NON-PROTEIN NITROGENOUS

COMPOUNDS

CREATININE

Larah Francesca A. Fernandez, RMT, MSMT

Intended Learning Outcomes
1. Apply the correct technique in the performance of NPN compound
analysis, particularly creatinine.
2. Analyze laboratory results in terms of acceptability, accuracy,
precision, and reliability.
3. Identify sources of error that may lead to false elevation or
decrease in creatinine levels.
4. Correlate laboratory results with clinical significance.
CREATININE
• Chemical formula: C4H7N3O
• Anhydride of creatine (C4H9N3O2)
• End product of muscle metabolism
• Derived from creatine and creatine phosphate in the muscle
• 99% excreted in the urine; <1% reabsorbed
Creatine – synthesized primarily in the liver (from arg, gly, and met)

**Creatine 🡪 muscle 🡪 creatine phosphate (high energy

source)
**Creatine (C4H9N3O2) loses water and creatine phosphate
(C4H10N3O5P) loses phosphoric acid (H3PO4) 🡪 creatinine (C4H7N3O)
GLOMERULAR FILTRATION RATE (GFR)
• Considered as the best overall indicator of the level of kidney
function
• Gauged by measuring excretion of substance that is only
minimally reabsorbed and secreted by the renal tubules

• Creatinine – most widely used marker for GFR, since:

✔Produced at a constant rate
✔Not bound to protein
✔Not reabsorbed by the renal tubules
✔Only small amount is secreted by the tubules (10%)
GLOMERULAR FILTRATION RATE (GFR)
• Considered as the best overall indicator of the level of kidney
function
• Gauged by measuring excretion of substance that is only
minimally reabsorbed and secreted by the renal tubules

• Creatinine – most widely used marker for GFR, however:

● Substantial individual variation depending on muscle mass
● Meat ingestion affects total daily production
● Alkaline picrate method of creatinine measurement is affected
by a lot of interferences
● Partially secreted by renal tubules – secretion is blocked by
various drugs
Creatinine Clearance (CrCl)
• Measure of the amount of creatinine removed from the blood by
the kidneys in a specified amount of time
• Reported in ml/min
• Provides reasonable approximation of glomerular filtration rate
• Plasma concentration is inversely proportional to GFR
Creatinine Clearance (CrCl) = (urine creatinine) (urine volume)
(plasma creatinine) (time)

Reference interval:
Male: 97-137
mL/min
Female: 88-128
mL/min
 DISEASE CORRELATIONS
• Assess renal filtration function ✓ More reliable kidney
• Determine sufficiency of kidney function test – not affected
by diet, dehydration and
function protein metabolism
• Determine severity of kidney
damage ● Plasma creatinine is an
• Monitor progression of kidney insensitive marker for mild or
disease early renal dysfunction (renal
■ ↑ creatine, ↑ urinary creatinine, N plasma damage must be >50%)
• Measure muscle
creatinine: diseases of
completeness such24-h
as
muscular dystrophy,
urine collection poliomyelitis,
(urine creatinine)
hyperthyroidism, trauma
METHODS OF DETERMINATION
1. Jaffe’s Method
• Jaffe reagent (alkaline picrate): picric acid, 10% sodium hydroxide

Creatinine + alkaline picrate 🡪 creatinine

picrate (red-orange chromogen)

• Positive interferences: ascorbate, glucose, uric acid, ketoacids, glutathione,

cephalosporin

a. Jaffe with adsorbent

• Lloyd’s reagent (sodium aluminum silicate)
• Fuller’s earth reagent (aluminum magnesium silicate)
b. Kinetic Jaffe
• Measures the rate of change in absorbance
• (+): cephalosporins and ketoacids
• (-): bilirubin and hemoglobin
METHODS OF DETERMINATION
2. Enzymatic Methods – no interference from glucose and other Jaffe
chromogens
a. Creatininase-CK
• Requires large volume of pre-incubated sample
• Not widely used

creatinine aminohydrolase
Creatinine + H2O 🡪 creatine
creatine kinase
Creatine + ATP 🡪 creatine phosphate + ADP
pyruvate kinase
ADP + phosphoenolpyruvate 🡪 ATP + pyruvate
LDH
Pyruvate + NADH 🡪 lactate + NAD+
METHODS OF DETERMINATION
2. Enzymatic Methods
b. Creatininase-H2O2
• No interference from acetoacetate or cephalosporins
• Positive interference: lidocaine

creatinine aminohydrolase

Creatinine + H2O 🡪 creatine

creatinase

Creatine + H2O 🡪 sarcosine + urea

sarcosine oxidase

Sarcosine + H2O + O2 🡪 glycine + formaldehyde +

H2O2
peroxidase
METHODS OF DETERMINATION
3. Isotope Dilution Mass Spectrometry (IDMS)
- Highly specific
- Detection of characteristic fragments following ionization
- Quantification using isotopically-labeled compound
- Accepted reference method
SPECIMEN REQUIREMENTS

• Plasma
• Serum
• Urine

Patient and specimen preparation:

• Non-fasting
• Avoid hemolyzed, icteric, and lipemic specimens
• Urine – refrigerated after collection ; frozen if >4 days
CREATININE DETERMINATION
Materials:
CREATININE DETERMINATION
Reagents:
1. Reagent 1 (R1) – picric acid
2. Reagent 2 (R2) – sodium hydroxide, disodium phosphate

Other components of run:

1. Standard – creatinine = 2 mg/dL or 176.8 umol/L (may vary)
2. Ellitrol 1 – normal control
3. Ellitrol 2 – pathologic control
4. Specimen (serum/plasma or urine)
CREATININE DETERMINATION
Principle:

Kinetic Jaffe – serum is mixed with alkaline picrate

solution and the rate of change in absorbance is measured
between two points.
Principle: Kinetic Jaffe
Assay Procedure:
1. Prepare and label test tubes: B, S, CN, CP, U
2. Prepare working reagent – mix reagent 1 with reagent 2 into a
separate tube
3. Deliver the desired volume (0.1mL) of the sample first on
separate tubes (standard, CN, CP, serum/unknown) – touch
the tip of the pipet directly to the bottom of the tube
Principle: Kinetic Jaffe
Assay Procedure:
4. Set up the spectrophotometer prior to adding the reagent into
the individual sample tubes
NOTE: this is a kinetic measurement – time is very critical

Assay Requirements:
1. Wavelength – 520nm
2. Optical path – 1cm
3. Temperature – 37°C
4. Measurement – read against distilled water
Principle: Kinetic Jaffe
Assay Procedure:
5. Add the required volume of the reagent – touch the tip of the
pipet to the side of the tube
6. Pipetting scheme:
Pipette into cuvettes B S CN CP U
Standard solution -- 0.1 mL -- -- --
Control normal serum -- -- 0.1 mL -- --
Control pathologic serum -- -- -- 0.1 mL --
Patient’s serum -- -- -- -- 0.1 mL
Working reagent -- 1.0 mL 1.0 mL 1.0 mL 1.0 mL
Principle: Kinetic Jaffe
Assay Procedure:
7. Cover the mouth of the test tube with parafilm
8. Mix by inverting 2-3x
9. Start incubation time
10.Read the absorbance at 7 seconds. (A1)
11.Continue the timer and read absorbance again at exactly 2
minutes (A2)
CALCULATIONS
Calculate creatinine concentration (serum/plasma):

1. A2 – A1 = ΔA

2.1. CU = CS (mg/dL) x ΔA sample

ΔA standard

2.2. CU = CS (umol/L) x ΔA sample

ΔA standard
Legend:
CS = concentration of standard
CU = concentration of unknown
A = absorbance
ΔA = change in absorbance
CALCULATIONS
Calculate creatinine concentration (24-urine):

C (mg/24hr) = mg/dL x vol of urine in 24 hr (mL) x 0.01

C (mmol/24hr) = mg/24hr x 0.00884

CALCULATIONS
Conversion Factor:

mg/dL x 88.402 = umol/L

umol/L x 0.0113 = mg/dL
 REFERENCE VALUES
Reference Interval Jaffe Method Enzymatic Method
Adult Male 0.9-1.3 mg/dL (80-115 umol/L) 0.6-1.1 mg/dL (55-96 umol/L)
Adult Female 0.6-1.1 mg/dL (53-97 umol/L) 0.5-0.8 mg/dL (40-66 umol/L)
Child 0.3-0.7 mg/dL (27-62 umol/L) 0.0-0.6 mg/dL (0-52 umol/L)
Adult Male (urine) 800-2000 mg/day (7.1-17.7 mmol/day)
Adult Female (urine) 600-1800 mg/day (5.3-15.9 mmol/day)
CASE STUDY
1. Given the following absorbance readings, compute for the
actual creatinine concentration of the serum sample, determine
the validity of the test, and interpret the results.
A1 A2
Standard 0.137 0.411 Control Expected Range
Level 1 control 0.348 0.598 Level 1 1.93 – 2.81 mg/dL
Level 2 control 2.136 2.816 Level 2 5.08 – 6.48 mg/dL
Unknown serum 0.543 1.678
Concentration of standard: 2.5 mg/dL
CASE STUDY
Compute for the concentration of Level 1 control:
a. Standard:
ΔA = A2 – A1
CL1 = CS (mg/dL) x ΔA L1 control
= 0.411 – 0.137
ΔA standard
= 0.274
= 2.5 mg/dL x ___0.250____
b. Level 1 control: 0.274
ΔA = A2 – A1
= 2.28 mg/dL
= 0.598 – 0.348
= 0.250
CASE STUDY
Compute for the concentration of Level 2 control:
a. Standard:
ΔA = A2 – A1
CL2 = CS (mg/dL) x ΔA L2 control
= 0.411 – 0.137
ΔA standard
= 0.274
= 2.5 mg/dL x ___0.680____
b. Level 2 control: 0.274
ΔA = A2 – A1
= 6.20 mg/dL
= 2.816 – 2.136
= 0.680
CASE STUDY
Compute for the concentration of unknown:
a. Standard:
ΔA = A2 – A1
CU = CS (mg/dL) x ΔA unknown
= 0.411 – 0.137
ΔA standard
= 0.274
= 2.5 mg/dL x ___1.135____
b. Unknown: 0.274
ΔA = A2 – A1
= 10.36 mg/dL
= 1.678 – 0.543
= 1.135
CASE STUDY
2. Given that the creatinine concentration of a 750mL 24-hour
urine, diluted 1:20, from the same patient is 4.83 mg/dL.
Calculate the concentration in mg/24hr.

• Actual conc = 4.83 x 20 = 96.6 mg/dL

• C (mg/24hr) = mg/dL x vol of urine in 24 hr (mL) x 0.01

= 96.6 x 750 x 0.01
= 724.5 mg/24hr
CASE STUDY
3. Given that the creatinine concentration of a 750mL 24-hour
urine, diluted 1:20, from the same patient is 4.83 mg/dL.
Calculate the creatinine clearance, using the serum creatinine
results obtained earlier.
• Actual conc = 4.83 x 20 = 96.6 mg/dL

• Creatinine Clearance (CrCl) = (urine creatinine) (urine volume)

(plasma creatinine) (time)
= ___(96.6 mg/dL) (1010 mL)___
(10.36 mg/dL) (1440 mins)
= 4.86 ml/min
Hypothetical Readings
Use this readings for LabSheet Ex. 9.3

Spectrophotometer Readings
9/17/20 520nm
2:00pm
CREATININE
A1 A2
Creatinine results of a male adult who frequently
goes to gym. Standard 0.488 0.736
CN 0.521 0.868
24-hour urine total volume = 1300 mL
CP 2.413 3.146
Dilution factor (urine) = 1:20
Unknown (serum) 0.398 0.586
Compute for the ff: Unknown (urine) 0.663 1.876
1. Concentration of unknown serum (mg/dL)
Concentration of standard = 2 mg/dL
2. Concentration of unknown urine (mg/dL)
3. Concentration of urine in mg/24hr
4. Creatinine clearance
Hypothetical Readings
Use this for LabSheet Ex. 9.3

Ellitrol Assayed Chemistry Control

Levels 1 and 2 EXP 2022-08-31
LOT No: 81640
Units Level 1 Level 2
Mean Range Mean Range
Creatinine
Alkaline picrate-kinetic mg/dL 2.37 1.93-2.81 5.78 5.08-6.48
 CILINICAL SIGNIFICANCE

Increased Serum Creatinine Decreased Serum Creatinine

1. Impaired renal function 1. Decreased muscle mass

2. Chronic Nephritis 2. Advanced and severe liver disease
3. Congestive heart failure 3. Pregnancy
 CILINICAL SIGNIFICANCE

Increased Creatinine Clearance Decreased Creatinine Clearance

1. High cardiac output 1. Impaired kidney function

2. Pregnancy 2. Shock, dehydration
3. Burns 3. Hemorrhage
4. Carbon monoxide poisoning 4. Congestive heart failure
Thank You!