ESTIMATION OF
ALKALINE
PHOSPHATASE
• Phosphatases are hydrolases.
• They hydrolyze a variety of organic phosphate esters transferring
phosphate groups from a donor substrate to an acceptor containing a
hydroxyl group.
• The active center of these enzymes contains a serine residue. Two
types are commonly estimated in serum:
A mixture of alkaline phosphatases with maximum activity at pH 8.5-10.5
Acid phosphatases with maximum activity between pH 5-6.
• There are also neutral phosphatases which dephosphorylate
Ser/Thr/Tyr residues of enzymes involved in various metabolic
pathways.
ALKALINE PHOSPHATASE (ALP)
• Alkaline phosphatases (EC [Link]) belong to class of enzymes called
Hydrolases.
• They are a family of isoenzymes.
• They are membrane-bound glycoproteins and zinc containing
metalloenzymes.
Tissue Distribution:
• present practically in all tissues of the body.
• High levels are present in:
Intestine: The small intestinal epithelium (I).
Liver: The bile canalicular and sinusoidal membrane of the liver (L)
Bone: The osteoblasts in the bone especially in infancy and childhood when
there is active bone growth (B).
Kidney: The tubular epithelium of the kidney (K).
The placenta and the lactating breast (P).
Sources of the enzyme in serum and
urine:
• In serum of normal adults most of the amount is contributed by liver,
and the rest coming from bone.
• The respective contributions of these two forms to the total activity
are markedly age dependent.
• The intestine and kidney contribute very little amount to the total ALP
activity.
• Placenta contributes a considerable amount during pregnancy.
FUNCTIONS:
• Alkaline phosphatases from different sources exhibit three types of
activity namely,
Hydrolytic R-P + H-OH → R-OH + H-P
Phosphotransferase R-P + R’-OH → R-OH + R’P
Pyrophosphatase R-P-P-R’ + H-OH → R-P + R’-P
• acts on a large variety of physiologic and non-physiologic substances.
• associated with calcification and mineralization process in bone and
probably in lipid transport in the intestine.
ISOENZYMES:
• group of true isoenzymes, encoded for by at least four different genes:
i. Tissue-nonspecific
ii. Intestinal
iii. Placental
iv. Germ-cell ALP.
• The isoforms derived from the tissue-nonspecific isoenzyme by post-translational
modification include the variants of the enzyme found in the liver, bone, kidney and in
the first trimester placenta.
• Some malignant tumours can produce a placental like form of the enzyme called the
Regan's isoenzyme.
ESTIMATION OF SERUM ALKALINE
PHOSPHATASE LEVELS by IFCC method
This method utilises 4-nitrophenyl phosphate as the substrate.
Under optimised conditions ALP present in the sample catalyses the following reaction.
ALP
AMP + 4-NPP + H2 O 4-nitrophenol + phosphate
Mg 2+ / Alkaline pH
At the pH of the reaction, 4-nitrophenol has an intense yellow colour.
The reagent also contains a metal ion buffer system to ensure that optimal concentrations of Zinc and
Magnesium are maintained.
The reaction is monitored by measuring the rate of increase in absorbance at 405 which is proportional to
the activity of ALP in the serum.
AMP -2-amino-2-methyl-1-propanol (AMP)
Procedure
Test Standard
Working reagent 1ml 1ml
Sample 0.020 ml -
Standard - 0.020 ml
• measure the initial absorbance of sample and standard.
• Measure the absorbance change exactly after 1, 2 and 3 min.
Calculate 1 minute absorbance change (ΔA/min).
• Calculation: ALP = Abs/min of Sample ×
Conc of standard
Abs/min of standard
• Measured in IU(International Units)
Normal reference range:
• The normal value for serum ALP in adult is 53-128 IU .
• In children the value is normally about 2-5 times greater particularly
during active growth
INTERPRETATION
Physiological Increase in ALP :
• Physiological bone growth elevates ALP in serum and hence in the
sera of growing children, enzyme activity is 1.5 -2.5 times that in
normal adult serum.
• 2-3 times increase above normal levels is seen in women in the third
trimester of pregnancy
Pathological Increase in ALP :
• The response of the liver to any form of biliary tree obstruction is to
synthesize more ALP in the hepatocytes adjacent to the biliary canaliculi.
• There is more elevation in extrahepatic obstruction (eg. by stone or by
cancer of the head of the pancreas) than in intrahepatic obstruction.
• Among the bone diseases, the highest levels of serum ALP are seen in
Paget’s disease
• Moderate increases are seen in Osteomalacia, Rickets and Fanconi’s
syndrome. High ALP levels are seen in osteogenic bone cancer.
ACID PHOSPHATASE (ACP)
• present in lysosomes, however, extralysosomal ACPs are also present
in many cells.
• The highest concentrations of ACP activity occur in
Prostate
bone (osteoclasts)
Spleen
Platelets
Erythrocytes.
It is of two types
• 1. Inhibited by d-rotatory tartarate ions: present in lysosomes and
prostate.
• 2. Tartarate resistant(TR-ACP) : present in bone ,erythrocytes and
leucocytes.
• Elevated Acid Phosphatase causes –
i. Carcinoma prostate – Increased osteoclast activity
ii. Paget’s disease
iii. Hyperparathyroidism
iv. Osteoclastoma
v. Gaucher’s disease (lysosomal storage disorder)
vi. Hairy cell leukemia
INTERPRETATION
• The normal value is 1.8-8.8 IU
• TR-ACP is a potentially useful marker of conditions with marked
osteolytic component such as
Paget’s disease
Hyperparathyroidism
Malignant invasion of bones by cancers.
To assess prognosis in prostatic carcinoma