SURNAME : ZIUMBWA

NAME : ELIJAH

REG NUMBER : R159164K

PROGRAM : MBChB 1

COURSE : BIOCHEMISTRY

DEPARTMENT : BIOCHEMISTRY

INSTITUTION : UNIVERSITY OF ZIMBZBWE

EXPERIMENT NUMBER : 16

EXPERIMENT TITLE : DETERMINATION OF ALKALINE PHOSPHATASE AND

ACID PHOSPHATASE IN HUMAN SERUM

DATE OF EXPERIMENT : 23 MARCH 2016

INTRODUCTION

Acid phosphatase (EC 3.1.3.2) is a phosphatase, a type of enzyme, used to

free attached phosphate groups from other molecules in breakdown of food.
It is mostly a phosphomonoesterase. It is stored in lysosomes and functions
when these fuse with endosomes, which are acidified while they function;
hence, it has an acid pH optimal. Enzyme activity is expressed in King
Armstrong units; where 1 KAU = 1mg phenol liberated by 100ml serum in 15
minutes at 370C. Typical values of ACP in serum range from 0.8-4.6 KAU and
for tartarate labile acid, 0-0.4 KAU. Different forms of acid phosphatase are
found in diverse organs, and their serum levels are used to evaluate the
success of the surgical treatment of prostate cancer. In the past, they were
also used to diagnose this type of cancer. Acid phosphatase catalyzes the
following reaction at an acidic optimal pH:
Orthophosphoric monoester + H2O → alcohol + H3PO4
(Nelson and
Cox, 2005)

Alkaline phosphatase (EC 3.1.3.1) is a hydrolase enzyme in control of

removing phosphate groups from various types of molecules, including
nucleotides, proteins, and alkaloids. As the name suggests, alkaline
phosphatases are best operative in an alkaline environment. It is sometimes
used synonymously as basic phosphatase. The pH optimum depends on the
substrate being hydrolyzed but it is usually between 8-10 units. The enzyme
needs magnesium ions as cofactors and therefore its action is inhibited by
substances that chelate divalent cations, for instance citrate. Ordinary values
of serum ALP in adults are 4-12 KAU and values go up to 24 KAU in children.
(Berg et al., 2006).

Alkaline phosphatase has turn out to be an advantageous tool in molecular

biology laboratories, since DNA generally has phosphate groups on the 5'
end. Eliminating these phosphates avoids the DNA from ligating (the 5' end
attaching to the 3' end), thus keeping DNA molecules linear up until the next
step of the process for which they are being prepared; also, removal of the
phosphate groups permits radio labeling (replacement by radioactive
phosphate groups) so as to measure the presence of the labeled DNA
through further steps in the process or experiment. For these purposes, the
alkaline phosphatase from shrimp is the most useful, as it is the easiest to
deactivate after it has completed its job. Another significant use of alkaline
phosphatase is as a label for immunoassays. Undifferentiated pluripotent
stem cells have elevated levels of alkaline phosphatase on their cell
membrane, consequently alkaline phosphatase staining is used to identify
these cells and to test pluripotency (i.e., embryonic stem cells or embryonic
carcinoma cells) (Seligman et al., 1956).

Alkaline phosphatase is frequently used in the dairy industry as a sign of

effective pasteurization. This is because the most heat stable bacterium
found in milk, mycobacterium par tuberculosis, is destroyed by temperatures
lower than those required to denature ALP. Therefore ALP presence is best
for signifying successful pasteurization (Seligman et al., 1956).

Alkaline phosphatase (ALP) is present in various tissues including liver, bone,

intestine, and placenta. Serum ALP is of concern in the diagnosis of 2 main
groups of conditions, hepatobiliary disease and bone disease related with
increased osteoblastic activity. An increase in ALP activity transpires with all
forms of cholestasis, predominantly with obstructive jaundice. The response
of the liver to any form of biliary tree blockade is to synthesize more ALP.
The chief site of new enzyme synthesis is the hepatocytes adjacent to the
biliary canaliculi. ALP also is elevated in disorders of the skeletal system that
include osteoblast hyperactivity and bone remodeling, such as Paget's
disease, hyperparathyroidism, rickets and osteomalacia, fractures, and
malignant tumors. A substantial rise in alkaline phosphatase activity caused
by augmented osteoblast activity following accelerated bone growth is
sometimes seen in children and juveniles (Murray et al., 2003).

Phosphatase enzymes are likewise used by soil microorganisms to access

organically bound phosphate nutrients. An assay on the rates of activity of
these enzymes may be used to determine biological demand for phosphates
in the soil. Some plant roots, particularly cluster roots, exude carboxylates
that perform acid phosphatase activity, helping to summon phosphorus in
nutrient-deficient soils. Certain bacteria like nocardia, can destroy this
enzyme and use it as a carbon source (Champe et al., 2006).

There are numerous methods presently available for the quantitative

approximation of acid and alkaline phosphatase activity in biologic
provisions. In all the techniques, a phosphoric acid ester functions as a
substrate and calorimetric determinations are made either of the inorganic
phosphate or of the organic moiety of the substrate released by enzymatic
hydrolysis (Seligman teal, 1956). In this experiment, the substrate employed
will be phenyl phosphate. At the suitable pH, the ALP or ACP will
quantitatively hydrolyze the substrate to produce inorganic phosphate and
phenol. This phenol is reacted with Folin-Ciocalteu reagent to give a blue-
grey color which is measured at 600nm in a spectrophotometer.

AIMS AND OBJECTIVES

To determine the activity and concentration of alkaline phosphatase and acid
phosphatase in human serum.

MATERIALS
As per MBChB/BDS1 practical schedule, 2015-2016, page 60-61.

METHOD
Four centrifuge tubes were used, A and B for acid phosphatase; C and D for
alkaline phosphatase. A and C were the controls whereas B and D where the
experimental. 2ml of Acid Phosphatase buffer and 2ml of phenyl
phosphate(substrate) were added to tubes A and B. 0.2ml of plasma was
added to tube A and 1.8ml of Folin- Ciocalteu’s reagent was immediately
added. Tube B was placed in a 37 0C water bath for 3 minutes then 0.2ml of
plasma were added whilst still immersed in the water bath. It was then left in
the water bath for an hour then 1.8ml of Folin – Ciocalteu’s reagent was
added and again left in the water bath for a further 5 minutes. Both test
tubes were centrifuged and 4ml supernatants extracted. The same
procedure was performed for tubes C and D except that alkaline
phosphatase buffer was used and tube D was placed in the water bath for 15
minutes as compared to the hour for tube B. A series of standards and blanks
were prepared as follows:
Table 1: Volumes of different solutions added to each test-tube

Blank STD 1 STD 2 STD 3 STD 4

Standard 0 1 2 3 4
phenol
reagent (ml)

Water (ml) 4 3 2 1 0
2ml of sodium carbonate were added to tubes A – D and to each standard
tube and the blank. The tubes were then placed in a water bath for 10
minutes. 4ml of distilled were then added to each of the tubes and the
absorbance for each tube read at 600nm.

RESULTS

Table 2: Absorbance readings at 600nm

Tube Absorbance(600nm) Amount of phenol (mg)

STD1 0.120 0.015
STD2 0.207 0.030
STD 3 0.377 0.045
ACP A 0.087 0.012
ACP B 0.122 0.016
Net ACP liberated
phenol: 0.004
ALP C 0.683 0.089
ALP D 0.573 0.074
Net ALP liberated
phenol: 0.015
CALCULATIONS
Net ACP liberated phenol =B–A
= 0.016 – 0.012
= 0.004 mg
Net ALP liberated phenol =C–D
= 0.089 – 0.074
= 0.015
For ACP
4ml of supernatant contained 0.004 mg phenol
0.2ml serum liberated 0.004 mg phenol
Total mg phenol = 0.004 mg x 6/4
= 0.006 mg
Therefore 100ml serum would produce: 100/0.2 x 0.006 = 3mg

For ALP
4ml of supernatant contained 0.015mg phenol
0.2ml serum liberated 0.015mg phenol
Total mg phenol = 0.015 x 6/4
= 0.0225 mg
Therefore 100ml serum would produce: 100/0.2 x 0.0225 = 11.25mg

In the sample;
1KAU = 1mg
ACP = 3 mg/4 = 0.75 KAU
ALP = 11.25mg = 11.25KAU

DISCUSSION

Graph 1 shows direct relationship between absorbance and the liberated

phenol from the reactions. As absorbance increases the amount of phenol
liberated also increases. This means that more phenol is formed as enzyme
activity increases and so the absorbance of the solutions also increases.

Normal values of ACP in serum are in the range of 0.8 – 4.6 KAU and tartrate-
labile is 0 – 0.4 KAU (Seligman et al., 1956). The value for ACP found in this
experiment was 0.75 KAU and it lies within the range, meaning that the
function of Acid Phosphatase is normal.
Normal values for serum ALP in adults are 4-12 KAU and in children values
can go up to 24 KAU. The value obtained in this experiment for ALP was
11.25 KAU. This value is within the typical range indicating that the ALP in
the serum was of a normal individual and was not related to any pathology
concerning ALP.

The serum was preserved at low temperature and only taken when required.
This was done to prevent red blood cell hemolysis. Incubation of the test
tubes in a 370 C water bath was to allow the enzymes to work at their
optimal temperature which is the body temperature.

The substrate employed is phenyl phosphate. At the appropriate pH the ALP

or ACP will quantitatively hydrolyse the substrate to yield inorganic
phosphate and phenol. Phenol reacts with the Folin – Ciocalteu’s reagent to
give a blue-grey colour whose intensity increases with increasing phenol
concentration. Buffers were added in order to measure the enzyme activities
separately.

The Folin-Ciocalteu’s reagent is a mixture of phosphomolybdate and

phosphotungstate used for the colorimetric assay of phenolic
and polyphenolic antioxidants. It works by measuring the amount of the
substance being tested needed to inhibit the oxidation of the reagent. Folin
& Ciocalteu’s phenol reagent does not contain phenol. Rather, the reagent
will react with phenols and nonphenolic reducing substances to form
chromogens that can be detected spectrophotometrically. It can likewise be
used as a spray reagent in chromatographic procedures. The color change is
due to the transfer of electrons at basic pH to reduce the
phosphomolybdic/phosphotungstic acid complexes to form chromogens in
which the metals have lower valence. As explained in the theory, the Folin-
Ciocalteu’s reagent reacted with the phenol liberated from ACP/ALP, to give a
blue-grey color. This color is then measured in the spectrophotometer and
the wavelength used was 600nm. (Murray et al., 2003)

CONCLUSION

The amount of serum ACP found in this experiment was 0.75 KAU; and this
lies within the normal range of serum ACP. The amount of serum ALP found
in this experiment was 11.25 KAU which lies in the normal range of serum
ALP.
ANSWERS TO QUESTIONS

1. Alkaline phosphatase (EC 3.1.3.1) is a hydrolase enzyme responsible

for removing phosphate groups from many types of molecules,
including nucleotides, proteins, and alkaloids. The process of removing
the phosphate group is called dephosphorylation.
Acid phosphatase (EC 3.1.3.2 is a phosphatase, a type of enzyme, used
to free attached phosphate groups from other molecules during
digestion.
2. Liver disease or blockage of the bile ducts is the most likely to cause
elevation of serum ALP because ALP is made mostly in the liver.
3. Serum alkaline phosphatase activity is higher in children because ALP
is also made in the bone therefore rapid bone growth which occurs in
children would naturally cause an increase in serum ALP.
4. Serum ALP requires magnesium ions as cofactors hence activity will be
inhibited by citrate, oxalate and EDTA which chelate divalent cations.
5. Isozymes are distinct enzyme forms that catalyze the same reaction.
(Murray et al., 2003). Prostatic acid phosphatase has got a tartaric acid
binding site and therefore it is tartarate labile while other acid
phosphatase isozymes do not.
6. Prostatic acid phosphatase, found in the prostate, contributes to most
of the tartarate labile acid phosphatase and females do not have a
prostate therefore they have a decreased tartarate labile acid
phosphatase.

REFERENCES

1. Berg, J.M., Tymoczko, J.L., Stryer, L. (2005), Biochemistry. 5th Edition.

(W.H. Freeman And Company), page 148

2. Champe, P. C., Harvey, R.A. and Ferrier, D.R. (2005) Lippincott illustrated
reviews of Biochemistry. 3rd Edition (Philadelphia: USA), pg 126-
3. Murray R.K, Granner D.K, Mayes P.A, Rodwell V.W. (2003) Harper’s
Illustrated Biochemistry, 26th Edition, McGraw-Hill Companies, page 103

4. Nelson, D.L., and Cox, M.M. (2005). Lehninger, Principles of

Biochemistry. 4th Edition (W.H Freeman: New York), pg 1053

5. Seligman A. M, Chauncey H. H, Nachlas M. M, Manheimer L. H, Ravin

H.A, The Colorimetric Determination Of Phosphatases In Human
Serum, November 16, 1956

6. Wilson, K and Walker, J. (1994) Principles and Techniques of Practical

Biochemistry. (Cambridge University Press: UK), pg 160-164