or acquired, idiopathic-damage to the pituitary gland or hypothalamus, deficiency of other pituitary
hormones
 GH deficiency occurs in both children and adults. In children, it may be familial or it may be due to tumors, such
as craniopharyngiomas. In adults, it is a result of structural or functional abnormalities of the pituitary however, a
decline in GH production is an inevitable consequence of aging and the significance of this phenomenon is poorly
understood.

Image taken from healthism.co

Image taken from businessinsider.com

Diagnostic method for Dwarfism
 Insulin tolerance test is the gold standard for Growth hormone deficiency, in which failure of blood sugar level to
decrease in response to insulin is a positive result.

2. Thyroid stimulating Hormone

 Synthesized in thyrotrophs and composed of two non-covalently linked alpha & beta sub-units
 α-unit is identical to FSH, LH & hCG while β-unit attaches to thyroid receptors & stimulate:
o Stimulates growth and vasculature of the thyroid gland and Uptake and organification of iodine
o Promotes release of stored thyroid hormones
Diagnostic method for TSH
 Pituitary production of TSH is measured by a method referred to as IRMA (immunoradiometric assay).
 Normally, low levels (less than 5 units) of TSH are sufficient to keep the normal thyroid gland functioning properly.
 When the thyroid gland becomes inefficient such as in early hypothyroidism, the TSH becomes elevated. This rise
in TSH represents the pituitary gland's response to a drop in circulating thyroid hormone; it is usually the first
indication of thyroid gland failure
 LATS (long acting thyroid stimulating substance), detectable in the serum of thyrotoxic patients which imitates the
biologic action of TSH
3. Gonadotropins
 These are secreted by the gonadotropic cells and control the functional activity of the gonads.
 Gonads are the organs that produces gametes, for males, it‘s the testes while ovary for the females.
 Pituitary gonadotropin is controlled by feedback from the gonadotropic hormones
 Females, LH and FSH is regulated by estrogen and inhibin
 Males, LH and FSH is regulated by testosterone and inhibin

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 Physiological Action
MALES FEMALES
FSH LH FSH LH
o Stimulates o Responsible for o Stimulates o Promotes and
spermatogenesis production of growth & maintaince the
nd
o Stimulates testosterone by the maturation of 2 portion of the
testicular growth Leydig cells of the ovarian follicles menstrual cycle
o production of testes o Promotes o Cause release of
ABP by the  Maturation of secretion of ova from ovarian
Sertoli cells spermatozoa estrogen by follicles
requires both LH & maturing follicles o Transformation of
FSH (in the presence the follicle into the
of LH) corpus luteum
o Promote after ovulation
endometrial o Secretion of
changes progesterone by
characteristics of the corpus luteum
st
the 1 portion of
menstrual cycle

Diagnostic Method for Gonadotropins

 Commercial RIA kits for both blood (serum or plasma) & urine samples
 Because of episodic, circadian & cyclic variation in secretion of gonadotropins, clinical evaluation requires
determinations in: pooled blood samples, multiple serial blood samples and Times sample of urine.

4. Prolactin/Leuteotropin
 Important in milk letdown reflex and maintenance of lactation
 Physiological increase is seen during: pregnancy, lactation and post pubertal women.
 The effect of PRL in males is less understood, although it may cause a deficiency of male sex hormones.
 Prolactin is secreted in circadian fashion where it is increased during sleep and the lowest concentration is at
10am to 12 noon.
Diagnostic Method for Prolactin
 The major circulating form is a non-glycosylated monomer and similar with other hormones assay, RIA was the
first practical method although today, because of biases caused by structural variation, sandwich techniques that
makes use of antibodies are used to recognize different epitopes on the prolactin polypeptide
 Monomeric prolactin accounts for more than 85% of total circulating hormone
 Although glycosylated or big prolactin constitutes a significant fraction
 IgG bound prolactin is now a common finding among normal individuals (macroprolactin)-decrease renal
clearance of Ig

5. Adrecorticotropic Hormone
 Secreted by the adenohypophysis as a derivative of pro-opiomelanocortin
 Acts primarily on the adrenal cortex stimulating its growth and the secretion of corticosteroids
 Controls the secretion of hormones from the adrenal cortex.
 Regulated by corticotropin-releasing hormone from the hypothalamus, and stress can also increase its release.
 ACTH synthesized in adenohypophysis is formed from the cleavage of a larger precursor molecule: POMC(pro-
opiomelanocortin)
 Lipotropin: described as having weak lipolytic effects; important as the precursor to beta-endorphin.
 Beta-endorphin and Met-enkephalin: Opioid peptides with pain-alleviation and euphoric effects
 Melanocyte-stimulating hormone (MSH): Known to control melanin pigmentation in the skin of most vertebrates.

Page | 87
Image taken from vivo.colostate.edu
Image taken from unlimitedchiroclub.com
Clinically important hormones of the Posterior Pituitary gland
1. Antidiuretic hormone
 Also known as argenine vasopressin and is regulated by the hypothalamus based on hydration status
 Primarily allows the kidney to conserve water
 Mechanism of ADH
o The main regulator of AVP secretion is Osmolality
 Osmoreceptors are located in the cell bodies of the hypothalamus
 2% increase in extracellular fluid osmolality will cause shrinkage of the osmoreceptors causing
stimulation of AVP
 Plasma osmolality above 280mOsm/kg is considered the osmotic threshold for AVP release.
o Pressure volume mechanism, AVP release is regulated by baroreceptors that respond to alterations in
blood volume
 A reduction in plasma volume or arterial pressure or both stimulates AVP secretion

Image taken from homeworkclinic.com

Diagnostic significance of ADH

Hypothalamic Nephrogenic Diabetes Psychogenic or Syndrome of Inappropriate
Diabetes Insipidus Insipidus Primary Polydipsia antidiuretic hormone
(SIADH)
 A.k.a Neurogenic,  Failure of the kidney to  Chronic and excessive  Plasma AVP
Central or Cranial respond to normal or intake of water concentrations are
diabetes insipidus increased supresses AVP increased without known
 Caused by a failure of concentration of AVP secretion stimuli
the pituitary gland to  Caused by mutations  Causes hypotonic  Result of:
secrete normal in the AVP receptors polyuria  Malignancy
amounts of AVP in and Aquaporin-2 water  Hypothalamic disease  Acute and chronic
response to channels affecting the thirst dse. Of the CNS
osmoregulatory  Acquired forms: centers of the brain  Side effects of
factors. Metabolic disorders, drugs
 Assoc. with neoplastic Drugs and Renal
disease, surgery, head disease
trauma, hypoxia,
granulomatous
disease, infections and
autoimmune dse.

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2. Oxytocin
 In childbirth: causes contraction of uterine muscles and in milk-letdown by forcing milk into ducts from the milk
glands.
 This is a nonpeptide that promotes uterine contractions and milk ejection and it contributes to the second stage of
labor in pregnancy
 Primary stimulus:
o Suckling – stimulation of the tactile receptors located around the nipples
o Stretch receptors in the uterus and vaginal mucosa
o Emotional stress may inhibit lactation

C. Thyroid Gland
 Located below the larynx and consists of two broad lobes connected by an isthmus.
 Consists of secretory parts called follicles filled with hormone-storing colloid (thyroglobulin)

1. Thyroid Hormone
 Follicular cells produce two iodine-containing hormones, thyroxine (T 4 ) (tetraiodothyronine) and triiodothyronine
(T3)
 Carriers protein of Thyroid hormones:
o TBG – thyroxine binding globulin (70-75%)
o TBPA – thyroxine binding prealbumin or TTR transthyretin (10-25%)
o TBA – thyroxine binding albumin (10%)
 Biological Function of thyroid hormones
o Increase basal metabolic rate and stimulate synthesis of Na/K ATPase
o Increase body temperature through the use of chemical energy for metabolic processes which is fueled
mainly by fatty acids
o Stimulate neural development and promote sexual maturation
o Increase adrenergic activity and stimulate protein synthesis and carbohydrate metabolism
Diagnostic significance

Clinical Condition TT4 TT3 FT4 TSH

1° Hypothyroidism ↓ ↓ ↓ ↑

2° Hypothyroidism ↓ ↓ ↓ ↓

1° Hyperthyroidism ↑ ↑ ↑ ↓

2° Hyperthyroidism ↑ ↑ ↑ ↑

1° Increased TBG ↑ ↑ N N

1° Decreased TBG ↓ ↓ N N

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Primary Hyperthyroidism Secondary Primary Hypothyroidism Secondary
Hyperthyroidism Hypothyroidism
- Hypermetabolic condition - Over stimulation of -Increased TSH -Central thyroid disease
caused by excessive production the thyroid -Decreased T3T4 -Serum T4 and T3 is low
of thyroid hormones - Over production of -Signs and symptoms : -TSH concentration are
thyroid stimulating *Fatigue either low or within
hormone *Weight gain reference interval
-Causes: *Decreased mental and -Cause:
*Carcinoma *physical output *Pituitary or
*TSH-secreting tumors *Cold intolerance hypothalamic disease
*TRH-secreting tumors
- Grave‘s disease - - Hashimoto‘s disease -
Autoimmune disease that leads Autoimmune disorder in which
to generalized over activity of the immune system creates
the thyroid gland antibodies that damage the
thyroid gland

Diagnostic Method for Thyroid Hormones

 Serum T4 and T3 is measured by radioimmunoassay or cheliminometric assay and ELISA
o Current kits are still unavailable to accurately measure free T4 and T3
o measurement of thyroxine is used in conjunction with TSH
o T3 measurement uses T3 specific antibody: reference ranges for T3 varies from laboratories
 Thyroglobulin is synthesized and secreted exclusively by thyroid follicular cells
o It is an ideal tumor marker for thyroid cancer and should be undetectable after successful treatment
o Thyroglobulin is measured by radioimmunoassay or enzyme linked immunoassay
 Thyroid autoimmunity is a test to detect antibodies to the TSH receptor
o TSAb,TSI – Thyroid stimulating antibody
o TRAb, TSHR-Ab – TSH receptor antibody
 Other tools
o Nuclear medicine, Thyroid ultrasound and Fine needle biopsy

Image taken from pinterest.com

Image taken from endocrineweb.com
Image taken from radiologyinfo.org
2. Calcitonin
 Produced by the C-cells (parafollicular) of the thyroid and its production is stimulated by hypercalcemia
 Lowers plasma Ca level and promotes Ca deposition in bones and urinary excretion of calcium
+2 +2

 Opposes the effects of parathyroid hormones w/c acts primarily to increase blood level of calcium

Page | 90
 Activity No. 18: THYROID GLAND
Thyroid Function Test: TSH

OBJECTIVE: At the end of the activity, the students are expected:

 To be able to appreciate how to perform an Enzyme immunoassay for the determination of TSH in serum by
viewing a simulation video which is recorded by the laboratory instructor.
 To correlate variations in TSH values to clinical conditions

INTRODUCTION:
Thyroid Stimulating Hormone (TSH) is secreted by the anterior lobe of the pituitary gland and induces the production
and release of thyroxine and triiodothyronine from the thyroid gland. It is a glycoprotein with a molecular weight of
approximately 28,000 daltons, consisting of two chemically different subunits, alpha and beta. The determination of
serum or plasma levels of TSH is recognized as a sensitive method in the diagnosis of primary and secondary
hypothyroidism.

MATERIALS:
 Vacutainer set, centrifuge, test tubes, applicator sticks, anticoagulants

PRINCIPLE: The TSH ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. The
assay system utilizes a unique monoclonal antibody directed against a distinct antigenic determinant on the intact TSH
molecule. Mouse monoclonal anti-TSH antibody is used for solid phase immobilization (on the microtiter wells). A goat
anti-TSH antibody is in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test sample is allowed to
react simultaneously with the two antibodies resulting in the TSH molecules being sandwiched between the solid phase
and enzyme-linked antibodies. The wells are washed to remove unbound labeled antibodies. A solution of TMB reagent is
added for blue color development. The color development is stopped with the addition of Stop solution, changing the color
to yellow. The concentration of TSH is directly proportional to the color intensity of the test sample. Absorbance is
measured spectrophotometrically at 450nm.

ASSAY PROCEDURE: (LumiQuick Diagnostics, Inc.): The instructor will make a recorded video of the actual
laboratory performance of the procedure and explanation of the principle of the test. The video will be uploaded for the
students to be able to familiarize themselves with the procedure.
1. Secure the desired number of coated wells in the holder.
2. Dispense 100μL of standard, specimen, and control into appropriate wells.
3. Dispense 100μL of Enzyme Conjugate Reagent into each well.
4. Thoroughly mix for 30 seconds. It is very important to mix them completely.
5. Incubate at room temperature (18 - 25C) for 60 minutes.
6. Remove the incubation mixture by flicking the plate contents into a waste container.
7. Rinse and flick the microtiter wells 5 times with distilled or deionized water.
8. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
9. Dispense 100μL of TMB Reagent into each well. Gently mix for 10 seconds.
10. Incubate at room temperature for 20 minutes.
11. Stop the reaction by adding 100μL of Stop solution to each well.
12. Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
13. Read absorbance at 450 nm with a microtiter well reader within 15 minutes.

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
determine whether the values are normal or outside the reference range. This will be written on a separate bond paper.
Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

GUIDE QUESTIONS: The instructor will make an online forum where the students can type and post their answers to the
QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail or express couriers.
1. Describe completely the Thyroid Stimulating Hormone.
2. Apart from ELISA, describe other methods for determining TSH level in blood.
3. Differentiate Primary Hypothyroidism from Secondary Hypothyroidism. What are the clinical manifestations of
Hypothyroidism?
4. What is LATS? What is the clinical significance of this substance?

Page | 91
 Activity No. 18: Thyroid Function Test: Triiodothyronine (T3)

OBJECTIVE: At the end of the activity, the students are expected:

 To be able to appreciate how to perform an Enzyme immunoassay for the determination of T3 in serum by
viewing a simulation video which is recorded by the laboratory instructor.
 To correlate variations in T3 & T4 values to clinical conditions

INTRODUCTION:
The thyroid gland exerts powerful and essential regulatory influences on growth, differentiation, cellular
metabolism, and general hormonal balance, as well as on the maintenance of metabolic activity and development of the
skeletal and organ system.
The hormones thyroxine (T4) and 3,5,3‘-triiodothyronine (T3) circulate in the blood stream, mostly bound to the
plasma protein, thyroxine binding globulin (TBG). The concentration of T3 is much less than that of T4, but its metabolic
potency is much greater.
T3 determination is an important factor in the diagnosis of thyroid disease. Its measurement has uncovered a
variant of hyperthyroidism in thyrotoxic patients with elevated T3 levels and normal T4 levels. It is useful in monitoring
patients under treatment for hyperthyroidism and those who have discontinued anti-thyroid drug therapy. It is especially
valuable in distinguishing between euthyroid and hyperthyroid subjects. In women, T3 levels are elevated during
pregnancy, during estrogen treatment, and contraceptive hormone therapy.

MATERIALS:
 Vacutainer set, centrifuge, test tubes, applicator sticks, anticoagulants
PRINCIPLE OF THE TEST: Enzyme Immunoassay (EIA)
In the T3 EIA, a second antibody (goat anti-mouse IgG) is coated on microtiter wells. A measured amount of
patient serum, a certain amount of mouse monoclonal anti-T3 antibody and a constant amount of T3 conjugated with
horseradish peroxidase are added to the microtiter wells. During incubation, the mouse anti-T3 antibody is bound to the
antibody on the wells, and T3 & conjugated T3 compete for the limited binding sites on the anti-T3 antibody. After a 60-
minute incubation at room temperature, the wells are washed to remove unbound T3 conjugate. A solution of TMB
reagent is then added and incubated for 20 minutes, resulting in the development of blue color. The color development is
stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450nm. The
intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of
unlabeled T3 in the sample.

ASSAY PROCEDURE (LumiQuick Diagnostics Inc.): The instructor will make a recorded video of the actual laboratory
performance of the procedure and explanation of the principle of the test. The video will be uploaded for the students to
be able to familiar themselves with the procedure.
1. Secure the desired number of coated wells in the holder.
2. Pipette 50 μL of standard, samples, and control into appropriate wells.
3. Dispense 50 μL of the Antibody Reagent into each well. Mix thoroughly for 30 seconds.
4. Add 100 μL of Working Conjugate Reagent into each well. Mix thoroughly for 30 seconds.
5. Incubate at room temperature for 60 minutes.
6. Remove the incubation mixture by flicking plate contents into a waste container.
7. Rinse and flick the microtiter wells 5 times with distilled or deionized water.
8. Strike the wells onto an absorbent paper to remove residual water.
9. Dispense 100 μL TMB reagent into each well. Gently mix for 10 seconds.
10. Incubate at room temperature in the dark for 20 minutes without shaking.
11. Stop the reaction by adding 100 μL of Stop Solution to each well.
12. Gently mix for 30 seconds.
* It is important to make sure that the blue color changes to yellow color completely.
13. Read OD at 450 nm with a microtiter reader within 15 minutes.

EXPECTED VALUES AND SENSITIVITY:

The range in normal individuals is 0.6 – 1.85 ng/mL. In general, total serum T3 levels will tend to parallel the
variations in serum levels of the major binding protein, TBG. Elevated T3 levels may be encountered in hypothyroid
individuals receiving replacement therapy. The minimum detectable concentration of T3 by this assay is estimated to be
0.2 ng/mL.

Learning Output: The students will be shown the absorbance reading from a set of samples. The students will then
determine whether the values are normal or outside the reference range. This will be written on a separate bond paper.
Page | 92
Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

Activity No. 19: Thyroid Function Test: Thyroxine (T4)

OBJECTIVE: At the end of the activity, the students are expected:

 To be able to appreciate how to perform an Enzyme immunoassay for the determination of T4 in serum by
viewing a simulation video which is recorded by the laboratory instructor.
 To correlate variations in T3 & T4 values to clinical conditions

INTRODUCTION:
L-Thyroxine (T4) is a hormone that is synthesized and stored in the thyroid gland. Proteolytic cleavage of follicular
thyroglobulin releases T4 into the bloodstream. Greater than 99% of T4 is reversibly bound to three plasma proteins in
blood: thyroxine binding globulin (TBG) binds 70%; thyroxine binding pre-albumin (TBPA) binds 20%; and albumin binds
10%. Approximately 0.03% of T4 is in the free, unbound state in blood at any one time.
Measurement of total T4 by immunoassay is the most reliable and convenient screening test available to
determine the presence of thyroid disorders in patients. Increased levels of T4 have been found in hyperthyroidism due to
Grave‘s disease and Plummer‘s disease and in acute and subacute thyroiditis. Low levels of T4 have been associated
with congenital hypothyroidism, myxedema, chronic thyroiditis (Hashimoto‘s disease), and with some genetic
abnormalities.

MATERIALS:
 Vacutainer set, centrifuge, test tubes, applicator sticks, anticoagulants

PRINCIPLE OF THE TEST: Enzyme Immunoassay (EIA)

In the T4 EIA, a certain amount of anti-T4 antibody is coated on microtiter wells. A measured amount of patient
serum and a constant amount of T4 conjugated with horseradish peroxidase are added to the microtiter wells. During
incubation, T4 & conjugated T4 compete for the limited binding sites on the anti-T4 antibody. After a 60-minute incubation
at room temperature, the wells are washed to remove unbound T4 conjugate. A solution of TMB reagent is then added
and incubated for 20 minutes, resulting in the development of blue color. The color development is stopped with the
addition of stop solution, and the absorbance is measured spectrophotometrically at 450nm. The intensity of the color
formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled T4 in the
sample.

ASSAY PROCEDURE (LumiQuick Diagnostics Inc.): The instructor will make a recorded video of the actual laboratory
performance of the procedure and explanation of the principle of the test. The video will be uploaded for the students to
be able to familiarize themselves with the procedure. The results will then be showed and will be used by the students for
the computation.

1. Secure the desired number of coated wells in the holder.

2. Pipette 25 μL of standard, samples, and control into appropriate wells.
3. Dispense 100 μL of Working Conjugate Reagent into each well. Mix thoroughly for 30 seconds.
4. Incubate at room temperature for 60 minutes.
5. Remove the incubation mixture by flicking plate contents into a waste container.
6. Rinse and flick the microtiter wells 5 times with distilled or deionized water.
7. Strike the wells onto an absorbent paper to remove residual water.
8. Dispense 100 μL TMB reagent into each well. Gently mix for 10 seconds.
9. Incubate at room temperature in the dark for 20 minutes without shaking.
10. Stop the reaction by adding 100 μL of Stop Solution to each well.
11. Gently mix for 30 seconds.
* It is important to make sure that the blue color changes to yellow color completely.
12. Read OD at 450 nm with a microtiter reader within 15 minutes.

EXPECTED VALUES AND SENSITIVITY:

The T4 EIA was performed in a study of 200 euthyroid patients in one geographic location and yielded a range
of 4.8 – 12.0 μg/dL. It is recommended that laboratories adjust values to reflect geographic and population differences
specific to the patients they serve. The minimum detectable concentration of Thyroxine by this assay is estimated to be
0.4 μg/mL.

Page | 93
LEARNING OUTPUT: The students will be shown the absorbance reading from set samples. The students will then
determine whether the values are normal or outside the reference range. This will be written on a separate bond paper.
Student can scan or take a picture of their output and send vial email. For offline students, output can be submitted
through text message or via courier.

GUIDE QUESTIONS: (for T4 & T3) The instructor will make an online forum where the students can type and post their
answers to the QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail or express
courier or through text message.

1. Describe in detail how T3 and rT3 are formed from the deiodination of Thyroxine.
2. Describe other methods which are employed for the measurement of T3 and T4 in blood.
3. Discuss the clinical significance of T3 and T4 assays.

Page | 94
D. Parathyroid Glands
 Located at the posterior of the thyroid which consists of tightly packed secretory cells covered by thin connective
tissue

1. Parathyroid Hormone
 Release is stimulated by hypocalcemia and increased plasma calcium levels
 The parathyroid hormone responds to decrease in free calcium concentration within seconds.
 It stimulate bone resorption of osteoclasts and promotes tubular reabsorption of Ca and intestinal absorption of
+2

calcium
 During a time of calcium deprivation, the increase in PTH rapidly alters both renal and skeletal metabolism

Diagnostic Significance Image taken from pinterest.pt

Hyperparathyroidism Hypoparathyroidism
-Primary -2° hyperparathyroidism -Due to injury of the PTG
hyperparathyroidism *Due to vitamin D deficiency -Persistent hypocalcemia
*80% due to parathyroid *Seen in chronic renal -Tetany and altered neuromuscular activity
adenoma failure -Deficiency of blood calcium causes neurons and muscle
*Seen in kidney and bone fibers to depolarize and produce action potentials
disease spontaneously
-Twitches, spasms and tetany of skeletal muscle

Page | 95
F. Adrenal Glands
 2-3 cm wide, 4-6 cm long and 1 cm thick and situated on top of the kidneys enclosed in a layer of fat
 because each glands sits atop of the kidney, the adrenal glands are also referred to as the suprarenal glands

Image taken from onlinesciencenotes.com

Image taken from urology.weillcornel.org

Structure
Adrenal medulla Adrenal Cortex
- comes from a neural crest origin and stores and secretes - cortex-makes up 80-90% of adrenal gland
catecholamines - have very rich arterial supply that forms a subcapsular
plexus and empties into a central vein.
- further divided into three layers: Zona glomerulosa
(Outermost layer), Zona fasciculate (Middle layer) and Zona
reticularis (Innermost layer)
Catecholamines: Epinephrine, Norepinephrine, Dopamine Adrenal steroid hormones: Glucocorticoids,
Mineralocorticoids, Sex steroids

1. Aldosterone
 Release is stimulated by a decrease in serum SODIUM levels
 Promotes tubular reabsorption of sodium in the kidneys
 Promotes excretion of potassium and hydrogen
 Production is controlled by RAA (renin-angiotensin-aldosterone) system

Diagnostic Significance
Hypoaldosteronism Hyperaldosteronism
Decreased Sodium and Hypernatremia
chloride Hypokalemia
Cortisol Hypertension
Urinary steroids Metabolic alkalosis
Increased
ACTH

Aldosterone Assay
 Makes use of fasting plasma, and levels in pregnant
patients are three to four time higher
 Urine sample is to be used must be collected for a 34 hour
specimen in 10g boric acid to maintain pH <7.5
Image taken from researchgate.net

2. Cortisol
Page | 96
  A glucocorticoid, influences the metabolism of glucose, CHON, & fat
 Has Anti-inflammatory and immunosuppressive effects
 Negative feedback mechanism controls the release of cortisol (CRH – hypothalamus; ACTH -anterior pituitary)
 Respond to conditions that stress the body & require a greater supply of energy

Image taken from sciencedirect.com

Diagnostic significance
Hypercortisolism Hypocortisolism
-Hypercortisolism : Cushing‘s syndrome causes -Hypocortisolism : Addison‘s disease
-Pituitary gland tumor -Darkening areas of skin (hyperpigmentation)
-Ectopic ACTH-secreting tumor -Severe fatigue
-Primary adrenal gland disease -Unintentional weight loss
-Familial Cushings disease -Gastrointestinal problems, such as nausea, vomiting and
abdominal pain
-Lightheadedness or fainting
-Salt cravings
-Muscle or joint pains
Methods for Cortisol determination
 Method employed is dexamethasone suppression test where exogenous steroid is used to suppress innate
cortisol production
 Measure of cortisol:
o Normal : decreased cortisol (suppressed)
o Cushing‘s syndrome : increased cortisol (not suppressed)
3. Catecholamines
 Produced in chromaffin cells
 Epinephrine,norepinephrine and dopamine
 Homovanillic acid (HVA) – metabolite of dopamine
 Metanephrines and Vanillylmandelic acid (VMA) – epinephrine metabolite
 Epinephrine: Most people know epinephrine by its other name—adrenaline. This hormone rapidly responds to
stress by increasing your heart rate and rushing blood to the muscles and brain. It also spikes your blood sugar
level by helping convert glycogen to glucose in the liver
 Norepinephrine: Also known as noradrenaline, this hormone works with epinephrine in responding to stress.
However, it can cause vasoconstriction (the narrowing of blood vessels). This results in high blood pressure.

Diagnostic significance
 Pheochromocytoma
o Tumor in adrenal medulla: excess epinephrine
o Hypertension, headache
o Increased heart rate
 Neuroblastoma - Malignant tumor of adrenal medulla seen in children which may lead to abdominal mass
Methods for determination
 Makes use of urine or plasma samples where urinary metanephrine is best for screening test of
pheochromocytoma and urinary VMA and HVA

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 Activity No. 20: The Adrenal Gland

OBJECTIVE: At the end of the activity, the students are expected:

 To differentiate the adrenal cortex from the adrenal medulla
 To learn the mechanism of action and clinical significance of the different hormones that come from the adrenal
gland

MATERIALS:
 Charts, Reference Materials

PROCEDURE:
 In a tabulated form, enumerate the hormones produced by the Adrenal gland (Adrenal cortex and Adrenal medulla)
their mode of action, target organ/s and clinical significance.
 In another table, indicate the site of origin, mode of action, target organ and clinical significance of the other
clinically important hormones to be enumerated.

DRAW AND LABEL: Drawings must be placed on a short bond paper with .5‖ margins in all sides bearing the name and
section of the student. Students will scan or will take a picture of their drawings to be submitted online. For offline
students, submit via mail or express courier (―padala‖) addressed to: Instructor’s name, School of Natural Sciences,
University of Baguio, Baguio City.
Adrenal gland ( Adrenal cortex and Adrenal medulla )

GUIDE QUESTIONS: The instructor will make an online forum where the students can type and post their answers to the
QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or through
text message.

1. Differentiate the Adrenal cortex from the Adrenal medulla in terms of:
A. Histological structure
B. Mechanism that controls hormonal secretion
C. Hormones produced

2. Completely describe the Renin-Angiotensin-Aldosterone (RAA) system.

3. Describe a test for the laboratory diagnosis of each of the following:

A. Hyperaldosteronism (or Hypoaldosteronism)
B. Hypercortisolism (or Hypocortisolism)
C. Pheochromocytoma

4. Describe the sampling requirements for the laboratory diagnosis of:

A. Hyperaldosteronism (or Hypoaldosteronism)
B. Hypercortisolism (or Hypocortisolism)
C. Pheochromocytoma

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G. Pancreas
 Secretes hormones as an endocrine gland, and digestive juices to the digestive tract as an exocrine gland.
 Endocrine cells : islets of Langerhans
o alpha cells - glucagon
o beta cells - insulin
o delta cells - somatostatin
o F cells – pancreatic polypeptide

1. Glucagon
 It stimulates the conversion of stored glycogen (stored in the liver) to glucose, which can be released into the
bloodstream. This process is called glycogenolysis.
 it promotes the production of glucose from amino acid molecules. This process is called gluconeogenesis.
 It reduces glucose consumption by the liver so that as much glucose as possible can be secreted into the
bloodstream to maintain blood glucose levels.

2. Insulin
 Release is stimulated by hyperglycemia
 Decreases BGL by stimulating liver glycogenesis, increasing CHON synthesis, and stimulating adipose cells to
store fat.

Diagnostic significance

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 Assessment No. 8

True or False. Please answer the following questions; write your answers on the space provided.

1. SIADH results in excessive secretion of vasopressin (ADH) from the posterior

pituitary, causing fluid retention and low plasma osmolality, sodium, potassium,
and other electrolytes by hemodilution.
2. Thyroid hormones are derived from the enzymatic modification of tyrosine
residues on thyroglobulin.
3. Total serum T4 and T3 are dependent upon both thyroid function and the
amount of thyroxine-binding proteins such as thyroxine-binding globulin (TBG).
4. Stress, exercise, and an upright position induce catecholamine elevation, and
therefore, patients must be resting supine for at least 1 hour prior to blood
collection.
5. Plasma and urinary catecholamines are measured in order to diagnose
pheochromocytoma.
6. Cushing‘s disease refers to adrenal hyperplasia resulting from misregulation of
the hypothalamic–pituitary axis.
7. Congenital adrenal hyperplasia (adrenogenital syndrome) results from a
deficiency of an enzyme required for synthesis of cortisol.
8. Serum cortisol can be decreased by factors such as stress, medications, and
cortisol-binding protein and the cortisol level of normal patients will overlap those
seen in Cushing‘s syndrome because of pulse variation.
9. When cortisol levels become elevated, cortisol-binding protein becomes
saturated, and free (unbound) cortisol is filtered by the glomeruli. Most is
reabsorbed, but a significant amount reaches the urine as free cortisol.
10. Cortisol is the most abundant adrenal hormone, and abnormal levels have
pronounced effects on carbohydrate and lipid metabolism.
11. Approximately 90% of patients with acromegaly will have an elevated fasting
GH level, but 10% will not.
12. Because GH is the most abundant pituitary hormone, it may be used as a
screening test for pituitary failure in children.
13. In children, a deficiency of GH can be ruled out by demonstrating normal or
high levels on two successive tests.
14. In women, serum or urine LH and FSH are measured along with estrogen and
progesterone to evaluate the cause of menstrual cycle abnormalities and
anovulation.
15. Prolactinoma can result in anovulation because high levels of prolactin
suppress release of LHRH
16. The most common cause of Cushing‘s syndrome is the administration of
medications with cortisol or glucocorticoid activity
17. Catecholamines are metabolized to metanephrines and VMA.

18. Urinary catecholamines are decreased by exercise and dietary ingestion.

19. Type 1 diabetes appears in younger people.

20. Insulin antagonizes the effects of glucagon.

Page | 100
Reference:

Anderson, Shauna and Susan Cockyane (2007). Clinical Chemistry: Concepts and Applications. USA: Waveland
Press Inc.

Ashwood E., D. Bruns and C. Burtis (2012). Tietz’s Textbook of Clinical Chemistry and Molecular Diagnostics 5th ed.
Philadelphia: W.B. Saunders Co.
nd
Daniels, Rick. (2010). Delmar's Guide to laboratory and diagnostic tests. (2 ed.) Delmar Cengage Learning.

McPherson, Richard. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Elsevier
Saunders, 2011.

Page | 101
 Chapter VI. TOXICOLOGY
Republic Act No. 9165 June 7, 2002, This Act shall be known and cited as the "Comprehensive Dangerous
Drugs Act of 2002". It is the policy of the State to safeguard the integrity of its territory and the well-being of its
citizenry particularly the youth, from the harmful effects of dangerous drugs on their physical and mental well-being,
and to defend the same against acts or omissions detrimental to their development and preservation. In view of the
foregoing, the State needs to enhance further the efficacy of the law against dangerous drugs, it being one of
today's more serious social ills.

At the end of this chapter, you should be able to:

a. Discuss the mechanism of action of various toxins in the body.
b. Associate clinical signs & symptoms to particular types of toxicity/poisoning.
c. Describe the effects of varying blood alcohol concentrations on behavior.
d. Describe the methods employed for alcohol determination.
e. Classify the drugs of abuse as to mode action
f. Describe the techniques for drug analysis.
g. Accurately perform a screening drug test following the proper protocol.

Section 1. DRUGS OF ABUSE

Nature of Drugs and Drug abuse

Drugs are any substance that produces physiological or psychological change within a short period of time after
ingestion of a specified dose. Different types of drugs affect your body in different ways, and the effects associated with
drugs can vary from person to person. How a drug effects an individual is dependent on a variety of factors including body
size, general health, the amount and strength of the drug, and whether any other drugs are in the system at the same
time.

Nature of Drug dependence

When an individual becomes strongly attached to a drug. Dependency is subdivided into two categories:
physiological and psychological. Drugs can have short-term and long-term effects. These effects can be physical and
psychological, and can include dependency. Psychological dependence is when a person develops an uncontrollable
―craving‖ (mental or emotional need) for a drug, the craving is a desperate need to continue. In physiological dependency,
the body continually needs to have the drug, a person experience sickness if drug is discontinued.

Six Categories of Drugs of abuse

A. Opiates and Narcotic B. Stimulants C. Hallucinogens

Drugs
*Morphine is the primary *Stimulants are taken to *Cause a significantly altered mental state, often including
active drug in opium that make one feel more hallucinations
came from the dried sap energetic, strong, or awake *Marijuana is one of the oldest
of the opium poppy plant. *Abused stimulants: *Physiologically active ingredients: Cannabinoids, found in the
Amphetamine, resinous leaf coating of Cannabis sativa
methamphetamine, and *The most active cannabinoid
cocaine is THC
*Methamphetamine is the
drug most commonly
Livescience.com produced in clandestine labs
*Opium can be smoked
directly or chemically
processed to isolate pure
morphine Medicalnewstoday.com
*Opiates are
*Cocaine - very powerful
psychologically addictive
stimulant; enormously Livescience.com
drugs; withdrawal causes
psychologically addicting
Page | 102
severe physiological *Cocaine hydrochloride is *Metabolites of Marijuana:
symptoms usually inhaled through the -Delta-9-carboxy-THC
*Codeine - second most nose -111-hydroxy-delta-9-THC
abundant component of *Its free base form (―crack‖) -Detectable in urine from 1 to 4 weeks after last use
opium, used as a strong is vaporized by heat in a *Hashish – more potent form of marijuana made from the
painkiller and cough pipe and inhaled into the flowering tops of the plant
suppressant lungs *Hash oil: made by heating the plant material in a solvent,
then evaporated leaving a thick oily material (almost pure
resin)
*Hash oil can be mixed with tobacco or other vegetable
material
*LSD is an extremely potent hallucinogen with normal dose of
Sunrisehouse.com 30-50 mg, this causes visual hallucinations, brilliant colors
and the perception that one is wise
Webmd.com

Medicalexpress.com

*Naturally occurring hallucinogens: Peyote, the bud of a

particular cactus whose main ingredient is mescalin
*Magic mushrooms: Genus Psilocybe; Active components:
psilocin and psilocybin

e.wikipedia.org

D. Depressants, Hypnotics and E. Club Drugs F. Athletic Performance

Tranquilizers Enhancers
*Alcohol (depressant) - most abused *prepared by clandestine labs, or obtained *Athletes trying to gain a
substance legally from other countries competitive edge may abuse
*MDMA - ―love drug‖ or ―Ecstasy‖ stimulants and painkillers
*GHB, gamma hydroxybutyrate, *The first drug controlled
*GHB and related compound GBL: because of their abuse by
used for their hypnotic or depressant athletes were anabolic steroids
effects *Anabolic steroids promote cell
growth
resulting in growth of muscle
tissue and
sometimes bone size and
Page | 103
 strength

Theconversation.com

*Ketamine - anesthetic and animal

Laressio.com tranquilizer; causes anterograde amnesia
*Rohypnol, GHB, and ketamine have been
*Barbiturates - physiologically implicated in cases of drug-facilitated
active depressants, resulting in a sexual assaults ―date-rape‖ drugs
physical & mental state similar to
alcohol-induced intoxication
En.wiki.com

Drugtestsinbulk.com

*Condensation product of urea and

malonic acid
*Fat soluble : easily crosses BBB
*Low doses: sedation, drowsiness and Statnews.com
sleep
*Higher doses : anesthesia
*Very high doses : stupor, coma and
death
*Toxicity : depression, cyanosis,
hypothermia, hypotension
*Valium (a benzodiazepine) - a
tranquilizer drug designed to relieve
anxiety
*Rohypnol or ―roofies‖ is a
benzodiazepine and a major drug of
abuse at raves and
the club scene

Testing for Drugs of Abuse

Drug testing centers over the years had an increasing capability. Drug testing has been required by government
agencies, industrial agencies and sports agencies. Forensic drug testing has been used for the application of drug test in
questions of law. The following are specimen requirements and the different methods used for drug testing:
 Urine specimen – the usual sample used to test a number of drugs of abuse
o Diasadvantage:
 Detects only fairly recent drug use
 Will not differentiate casual use from chronic drug abuse
 Does not determine degree of impairment
 Does not determine dose of drug taken
 Will not state exact time of use
 Meconium
o first stool of the newborn
o begins to form during second trimester and continues to accumulate until birth
o provides evidence of maternal drug use anytime during the last two trimesters

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  Hair
o obtained easily; not easily tampered
o prior drug use may be detected for several months
o hair growth : 0.3 to 0.4 mm/day
o mechanism of drug deposition in hair
 transfer from blood to growing hair shaft
 transfer from sweat
 environmental contamination
 Sweat
o sweat-patch collection devices, worn for several days to several weeks
o the drug, if present accumulates on the absorbent pad in the patch
o advantage : monitoring of drug use in correctional institutions or in drug rehab programs
 Saliva
o easy to obtain, less invasion of privacy and ease of adulteration
o ultrafiltrate of plasma and used for recent drug use
o detection of drug level is shorter compared with urine

Laboratory Considerations
Specimen collection
o urine : sample of choice; represents the net load of the drug over a long period
o guard against specimen exchange and/ or adulteration
o urine pH, specific gravity and creatinine
o blood : represents transient passage of the drug thru the circulation
o only a quick picture of the drug level at a specific time
Processing and handling – confidentiality and chain of custody
Analytical methods
a. screening assay – good sensitivity with marginal specificity
b. confirmatory assay – high sensitivity and specificity
- qualitative and quantitative info
- different from screening procedure

Techniques for Drug Analysis

 IMMUNOLOGIC
I. Enzyme Multiplied Immunoassay Technique (EMIT)
o Drug to be measured : hapten part of an antigen
o Serum + antibody mixture + enzyme labeled drug + substrate
o Measure enzyme activity : drug concentration
o More rapid than RIA

II. Enzyme-Linked Immunosorbent Assay (ELISA)

o Drug to be measured is the hapten
o Specific antibodies bound to a solid state carrier
o Separation of the bound drug from the unbound

III. Fluorescence Immunoassay

o Drug bound to a fluorogenic substrate (Umbelliferyl-β-galactoside)
o Fluorogenic drug reagent + antibody + beta-galactosidase --- incubated with serum sample
o Drug and FDR compete for binding
o Fluorescent product = Umbelliferone

IV. Radioimmunoassay
o Drug (hapten) + protein + antibody
o Incubation of serum + antibody + radiolabeled drug (competition for antibody binding sites)
o 2 fractions: Free fraction (unbound drug) and Antibody-bound drug

 CHROMATOGRAPHIC
I. Chromatography
o Adsorption of drug to a solid support and elution by means of a mobile , liquid phase
o TLC – screening for drug identification
Page | 105
 Thin Layer Chromatography
o use solid phase support medium and liquid mobile phase separation system
o drugs are separated on the basis of their ability to dissolve in the solvent system and its strength of
interaction with the support phase
o color reactions are then used to locate and identify the specific substance
o urine sample adjusted to pH 8.5

HPLC and GLC- primarily for quantitating serum drug levels in TDM and also for confirming drug identification
Gas Chromatography/ Mass Spectrometry
o most specific and sensitive: gold standard
o parent drug and metabolites may be detected
o sample is placed in a solvent to extract the substance
o extract is concentrated and injected into a gas chromatograph
o fractionation pattern is determined
o mass spectrometry: determine molecular weight and structural characteristics to identify the drug

 SPECTROPHOTOMETRIC
o Spectral scan : tentative identification of drugs
o Protein precipitation or extraction
o Quantitative analysis

Assessment No. 9

Question: On the space provided, differentiate a screening from a confirmatory drug testing laboratory as to
service capabilities and test processes.

Question: In your opinion, was the War on Drugs by the President successful?

Reference:
th
Hayes, Wallace A (2007). Principles and Methods of Toxicology (5 ed.). USA: Informa Healthcare.
th
Katzung, B.G., ed. (2009) Basic and clinical pharmacology. (11 ed.). Boston : McGraw-Hill.

Page | 106
 Activity No. 21: Drug Testing: MET and THC

OBJECTIVE: At the end of the activity, the students are expected:

 To be able to appreciate how to properly perform the screening test for drugs of abuse based on Thin Layer
Chromatography by viewing a simulation video which is recorded by the laboratory instructor.
 To describe the principle underlying Thin Layer Chromatography as a screening test for drugs of abuse
 To appreciate the significance of monitoring ―chain of custody‖ in drug testing

MATERIALS:
 Specimen: Urine
 Drug Testing kits

PROCEDURE: The instructor will make a video of the actual laboratory performance of the procedure and explanation of
the principle of the tests. The video will be uploaded for the students to be able to familiarize themselves with the
procedure. The video will explain the complete process of screening drug tests and the results of such test.

LEARNING ACTIVITY: From the test procedure, the instructor will show results from a set of specimen and the students
will determine whether the samples have a positive, negative or invalid results.

GUIDE QUESTIONS: The instructor will make an online forum where the students can type and post their answers to the
QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or through
text message.

1. Discuss the principle associated with screening drug testing (TLC) and confirmatory drug testing (GC-MS)
2. In tabular form, characterize the different drugs of abuse based on:
a. Other names (common names/chemical name)
b. Mode of action
c. Detection Limit & Detection Time in Urine
3. Discuss the concept of ―Chain of Custody‖ in drug testing.
4. Classify drug testing laboratories based on ownership, institutional character and service capability.
5. Explain the difference between a mandatory drug testing and a random drug testing
6. Enumerate and give the function of each personnel of a drug testing laboratory.

Page | 107
 Section 2. THERAPEUTIC DRUG MONITORING

 It involves the analysis, assessment and evaluation of circulating concentrations of drugs in serum, plasma or
whole blood.
 Ensures that a given drug produces maximal therapeutic benefit and minimal side effects
 Only the free fraction of the drugs can interact with the site of action and result in a biologic response.
 Mixed Function Oxidase (MFO) system – biochemical pathway responsible for the greatest portion of drug
metabolism.

Indications for TDM:

1. Adverse effects of overdosing and under-dosing
2. The margin of safety is narrow.
3. There is a poor relationship between the dose of drug and circulating concentrations but a good correlation
between circulating concentrations and therapeutic and toxic effects.
4. There is a change in the patient‘s physiologic state that may unpredictably affect circulating drug concentrations.
5. An unfavorable drug interaction occurs or is anticipated.
6. Monitoring patient compliance

Routes of Administration:
 Routes: intravenous, intramuscular, subcutaneous, inhalation, suppository and transcutaneous.
Absorption:
 Tablets and capsules require dissolution before being absorbed; liquid solutions are rapidly absorbed.
 Most drugs are absorbed by passive diffusion
 Weak acids are absorbed in the stomach; weak bases in the intestine.
 Factors affecting absorption: changes in intestinal movement, pH, inflammation and the presence of food or other
drugs.

CARDIOTROPICS – used to treat Class I – rapid sodium channel Quinidine

congestive heart failure and arrhythmias blockers Procainamide
- Act on the conduction system of cardiac Lidocaine
muscle Phenytoin
- Net effect: slow down electrical
conduction
Class II – beta adrenergic blockers Propranolol
Class III – potassium channel Amiodarone
blockers
Class IV – calcium channel Verapamil
blockers
CARDIOTROPICS ACTION TOXICITY
Lidocaine - local anaesthetic - convulsions, coma, respiratory
- acute control & prevention of ventricular depression, bradycardia,
arrhythmias after MI hypotension
Quinidine - for supraventricular and ventricular arrhythmias Cinchonism- vertigo, tinnitus,
Common formulation: Quinidine sulfate & Quinidine headache, visual disturbances and
gluconate disorientation

Procainamide - for supraventricular or ventricular arrhythmias Reversible lupus-like syndrome w/

Metabolite : n-acetylprocainamide elevated ANA titers, urticaria, rash,
agranulocytosis and nephrotic
syndrome
Propranolol - Antagonizes effects of epinephrine on the heart,
arteries and arterioles of skeletal muscles and on
the bronchus
- causes vasodilation
- used for treatment of hypertension and angina

Page | 108
 pectoris
Amiodarone - for life-threatening ventricular arrhythmias
Verapamil - angina, hypertension, supraventricular
arrhythmias
DIGITAL GLYCOSIDES ACTION TOXICITY
+ +
Digoxin - Inhibits membrane Na -K -ATPase Toxic effects: nausea, vomiting,
+ + +
- - Decreases K and Mg and increases Ca visual disturbances, premature
- Rapid onset of action : 1-2 h after oral ventricular contractions and
administration atrioventricular node blockage.

Digitoxin Active metabolite: Digoxin

- Slower onset of action (1-4 hours oral)
ANTIBIOTICS ACTION TOXICITY
Aminoglycosides - Treatment of gram (-) bacterial infections - nephrotoxicity and ototoxicity
Examples: Gentamicin, - Administered IM or IV
tobramycin, amikacin
and kanamycin
Vancomycin - Effective against gram (+) cocci & bacilli - Toxic side effects occur in the
- Administered by IV infusion therapeutic range (5 – 10 μg/mL)
- Trough levels are monitored
- Toxic effects: ―red-man
syndrome‖, nephrotoxicity and
ototoxicity
ANTI-SEIZURE DRUGS ACTION TOXICITY
Phenobarbital - Slow-acting barbiturate that controls several types Nystagmus, ataxia, stupor,
of seizures respiratory depression, hypotension
- A potent inducer of the hepatic MFO system and coma
- Only trough levels are evaluated unless there is
toxicity
Phenytoin (Dilantin) - a short-term prophylactic agent in brain injury Major toxicity: initiation of seizures
- Toxicity is seen at the level of the therapeutic Other toxic effects: hirsutism,
range gingival hyperplasia, Vit. D & folate
deficiency
Valproic acid (Depakene) - used for treatment of petit mal and absence Toxic level: > 120 µg/mL - nausea,
seizure lethargy and weight gain
- Enhance the activity of GABA-mediated inhibitory > 200 µg/mL - pancreatitis,
system hallucinations, hyperammonemia
(hepatic dysfunction)
Other toxic effects: sedation, gastric
disturbances, ataxia
Carbamazepine - used for the treatment of various seizure Toxicity : drowsiness, ataxia,
(Tegretol) disorders and trigeminal neuralgia (tic douloureux) dizziness,nausea and vomiting
- causes a reduction of excitatory synaptic Rare : - aplastic anemia,
transmission in the spinal trigeminal nucleus thrombocytopenia
- chemically related to imipramine (TCA) agranulocytosis

Primidone - for generalized tonic clonic, simple partial and

complex partial seizures
Metabolites: Phenobarbital and
phenylethylmalonamide
Ethosuximide - for treatment of petit mal (absence) seizures Toxicity : GI : nausea, vomiting,
(Zerontonin) - chemically related to imipramine (TCA) gastric distress

ANTI-ASTHMA DRUGS ACTION TOXICITY

Theophylline - used for treatment moderate or severe asthma * 15-20 ug/mL: Nausea, vomiting,
attacks headache and anxiety
Page | 109
 - bronchodilator, vasodilator * 20-40 ug/mL: Tachycardia,
- stimulates respiration & strengthens action of arrhythmias
cardiac muscles * >40 ug/mL: Seizures, cardiac
- CNS stimulant arrest
Drug interactions: Phenytoin, Carbamazepine,
Erythromycin
ANTI-INFLAMMATORY ACTION TOXICITY
DRUGS
Corticosteroids - Block the cyclo-oxygenase pathway - Fluid retention, weight gain,
- Anti-inflammatory osteoporosis, gastrointestinal
bleeding and mental changes
NSAIDS
Acetylsalicylic acid Function: decreases thromboxane and Chronic toxicity: tinnitus, muffled
(Aspirin) prostaglandin formation through inhibition of hearing
cyclooxygenase Acute toxicity : acidosis
- Commonly used analgesic, antipyretic and anti- Reye‘s syndrome: hepatotoxicity
inflammatory drug
- Lower doses: anti-thrombotic
- Direct stimulator of the respiratory system and an
inhibitor of the Kreb‘s cycle
Method for testing: Trinder assay (using ferric
nitrate)
Acetaminophen - Analgesic and anti-pyretic Acute toxicity : nausea, vomiting
/Paracetamol Metabolites : glucoronide and sulfate conjugates and abdominal pain
Reference method: HPLC Chronic toxicity : anemia, renal
damage, GI disturbances
IMMUNOSUPPRESSIVES ACTION TOXICITY
Cyclosporine - used for suppression of host-versus-graft * Adverse effect : nephrotoxicity
rejection * Other effects : neurologic,
- Drug of choice for maintenance of kidney, liver, dermatologic, hepatotoxic
heart and heart-lung allografts
Tacrolimus - Currently used in transplant surgery to prevent - Similar toxicity profile to
organ rejection ciclosporin
- High levels is associated with
thrombus formation
Rapamycin (Sirolimus) - Similar to tacrolimus - major side effects are lipid
abnormalities and
thrombocytopenia

Mycophenolate mofetil - Rapidly converted in the liver into its active form,
mycophenolic acid (MPA)
- A lymphocyte proliferation inhibitor
- It decreases renal allograft rejection
Leflunamide (LFM) - Inhibits lymphocyte proliferation
- for treatment of rheumatoid arthritis
PSYCHOACTIVE DRUGS ACTION TOXICITY
Lithium (as Lithium - used for the treatment of manic depression *1.2 – 2.0 mmol/L – apathy,
citrate or Lithium - used for prophylaxis and treatment of bipolar lethargy, speech difficulties
carbonate) disorder * > 2.0 mmol/L – seizures, muscle
rigidity and coma
Others: Renal toxicity &
hypothyroidism
TRICYCLIC ANTI- - Block reuptake of adrenergic and dopaminergic Toxicity: excess CNS stimulation -
DEPRESSANTS neurotransmitters seizures, coma, hypotension,
Examples: imipramine, - Used for the treatment of depression, insomnia, respiratory depression
amitriptyline, doxepin, extreme apathy and loss of libido
desipramine and
nortriptyline

Page | 110
Fluoxetine (Prozac) - Blocks the re-uptake of serotonin in central Side effects: attempted suicide,
serotonergic pathways decreased libido and sexual
function

CHEMOTHERAPEUTIC ACTION TOXICITY

AGENTS
Methotrexate - Inhibits DNA synthesis by blocking dihydrofolate - Hematologic effects (leucopenia,
reductase thrombocytopenia), GI effects
- used for the treatment of Psoriasis, Refractory (ulceration), cirrhosis
rheumatoid arthritis and Malignant neoplastic
diseases
Busulfan - used for the treatment of leukemias and Side effects: nausea & vomiting
lymphomas prior to BM transplantation Heavy doses: excessive bone
- Depresses granulocyte formation; used in the marrow depression
treatment of myelocytic leukemias Others: hepatic veno-occlusive
disease

Assessment No. 10
Question: On the space provided, give at least one type of drug for each route of administration and explain its
indications and contra indications.

Reference:
th
Hayes, Wallace A (2007). Principles and Methods of Toxicology (5 ed.). USA: Informa Healthcare.

Page | 111
 th
Katzung, B.G., ed. (2009) Basic and clinical pharmacology. (11 ed.). Boston : McGraw-Hill.

Section 3. TOXIC AGENTS

 Routes of exposure: ingestion, inhalation and transdermal absorption
 Absorption in the GIT is by passive diffusion: this process requires that the substance cross cellular barriers
 Toxins that are not absorbed from the GIT do not produce systemic effects but may produce local effects –
diarrhea, bleeding and malabsorption of nutrients.

A. Alcohols - common CNS depressants

 Cause disorientation, euphoria, confusion and may progress to unconsciousness, paralysis and even death

1. Ethanol
 Most commonly abused depressant
 Toxic metabolite: acetaldehyde
 Increase in GGT, AST, AST/ALT ration (>2.0), increased HDL and MCV
 Serum, plasma and whole blood are acceptable specimen (venipuncture site should be cleaned with alcohol-
free disinfectant – benzalkonium chloride)
 The specimen must be capped all the time to avoid evaporation of alcohol
 Methods for testing: enzymatic, GC and osmometry
 Detection limit: 12 hours
 Fatal dose: 300 – 400 mL of pure alcohol consumed in <1 hour

Influence of Acute Ethanol Ingestion on Ethanol Levels and Behavior

BAC (mg/dL) Influence
10-50 mg/dL None to mild euphoria
50-100 mg/dL Mild influence on stereoscopic vision and dark adaptation
100-150 mg/dL Euphoria; disappearance of inhibition; prolonged reaction time
150-200 mg/dL Moderately severe poisoning; reaction time greatly prolonged; loss of
inhibition and slight disturbances in equilibrium and coordination
200-250 mg/dL Severe degree of poisoning; disturbances of equilibrium and
coordination; retardation of thought processes and clouding of
consciousness
250-400 mg/dL Deep, possibly fatal coma
(lifted from Henry’s Clinical Diagnosis and Management by Laboratory Methods, edited by McPherson and
Pincus)

Stages of Acute Alcoholic Influence / Intoxication

BAC (g/100 mL) INFLUENCE
0.01 – 0.05 Subclinical
0.03 – 0.12 Euphoria
0.09 – 0.25 Excitement
0.18 – 0.30 Confusion
0.25 – 0.40 Stupor
0.35 – 0.50 Coma
0.45+ Death
Modified from Dubowski KM, Gadsden RH Sr, Poklis A. The stability of ethanol in human whole blood controls: an
interlaboratory evaluation. J Anal Toxicol. 1997 Oct;21(6):486-91. All rights reserved
th
(lifted from Tietz Textbook of Clinical Chemistry and Molecular Diagnostics 5 edition by Burtis, Ashwood & Bruns)
OTHER ALCOHOLS
NAME/SYNONYMS SOURCES TOXIC SYMPTOMS OF TOXICITY METHODS FOR
METABOLITE DETERMINATION
Methanol / wood Methylated spirits Formic acid Ocular toxicity, metabolic GC-MS
alcohol Anti-freeze agents acidosis
Isopropyl alcohol / Antiseptic agent Acetone Acetonemia, acetonuria & Gas
rubbing alcohol hyperosmolarity without Chromatography
hyperglycemia & acidosis
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Ethylene glycol / Hydraulic fluic & Glycolic acid Deposition of calcium oxalate HPLC
1,2-ethanediol antifreeze agent Oxalic acid crystals in renal tubules

Activity No. 22: TEST FOR ETHANOL

Objectives:
At the end of the activity, you are expected to:
a. be able to appreciate how to determine the presence of ethyl alcohol in the given samples from a simulated
video procedure recorded by the laboratory instructor.
b. understand the metabolism of ethanol and its toxic effects in the system

Materials needed:
Samples: Serum and vomitus
Control: Ethanol
Reagents: Lugol‘s iodine, KOH, Benzoyl chloride, 10% NaOH, Dilute sulfuric acid, concentrated sulfuric acid,
dilute potassium dichromate, anhydrous sodium acetate, carbon disulfide and ammonium molybdate solution.

Procedures: The instructor will make a video of the actual laboratory performance of the procedure and explanation of
the principle of the tests. The video will be uploaded for the students to be able to familiarize themselves with the
procedure. The students should take note of the results from the different tests.

1) Lieben's lodoform Test

a. Gently warm the liquid (40-50C), add 1 mL of Lugol‘s solution and enough KOH solution to give the liquid
a distinct yellow to brownish color.
b. If ethyl alcohol is present, a yellowish white to lemon-yellow precipitate of iodoform will appear
immediately, or as the solution cools.
c. If the quantity of ethyl alcohol is very small, a precipitate will form on long standing. When iodoform is
deposited slowly, the crystals are very perfect.
Note:
This iodoform test is very delicate but not characteristic of ethyl alcohol, since other primary alcohols,
except methyl alcohol, and many secondary alcohols, as well as their oxidation products, aldehydes and
ketones, give iodoform under the same conditions. Acetic ether, aceto-acetic ether, lactic acid, etc., also
give iodoform.

2) Berthelot's Test
a. Mix 1 mL of the liquid with a few drops of benzoyl chloride and about 1 mL of 10% sodium hydroxide
solution, until the irritating odor of benzoyl chloride has gone.
b. The aromatic odor of ethyl benzoate will appear.

3) Chromic Acid Test

a. Warm 1 mL of the liquid with 4 drops dilute H2SO4 or HCl.
b. Add 1-2 drops of very dilute potassium dichromate solution.
c. The color of the liquid will change from red to green, and at the same time the odor of acetaldehyde will
be recognized
NOTE: This test is not characteristic of ethyl alcohol because many other volatile organic compounds behave
similarly.

4) Ethyl Acetate Test

a. Mix 1 mL of the liquid with the same volume of conc. H2SO4
b. Add a very small quantity of anhydrous sodium acetate and heat
c. Ethyl acetate will be recognized by its odor.

5) Vitali's Test
a. Thoroughly mix 1 mL of distillate in a glass dish with a small piece of solid KOH and 2-3 drops of carbon
disulfide.
b. Let this mixture stand for a short time without warming.
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 c. When most of the carbon disulfide has evaporated
d. Add a drop of NH4 molybdate solution and then an excess of dilute H2SO4.
e. If the distillate contains ethyl alcohol, a red color will appear.

LEARNING ACTIVITY: The students will make a tabulation of the results for the different tests performed.

GUIDE QUESTIONS: The instructor will make an online forum where the students can type and post their answers to the
QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or through
text message.

1. Give the physical and chemical characteristics of Ethyl alcohol.

2. Discuss the metabolism of ethanol in the body. What is the lethal dose of ethanol?
3. Describe the clinical manifestations or behavioral changes in relation to different blood alcohol concentrations.
4. Give the underlying principle of the following tests for ethyl alcohol:
A. Iodoform test
B. Berthelot‘s test
C. Vitali‘s test
5. Give the physical and chemical characteristics of methanol.
6. Describe the mechanism of action of methanol in cases of poisoning.
7. Describe the clinical manifestations of methanol toxicity.
8. Enumerate different sources of methanol poisoning. What is the lethal dose of methyl alcohol?

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B. Carbon Monoxide - colorless, odorless, tasteless gas
 Produced by incomplete combustion of carbon-containing substances
 Toxic effects are manifested by binding w/ heme proteins containing the divalent iron (cytochromes, myoglobin
and hemoglobin)
 Affinity with hemoglobin is 200x greater than that of oxygen
 It inhibits cellular respiration and electron transport
 Sources: gasoline engines, wood or plastic fires
 Net effect: hypoxia
 Indicator of toxicity: ―cherry red‖ color of the face
 Antidote: Hyperbaric oxygen therapy (HBOT) / pure oxygen therapy

Activity No. 23: TEST FOR CARBON MONOXIDE

Objectives:
At the end of the activity, you are expected to:
a. be able to appreciate how to determine the presence of Carbon Monoxide in the given samples from a
simulated video procedure recorded by the laboratory instructor.
b. describe the toxic effects of carbon monoxide in the body
c. describe the clinical manifestations related to carbon monoxide poisoning

Materials:
Sample: Whole blood
Control material: Normal Whole blood
Reagents: 10% Sodium Hydroxide solution, Lead acetate, 20% potassium ferrocyanide solution, dilute acetic
acid, 1% tannin solution, saturated copper sulfate solution, ammonium sulfide solution and 30% acetic acid.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the tests. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The students should take note of the results from the different tests.

1. Boiling Test
1.1. Blood containing carbon monoxide gives a brick -red coagulum, if boiled or warmed in a water bath.
1.2. Ordinary blood gives a grayish brown or brownish black precipitate.
2. Sodium Hydroxide Test
2.1. Carbon monoxide blood shaken with 1-2 volumes of sodium hydroxide solution (sp. gr. 1.3 = 26.8%) remains
red and in a thin layer is the color of red lead or vermilion.
2.2. Normal blood similarly treated is almost black and in a thin coating upon a porcelain plate is dark greenish
brown.
2.3. A procedure recommended consists in diluting the blood with 6-10 times its volume of water and using about
5 drops of sodium hydroxide solution to 10 cc. of diluted blood.
NOTE:
Even gentle warming with sodium hydroxide solution (10% NaOH) does not alter the red color of this
carboxyhemoglobin solution, whereas a solution of normal human blood becomes greenish to dark brown.
3. Basic Lead Acetate Test
3.1. Mix 4-5 volumes of basic lead acetate solution in a test-tube with diluted or undiluted carbon monoxide blood
and shake vigorously for a minute.
3.2. Such blood remains bright red but normal blood is first brownish and then chocolate to greenish brown.
4. Potassium Ferrocyanide Test
4.1. Mix undiluted blood (15 cc.) with an equal volume of 20% potassium ferrocyanide solution and 2 cc. of diluted
acetic acid.
4.2. Shake the mixture gently and a coagulum will gradually form.
4.3. That from normal blood is dark brown but from blood containing carbon monoxide bright red. This difference
disappears slowly but not entirely for weeks.
5. Tannin Test
5.1. Mix an aqueous blood solution 2-3 times its volume of 1% tannin solution and shake thoroughly.

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 5.2. A difference in color between normal and carbon monoxide blood can be recognized after several hours, most
distinctly after 24 hours.
5.3. Normal blood is gray but carbon monoxide blood is crimson-red. This difference is apparent even after
several months
6. Copper Sulfate Test
6.1. A drop of saturated copper sulfate solution added to 2 cc. of carbon monoxide blood mixed with the same
volume of water gives a brick-red precipitate.
6.2. The deposit from normal blood is greenish brown.
NOTE:
In all these precipitation tests (4, 5 and 6) the less easily decomposed carbon monoxide blood remains bright red
but the more easily decomposed normal blood in presence of the precipitants used and others is off color or
dark.
7. Ammonium Sulfide Test
7.1. Mix 0.2 cc. of ammonium sulfide solution and 0.2-0.3 cc. of 30% acetic acid with 10 cc. of 2% aqueous blood
solution.
7.2. Carbon monoxide blood gives a fine rose color but normal blood is greenish gray.
7.3. The former within 24 hours gives a red flocculent precipitate.

LEARNING ACTIVITY: The students will make a tabulation of the results for the different tests performed.

GUIDE QUESTIONS: The instructor will make an online forum where the students can type and post their answers to the
QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or through
text message.

1. Describe the mechanism of action of carbon monoxide in cases of poisoning.

2. Enumerate the sources of carbon monoxide poisoning.
3. Describe the symptoms associated with carbon monoxide poisoning

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C. Cyanide - exists as solid, gas or in solution; a super toxic substance
 Component of insecticides and rodenticides; common suicide agent
 Also a pyrolysis product – burning of plastics
 Binds to mitochondrial cytochrome oxidase causing an uncoupling of oxidative phosphorylation – depletion of
cellular ATP
 Characteristic ―odor of bitter almonds‖
 Non-toxic product: thiocyanate

Activity No. 24: TEST FOR CYANIDE

Objectives:
At the end of the activity, you are expected to:
a. be able to appreciate how to determine the presence of Cyanide in the given samples from a simulated video
procedure recorded by the laboratory instructor.
b. understand the mechanism of cyanide in cases of poisoning

Materials:
Sample: Seeds of apple, green potato, photographs or X-ray films
Control: 1% sodium cyanide
Reagents: 10% tartaric acid solution, 1% silver nitrate solution, saturated solution of picric acid, 10% sodium
hydroxide solution, 4% sodium hydroxide, 2% ferrous sulfate solution, Dilute hydrochloric acid, 1% ferric chloride solution,
5% potassium nitrite solution, dilute sulfuric acid, dilute ammonium hydroxide and 5% ammonium sulfide.

Procedure: The instructor will make a video of the actual laboratory performance of the procedure and explanation of the
principle of the tests. The video will be uploaded for the students to be able to familiarize themselves with the procedure.
The students should take note of the results from the different tests.

A. Preparation of the Samples:

1) Triturate 10 g of the sample. Add 10 mL of tartaric acid and transfer to a distilling flask.
2) Check the acidity of the mixture. Add more tartaric acid if necessary
3) Distill at a low rate. Collect about 25 mL of the distillate in a covered flask and use for the following
tests.
B. Picric acid test:
a. To 1 mL of the control and the distillate, add 20 drops of saturated picric acid solution and 12
drops of 10% sodium hydroxide.
b. Shake to mix and heat for 15 minutes on a water bath. Note the color of the solution
C. Prussian Blue test:
a. To 3 mL of the control and distillate, add 0.2 mL of 4% sodium hydroxide solution and 1 mL of 2% ferrous
sulfate solution.
b. Mix and warm for 5 minutes on a waterbath.
c. Acidify with dilute hydrochloric acid and then add 0.1 mL ferric chloride solution. Note the color of the
solution.
D. Sulfocyanate test:
1. To 3 mL of the control and distillate, add 0.1 ml of 4% sodium hydroxide solution.
2. Test the alkalinity of the mixture, adding sodium hydroxide as needed.
3. Add 0.1 mL ferrous sulfate and mix.
4. Warm the misture of a water bath for 5 minutes and acidify with dilute hydrochloric acid.
5. Add 0.1 mL ferric chloride and note the color of the solution.
E. Silver Nitrate test:
1. To 3 mL of the control and distillate, add 0.2 mL silver nitrate solution.
2. Note the color of the precipitate.
F. Vortmann’s Sodium Nitroprusside test:
1. To 1 mL of the distillate, add 4 drops each of potassium nitrite solution, ferric chloride solution and dilute
sulfuric acid

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 2. Boil the solution then cool.
3. Alkalinize with dilute ammonium hydroxide and filter.
4. To the filtrate, add ammonium sulfide. Note the resulting colored solution.

LEARNING ACTIVITY: The students will make a tabulation of the results for the different tests performed.

GUIDE QUESTIONS: The instructor will make an online forum where the students can type and post their answers to the
QFRs. For offline students, they can write it on an intermediate pad to be submitted via mail, express couriers or through
text message.

1. What are the sources of Cyanide poisoning? Give the lethal dose.
2. Describe the mechanism of action of cyanide in the body in cases of poisoning.
3. Describe the clinical manifestations which correlate to the toxicity of cyanide.

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D. Metals
1. Arsenic
 Component of ant poisons, rodenticides, paints and metal alloys
 Exposure to this chemical occurs in smelting industries and agriculture
 Common homicide or suicide agent
 Expresses its toxicity by high affinity binding to the thiol groups in proteins
 Characteristic ―odor of garlic‖ and ―metallic taste‖
 Use of hair and nails as specimens are important in the evaluation of long-term exposure (Ion emission
spectroscopy)
 Blood and urine specimens – for short-term exposure
 Acute fatal dosage: 120 mg
 Method for testing: Atomic absorption spectrophotometry

2. Cadmium
 Utilized in electroplating and galvanizing
 Significant environmental pollutant – pigment in paints and plastics

3. Iron
 Acute poisoning common in young children
 Toxic amount: >30mg/kg
 Once absorbed, removal is difficult
 Hepatic cell damage, shock, lactic acidosis
 Vomiting: initial manifestation
 Severe gastroenteritis, melena, abdominal pain and hematemesis
Diagnosis
o Serum iron concentration
o TIBC
Treatment
o Supportive treatment
o Emesis or gastric lavage
o Chelation therapy: deferoxamine

4. Lead
 A component of household paints and a potent enzyme inhibitor
 Combines with the matrix of bone and can persist in this area for a long time (Half-life: 32 years)
 Exposure to this metal results to encephalopathy, birth defects and compromised immunity
 Low-level exposure may cause behavioral changes – hyperactivity & attention deficit disorder, and also affect
intelligence quotient scores (decrease)
 Vit. D and heme synthesis pathway are affected
 Characteristic ―wrist drop or foot drop‖ manifestation
 Indicators of toxicity: increased urinary δ-ALA; presence of basophilic stippling in RBC
 EDTA and dimercaptosuccinic acid (DMA) are used for treatment as therapeutic chelators (to remove lead
from soft tissues and bone)
 Whole blood for quantitative testing; urine for assessment of recent lead exposure
 Methods for testing: graphite furnace, AAS, inductively coupled plasma emission spectrophotometry (ICPES),
anodic stripping voltammetry
 Toxicity dose: > 0.5 mg/day
 Fatal dose: 0.5 g

5. Mercury
 Binds with proteins and also an enzyme inhibitor
 Three forms: elemental (liquid), inorganic salts (mercurous or mercuric) or component of organic compounds
(alkyl mercury)
 Small drops of mercury on benchtops and floors can poison the environment in a poorly ventilated room
 Its presence in blood may result to loss of glomerular integrity
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  If inhaled or absorbed through the skin, it can pass through the blood-brain barrier, and can accumulate in the
CNS
 Method for testing: Reinsch test

Assessment No. 11

Please answer the following questions, write your answer on the space provided
Match the following:
A. Schedule I B. Schedule II C. Schedule III
_____ 1. Amphetamines _____ 2. Cocaine _____ 3. Phenobarbital

A. Narcotics B. Club drugs C. Depressants D. Stimulants

E. Hallucinogens F. Athletic Performance

_____ 1. Angel dust _____ 2. Mescaline _____ 3. Alcohol

_____ 4. Ketamine _____ 5. Roofies _____ 6. Crack

Match the following clinical significance of each toxic Metals.

A. Arsenic D. Lead
B. Iron E. Organophosphates
C. Mercury

_____1. Acute poisoning of this metal is common among young children that may result to hepatic cell damage, shock
and lactic acidosis.

_____2. This is a component of redenticides and weed killers.

_____3. This is a liquid metal usually seen in thermometers and light bulbs.

_____4. This is measured using cholinesterase assay.

_____5. This can be managed using chelating agents such as dimercaprol.

_____6. This metal may disrupt metabolic systems.

Match the following toxins with their clinical importance.

A. Carbon monoxide D. Methyl alcohol
B. Cyanide E. Ethyl alcohol
C. Isopropyl alcohol F. Ethylene glycol

_____7. This is also known as wood alcohol.

_____8. This is metabolized into acetone, CO and water.

_____9. This binds to hemoglobin.

_____10. This binds to ferric ion.

Reference:
th
Klaassen, Curtis (2007). Casarett and Doull’s Toxicology: The Basic Science of Poisons (7 ed.) USA: McGraw-Hill
Professional.

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Linton, Jeremy M. (2008) Overcoming problematic alcohol and drug use : a guide for beginning the change
process. New York : Routledge.

Richards, Ira. (2008). Principles and practice of toxicology in public health. Jones and Barlett Publishers.
IV. REFERENCES

A. Textbook
McPherson, Richard. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Elsevier
Saunders, 2011.

B. Books
Anderson, Shauna and Susan Cockyane (2007). Clinical Chemistry: Concepts and Applications. USA:
Waveland Press Inc.

Arneson, W. and J. Brickell (2007). Clinical Chemistry: A Laboratory Perspective. USA: F.A. Davis Co.

Ashwood E., D. Bruns and C. Burtis (2012). Tietz’s Textbook of Clinical Chemistry and Molecular
Diagnostics 5th ed. Philadelphia: W.B. Saunders Co.

Bertholf, Roger and Ruth Winecker (2007). Chromatographic Methods in Clinical Chemistry and Toxicology.
USA: Wiley.
th
Bishop, Michael (2013) Clinical Chemistry Procedures, Correlation. (7 ed.). Holland
th
Crook, Martin (2006). Clinical Chemistry and Metabolic Medicine (7 ed.). USA: Hodder Arnold Publication.
nd
Daniels, Rick. (2010). Delmar's Guide to laboratory and diagnostic tests. (2 ed.) Delmar Cengage
Learning.
th
Gardner, David and Dolores Shoback (2007). Greenspan’s Basic and Clinical Endocrinology (8 ed.).
McGraw-Hill Professional.

Hart, C. L. (2009) Drugs, society and human behavior. (13th ed.). Boston : McGraw-Hill.
th
Hayes, Wallace A (2007). Principles and Methods of Toxicology (5 ed.). USA: Informa Healthcare.
th
Katzung, B.G., ed. (2009) Basic and clinical pharmacology. (11 ed.). Boston : McGraw-Hill.
th
Klaassen, Curtis (2007). Casarett and Doull’s Toxicology: The Basic Science of Poisons (7 ed.) USA:
McGraw-Hill Professional.
th
Kronenberg, Henry M. et. al (2007). Williams Textbook of Endocrinology (11 ed.) Philadelphia: W.B.
Saunders.

Linton, Jeremy M. (2008) Overcoming problematic alcohol and drug use : a guide for beginning the change
process. New York : Routledge.
nd
Negrusz, Adam & Cooper, Gail. Clarke’s Analytical Forensic Toxicology, 2 edition. London, Pharmaceutical
Press
th
Pagana, Kathleen. (2011) Mosby's Diagnostic and Laboratory test reference. (10 ed.) Elsevier Mosby.

Richards, Ira. (2008). Principles and practice of toxicology in public health. Jones and Barlett Publishers.
nd
Scott M., A. Gronowski and C. Eby (2007). Tietz’s Applied Laboratory Medicine. (2 ed.) USA: Wiley-Liss.
th
Skidmore-Roth, Linda. (2012). Mosby's Drug Guide. (25 ed.). St. Louis: Mosby.

Suba, Sally C. (2008). Laboratory Guide in Clinical Blood Chemistry. Rex Book Store, Inc.

Page | 121
 th
Turgeon, Mary. (2012). Clinical laboratory science. (6 ed.) Elsevier Mosby.
th
Wu, Allan (2006). Tietz’s Clinical Guide to Laboratory Tests (4 ed.). Philadelphia: W.B. Saunders.

C. Electronic Sources

Clinical Chemistry Links. Institute for Quality in Laboratory Medicine retrieved from
http://www.dgrhoads.com/links.shtml

Clinical Chemistry Links. Clinical Laboratory Management Association retrieved from

http://www.dgrhoads.com/links.shtml

Clinical Chemistry Links. Clinical and Laboratory Standards Institute retrieved from
http://www.dgrhoads.com/links.shtml

Medical – KMC Systems. Clinical Chemistry Analyzers retrieved from

http://www.kmcsystems.com/invitro_clinical.asp#1

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 Module Evaluation

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II. The Assessment
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Thank you.

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