CLINICAL CHEMISTRY 2: DIAGNOSTIC ENZYMOLOGY

General Guidelines: (MUST READ!)

***As a prelim review, one will know whether a test measures increasing or decreasing absorbance by
looking at what is detected in the test. If the test measures the disappearance/loss of a substrate,
then decreasing absorbance. If it measures the product, then increasing absorbance. In the case
of NAD/NADP and NADH/NADPH, it is always NADH or NADPH that is being measured. Look carefully
where NADH is. If it is the substrate, then the test measures decreasing absorbance. If it is the product,
then the test measures increasing absorbance. (ALWAYS ANALYZE EACH TEST IF INCREASING
OR DECREASING)

Specificity – how specific an enzyme is to the organ. The more the tissue sources of an enzyme, the
lesser its specificity.

Sensitivity – the persistence of the enzyme in the circulation in order to be detected.

Damage or any disease affecting the tissue source of the enzyme will cause elevated levels of the
enzyme regardless if the specific disease is stated in the clinical significance or not. (UNLESS STATED
OTHERWISE)

ALPHA-AMYLASE (EC 3.2.1.1)

Systematic name and Class: Alpha-1,4-D-Glucan Glucanohydrolase / Class 3 (Hydrolase)

Function: Catalyzes the breakdown of starch (plant sources) and glycogen (animal sources). Amylase
is an important enzyme in the physiologic digestion of starches.

Starch is a polysaccharide consisting of both amylose and amylopectin. Amylose is a long,

unbranched chain of glucose molecules with alpha-1,4 glycosidic linkages. Amylopectin is a
branched chain polysaccharide with alpha-1,6 linkages at the point where it branches off. Glycogen
is similar in structure to amylopectin but is more highly branched.

Alpha-amylase attacks only the alpha-1,4 glycosidic bonds to produce degradation products
consisting of glucose, maltose and intermediate chains called dextrins (these dextrins contain the
branching alpha-1,6 linkage which is not attacked by amylase).

***Cellulose and other structural polysaccharides are not attacked by amylase. (Since they do
not have alpha-1,4 glycosidic bonds)

Chemical Reaction: Starch or Glycogen by the action of alpha-amylase will yield to form glucose,
maltose and dextrins.

Cofactors: Calcium ions = Activator

Other Activators: Chloride, Bromide, Nitrate, Cholate, Monohydrogen Phosphate

***Due to amylase having lots of inorganic activators, it is also known as a metalloenzyme.

Tissue Source:

Major tissue source = Acinar cells of pancreas (exocrine pancreas) and Salivary glands.

Minor tissue sources = Skeletal muscles, small intestines and fallopian tubes.
Unique Properties: It is the smallest enzyme with a molecular weight of only 50,000 to 55,000 Daltons
and is readily filtered by the kidneys but is not reabsorbed which is why it is present in the urine. It is
the only enzyme normally found in the urine.

Clinical Significance:

It is most commonly used for the diagnosis of ACUTE PANCREATITIS however disorders of
tissues other than the pancreas may also produce elevations in amylase levels. Therefore, an elevated
amylase level is a nonspecific finding (not specific for acute pancreatitis meaning it has low
specificity).

Amylase’s specificity for acute pancreatitis increases when results are coupled with serum lipase
levels and urinary amylase levels.

***In acute pancreatitis, serum amylase levels begin to rise 5-8 hours after the onset of an attack,
peak at 24 hours and return to normal levels within 3-5 days. (According to Henry’s = rise after 6-
48 hours. According to Hubbard = rise after 2-12 hours)

Elevated Serum Amylase occurs in the ff: Salivary gland lesions (mumps and parotitis) and
intra-abdominal diseases (perforated peptic ulcer, intestinal obstruction, cholecystitis, ruptured
ectopic pregnancy, mesenteric infarction and acute appendicitis), renal insufficiency and
diabetic ketoacidosis.

Hyperamylasemia (increased amylase in the blood) has also been noted in 1-2% of the
population. This commonly occurs in neoplastic (tumor) diseases and results in elevated amylase
levels.

Macroamylasemia is a condition that results when an amylase molecule combines with

immunoglobulins to form a complex that is too large to be filtered across the glomerulus. This
causes elevated serum amylase levels and abnormally low urinary amylase. Macroamylasemia must
be differentiated from other causes of hyperamylasemia.

Isoenzymes: Amylase has two isoenzymes: P-type and S-type (Ptyalin). P-type is derived from
pancreatic tissue while S-type is derived from salivary gland tissue as well as the fallopian tube
and the lungs. S-type amylase only works on a short duration because it is inactivated by the HCl of
the stomach. P-type amylase performs the major digestive action of starches once it reaches the
intestines.

S-type amylase especially S1, S2 and S3 migrate most quickly in the electrophoresis (since
enzymes are also proteins, they can be separated using an electrophoresis) while P-type amylase
such as P1, P2 and P3 are slower. In normal human serum, the isoenzymes of amylase migrate
in the beta to alpha-globulin regions of the electrophoresis.

In cases of acute pancreatitis, there is an increase in P-type amylase with P3 being the most
predominant isoenzyme. However, P3 has also been detected in cases of renal failure and is
therefore not specific for acute pancreatitis.

S-type amylase represents approximately 2/3 of serum amylase while P-type amylase
predominates in normal urine. (S-type predominates serum amylase)

S1, S2 and P2 are the amylase isoenzymes predominantly found in normal serum.
Laboratory Assays: There are four main methods for determining amylase activity:

1. Amyloclastic – A starch substrate is attached with iodine which forms a dark-blue color. As the
amylase hydrolyzes the starch molecule into smaller units, the iodine is released and a decrease
in color occurs. Therefore, the amyloclastic method measures the decrease in color or
absorbance which is directly proportional to the amylase concentration. (Measures
disappearance of starch substrate) ***DECREASING ABSORBANCE
2. Saccharogenic – A starch substrate is hydrolysed by amylase to its constituent carbohydrate
molecules that have reducing properties (since glucose and maltose and reducing sugars). The
amount of reducing sugars is measured which is directly proportional to amylase activity.
(Measures the appearance of the product) ***INCREASING ABSORBANCE
- The saccharogenic method is the classic reference method for determining
amylase activity and is reported in Somogyi units. Somogyi units is the # of
milligrams of glucose released in 30 minutes at 37C.
3. Chromogenic – A starch substrate is attached with a chromogenic dye to form an insoluble dye-
substrate complex. As amylase hydrolyzes the starch substrate, smaller dye-substrate
fragments are produced which are water-soluble. The increase in color intensity of the
soluble dye-substrate solution is directly proportion to amylase activity. (Measures the
increasing color or absorbance due to product formation) ***INCREASING ABSORBANCE
4. Continuous Monitoring – It is a coupled-enzyme system which measures the change in
absorbance of NAD at 340nm. The optimal pH of the method is 6.9. (INCREASING
ABSORBANCE)

Continuous Monitoring Coupled-Enzyme Reaction: (Amylase, Alpha-Glucosidase, Hexokinase, G6PD)

Salivary amylase is preferentially inhibited by wheat germ lectin. Therefore, salivary and
pancreatic amylase can be estimated by measuring total amylase in the presence and absence of
lectin. Total amylase + lectin = pancreatic amylase only. Total amylase without lectin = both
pancreatic and salivary amylase.

Source of Error: Amylase in serum and urine is stable. It is stable at room temperature for 1 week,
or at 4C for 2 months.

False increase for amylase: Contamination with saliva and Morphine and other Opiates

False decrease for amylase: Plasma Triglycerides (amylase values may be normal in acute
pancreatitis with hyperlipidemia as triglycerides inhibit amylase activity) and Most Anticoagulants
(EDTA, Citrate and Oxalate as they inhibit calcium which is a necessary cofactor for amylase)

***Heparin is the only anticoagulant which does not inhibit amylase levels.
LIPASE (EC 3.1.1.3)

Systematic Name and Class: Triacylglycerol Acylhydrolase / Class 3 (Hydrolase)

Function: It hydrolyzes the glycerol ester linkages of fats to produce alcohols and fatty acids.
Specifically, it catalyzes the partial hydrolysis of dietary triglycerides in the intestines to the 2-
monoglyceride intermediate, with the subsequent production of long-chain fatty acids.

The enzymatic activity of lipase is specific for the fatty acid residues at position/carbon 1 and 3
of the triglyceride molecule but substrate must be an emulsion for the activity to occur. Lack of
emulsification by bile renders lipase ineffective in the body.

Chemical Reaction: Triglycerides by the action of lipase yields to form 2-monoglyceride and 2 long
chain fatty acids.

Cofactors: Colipase and Bile Salt (Calcium is also needed but at high levels, can also be inhibitory)

Tissue Source: Lipase concentration is found primarily in the pancreas although a small amount is
also present in the stomach, small intestines, liver, WBCs, adipocytes and colostrum.

Clinical Significance:

Clinical significance of serum lipase is confined almost exclusively for the diagnosis of ACUTE
PANCREATITIS. Serum lipase increases 4-8 hours after the onset of the attack, peak at 24 hours
and decrease and eventually return to normal within 8-14 days. It is similar to amylase in this
respect but is considered more specific for pancreatic disorders than amylase measurements.

The extent of the elevation of lipase and amylase concentrations does not correlate with the
severity of the pancreatitis but it greatly increases the chance of having acute pancreatitis.

Elevated lipase levels may also be found in other intra-abdominal conditions but with less
frequency than elevations of serum amylase such as penetrating duodenal ulcers, perforated peptic
ulcers, intestinal obstruction and acute cholecystitis.

Isoenzymes: There are three lipase isoenzymes: L1, L2 and L3. Among them, L2 Is thought to be
the most clinically specific and sensitive.

Laboratory Assays: Optimum temperature of 40C and optimum pH of 8.8

1. Cherry-Crandall method (Titrimetric Method) – An olive oil substrate by the action of lipase
produced liberated fatty acids. These liberated fatty acids were then measured by titration
after a 24 hour incubation.

***Limitations of the Cherry-Crandall method include lack of stable and uniform substrates.

***Triolein is one of the substrates now used as a purer form of triglyceride for the method.
 2. Turbidimetric methods – These are simpler and more rapid methods than titrimetric assays. Fats
in a solution create a cloudy emulsion. As the fats are hydrolysed by lipase, the particles
disperse and the rate of clearing is used to measure and estimate the lipase activity.
(MEASURES DECREASING ABSORBANCE DUE TO HYDROLYSIS OF FATS, the substrate)
3. Colorimetric methods – These are methods based on coupled reactions with enzymes such
as peroxidase or glycerol kinase.

Sources of Error: Lipase is stable in serum with no loss of activity at room temperature for 1 week or
at 4C for 3 weeks.

HEMOLYSIS SHOULD BE AVOIDED BECAUSE HEMOGLOBIN INHIBITS ACTIVITY OF

SERUM LIPASE CAUSING FALSELY LOW VALUES. (Similar to bilirubin)

Heavy metals and Quinine inhibit lipase activity.

***Lipase has a MW of 45,000 Da and is therefore freely filtered in the glomerulus but it is also
normally completely reabsorbed by the kidney tubules and is absent from normal urine. (May be
found in the urine in cases of renal tubular reabsorption failure)

ASPARTATE AMINOTRANSFERASE (EC 2.6.1.1)

Systematic Name: L-Aspartate: 2-Oxoglutarate Aminotransferase

Previous Name and Class: Serum Glutamate Oxaloacetate Transaminase / Class 2 (Transferase)

Function: It is an enzyme belonging to the class of transferases and is commonly referred to as a

transaminase as it catalyzes the transfer of an amino group between the amino acid aspartate
and alpha-keto acids.

Chemical Reaction: Synthesis and degradation of amino acids which in this case is Aspartate. The
ketoacids formed by the reaction are ultimately oxidized by the tricarboxylic acid cycle (Kreb’s Cycle)
to provide a source of energy which in this case is Oxaloacetate. (Important in the biochemical pathway
of Kreb’s Cycle or Tricarboxylic Acid Cycle)

Cofactors: Pyridoxal Phosphate aka Vitamin B6 (specifically a coenzyme)

Tissue Source: It is widely distributed in human tissue.

Major tissue source = Cardiac Tissue, Liver and Skeletal Muscles

Minor tissue sources = Kidneys, Pancreas and Erythrocytes

Clinical Significance: It is used mainly for the diagnosis and evaluation of hepatocellular disorders
and skeletal muscle involvement.
 In acute myocardial infarction, AST begins to rise within 6-8 hours, peak at 24 hours and
generally return to normal within 5 days. However because of the wide tissue distribution of AST, it
is NOT USEFUL for the diagnosis of acute myocardial infarction.

ALT is elevated in the following conditions: pulmonary embolism, congestive heart failure
(probably due to inadequate blood supply to the liver causing hepatic damage), acute hepatocellular
disorders (viral hepatitis and cirrhosis), muscular dystrophies and inflammatory conditions.

Agents like ethanol causes release of mitochondrial AST from hepatocytes which will cause an
increase in AST levels. This happens especially in the case of alcoholic hepatitis as the ethanol intake
will induce hepatic mitochondrial damage resulting in the release of mitochondrial AST.

***Mitochondrial AST – predominant form of AST only in hepatocytes and has a longer half-life than
the cytoplasmic isoenzyme of AST and ALT.

The release of mitochondrial AST in alcoholic hepatitis frequently results in a disproportionate

elevation of AST over ALT yielding an AST/ALT ratio (AKA De Ritis Ratio) of 3-4:1 which only
happens in cases of alcoholic hepatitis. In the normal De Ritis Ratio in any given hepatic disease
(commonly viral hepatitis), ALT is always usually higher than the AST with the sole exception of the
case in alcoholic hepatitis.

Isoenzymes: Cell cytoplasm fraction and Mitochondrial fraction

The cytoplasmic isoenzyme is the overall predominant form occurring in the serum. (The
cytoplasmic AST has a half-life of 17 hours)

The mitochondrial isoenzyme may only be significantly increased in cases of CELLULAR

NECROSIS and ALCOHOLIC HEPATITIS. (The mitochondrial AST has a half-life of 87 hours)

***However, isoenzyme analysis of AST is not routinely performed in the clinical laboratory.

Laboratory Assays:

1. Karmen Method – It is a coupled enzymatic reaction using malate dehydrogenase as the

indicator enzyme and monitors the change in absorbance of NADH at 340nm. The
optimum pH of the reaction is 7.3 to 7.8. (DECREASING ABSORBANCE)

***Insert malate dehydrogenase as the enzyme in the second reaction…

Sources of Error: HEMOLYSIS should be avoided because it can dramatically increase serum AST
concentration because erythrocytes contain abundant intracellular AST.

AST activity is stable in serum for 3-4 days at refrigerated temperature.

ALANINE AMINOTRANSFERASE (EC 2.6.1.2)

Systematic Name: L-Alanine: 2-Oxoglutarate Aminotransferase

Previous Name and Class: Serum Glutamate Pyruvate Transaminase / Class 2 (Transferase)

Function: It is a transferase with enzymatic activity similar to that of AST.

Specifically, it catalyzes the transfer of an amino group from alanine to alpha-ketoglutarate

with the subsequent formation of glutamate and pyruvate. (IMPORTANT IN KREB’S CYCLE)

Chemical Reaction:

Cofactor: Pyridoxal Phosphate (Vitamin B6) = coenzyme

Tissue Source: ALT is distributed in many tissues with comparatively high concentrations in the liver.
It is considered to be the more liver-specific enzyme of the transferases.

Major tissue sources = Liver, Kidney, Heart.

Minor tissue sources = Skeletal Muscles, Pancreas, Spleen, Erythrocytes

Clinical Significance:

It is confined mainly for the evaluation of hepatic disorders. Higher elevations are found in
hepatocellular disorders than in extrahepatic or intrahepatic obstructive disorders.

In acute inflammatory conditions of the liver, ALT elevation are frequently higher than those
of AST and tend to remain elevated longer as a result of ALT’s longer half-life in serum which
is 24 hours (Bishop) or 47 hours (Henry’s) while AST only has a half-life of 16 hours (Bishop)
or 17 hours (Henry).

Cardiac tissues also contain a small amount of ALT activity but serum levels of ALT usually
remain normal in acute myocardial infarction. (NOT SURE IF ALT IS ELEVATED IN MYOCARDIAL
INFARCTION BUT ONE OF ITS TISSUE SOURCE IS HEART SO PERHAPS BUT ONLY SLIGHTLY
ELEVATED IF SO)

Laboratory Assay: (In the past, pyruvate dehydrogenase is also used as an indicator enzyme
converting pyruvate to Acetyl Coenzyme A with conversion of NADH to NAD. However, it is no longer
used)

1. Wroblewski-LaDue – It is a coupled enzymatic method using Lactate Dehydrogenase as the

indicator enzyme and measures the change in absorbance of NADH at 340nm which is
directly proportional to ALT activity. The optimum pH of the reaction is 7.3 to 7.8.
(DECREASING ABSORBANCE)
 2. Reitman-Frankel – It is a colorimetric assay which uses an alkaline medium to detect 2,4-
Dinitrophenyl hydrazone (brown color). (INCREASING ABSORBANCE)

Source of Error: ALT is stable for 3-4 days at 4C. It is affected by hemolysis (Henry’s) because
RBCs contain intracellular ALT.

TAKE NOTE! = Both ALT AND AST LEVELS may be low even in cases of hepatocellular damage
when there is Vitamin B6 (Pyridoxal Phosphate) insufficiency. In order to see the actual AST
and ALT levels, vitamin B6 must first be administered to the patient.

Henry’s Additional Discussion Regarding ALT and AST in Liver Diseases:

ALT and Cytoplasmic isoenzyme of AST = FOUND IN THE HEPATIC CYTOSOL.

Mitochondrial isoenzyme of AST = FOUND IN THE HEPATOCYTE MITOCHONDRIA.

In cases of alcoholic hepatitis, a patient was found to have normal to low serum levels of ALT
and AST which is unusual as hepatitis should mean that they would be increased. For the next 24
hours, he was treated for his condition and a repeat liver function profile showed marked elevation of
his ALT and AST. How did this happen?

Answer: Part of the treatment for alcoholic hepatitis is the administration of Vitamin B6 and the serum
assays for both ALT and AST requires Vitamin B6 which is commonly deficient in alcoholic
patients. (Pyridoxal phosphate or Vitamin B6 is really important to accurately test for ALT and AST)

In most forms of hepatocellular injury such as hepatitis, AST will be higher than ALT initially
because of the higher activity of AST in hepatocytes but within 24-48 hours, ALT will become higher
than AST due to its longer half-life.

In end-stage liver cirrhosis, levels of both ALT and AST are generally not elevated anymore and
may be low as a result of massive tissue destruction.

ALKALINE PHOSPHATASE (EC 3.1.3.1)

Systematic Name and Class: Orthophosphoric Monoester Phosphohydrolase (Alkaline Optimum) /

Class 3 (Hydrolase)

Function: It catalyzes the hydrolysis of various phosphomonoesters at an alkaline pH. It is a

nonspecific enzyme capable of reacting with many different substrates as long as they are
phosphomonoesters.
Chemical Reaction: Specifically, it functions to liberate inorganic phosphate from an organic
phosphomonoester with the concomitant production of an alcohol. The optimal pH for the reaction
is 9.0 to 10.0 but optimum pH varies with the substrate used.

Cofactor: Magnesium ion (Mg 2+), Zinc ion, Cobalt and Manganese

Inhibitors: Phosphate, Borate, Oxalate and Cyanide

Tissue Source: ALP is present on cell surfaces (cell membrane) in most human tissue.

Major tissue source = Intestines, Liver, Bone, Spleen, Placenta and Kidney.

In the liver, the enzyme is located on both the sinusoidal and bile canalicular membranes.

In the bone, the enzyme’s activity is confined to the osteoblasts (the cells involved in the production
of bone matrix).

Clinical Significance: Elevations of ALP are of most diagnostic significance in the evaluation of
hepatobiliary and bone disorders. In hepatobiliary disorders, elevations are more prominent in
obstructive conditions (blockage) than in hepatocellular disorders (cell damage). In bone disorders,
elevations are observed when there is involvement of osteoblasts.

Elevated ALP are seen in the following conditions: biliary tract obstruction, hepatocellular
disorders (only slight increase), Paget’s Disease or Osteitis deformans (highest ALP elevation) and
other bone and liver diseases.

A single elevated ALP level (low specificity) is difficult to interpret and must be evaluated with
other liver function tests for diagnostic significance.

Care must be taken to consider the age, gender and patient history of the patient before
interpreting elevated ALP results as ALP is often elevated in periods of physiologic bone growth,
pregnancy (and other pregnancy complications) and in healing bone fractures.

Low ALP levels are seen in the following conditions: Post-Blood Transfusions, Post-
Cardiopulmonary Bypass, Zinc Deficiency and Hypophosphatasia (due to absence of bone ALP).

Isoenzymes: The major isoenzymes of ALP that are found in the serum are those found in the liver,
bone, intestine and placenta.

Electrophoresis is considered the most useful single technique for ALP isoenzyme
analysis. For bone-related ALP, a direct immunochemical method is now available which
eliminates the need for electrophoresis.

As a summary for isoenzymes of ALP… (all info are important since outlined na ni)

3 Methods of ALP isoenzyme analysis: ELECTROPHORESIS (single most useful), Heat stability and
Selective Chemical Inhibition via the use of phenylalanine
4 Fractions of ALP isoenzymes: Liver (migrates the fastest), Bone (2nd), Placental (3rd), Intestinal (4th)

- There is no clear separation between liver and bone fractions.

- In order to enhance liver and bone ALP separation, neuraminidase and wheat germ
lectin are used.

Liver Fraction: Divided into Major Liver Band and Fast Liver or Alpha-1 Liver. Major liver band is
elevated in hepatobiliary disorders. Fast liver (faster between the two and migrates to alpha-1 region in
electrophoresis) is elevated in metastatic carcinoma of the liver.

- In cases of increased liver ALP, major liver band is the one usually elevated.
- Liver fraction serves as a valuable indicator of obstructive disease.

Bone Fraction: Normally elevated in children during growth period and adults older than age 50.

Intestinal Fraction: Normally found in people with blood type B or O and have a secretor gene.

- Not normally found in those with Blood type A because the intestinal ALP is bound to
their erythrocytes.
- Increase in intestinal ALP in those with Blood type B or O occurs after consumption of
a fatty meal.

According to Heat Stability: Placental (1st), Intestinal (2nd), Liver (3rd), Bone (4th)

- Placental ALP resists denaturation at 65C for 30 minutes.

- Placental ALP (PLAP) is a useful tumor marker in serum and CSF for most germ cell
tumors.
- If less than 20% of the activity remains after heating, then it is of the bone isoenzyme.
- If greater than 20% of the activity remains, then it is of the liver isoenzyme.

Based on Selective Chemical Inhibition (Phenylalanine): Inhibits Intestinal and Placental ALP

- It cannot differentiate intestinal ALP from placental ALP and liver ALP from bone ALP.
- Levamisole is used to inhibit liver and bone ALP.

Abnormal Fractions of ALP: Regan isoenzyme, Nagao isoenzyme and Kasahara isoenzyme (Both are
called Carcinoplacental ALPs due to their similarities with placental ALP)

Regan isoenzyme – migrates in the electrophoresis similar to the bone fraction.

- Most heat stable of all ALP isoenzymes, resists heat denaturation at 65C for 30 mins.
and is inhibited by phenylalanine.
- Has been detected in various carcinomas but only occurs in 3% - 15% of cancer
patients so it is not used to diagnose malignancy.
- Useful in monitoring effects of therapy as it disappears upon successful treatment.

Nagao isoenzyme – identical in most ways to the Regan isoenzyme but it has the additional property
of being inhibited by L-Leucine. It has been detected in metastatic carcinoma of pleural surfaces and
adenocarcinoma of the pancreas and bile duct.

Kasahara isoenzyme – seen in hepatoma and GIT tumors.

Laboratory Assay: Based on the non-specificity of ALP with regard to substrates, a variety of
methodologies can be used. The major differences between them is only on the type of substrate,
concentration, buffer used and pH of the reaction.

1. Bowers and McComb – A continuous monitoring technique which calculates ALP activity based
on the molar absorptivity of p-nitrophenol (the product). (INCREASING ABSROBANCE)

- P-nitrophenylphosphate (colorless) is hydrolysed to p-nitrophenol (yellow) and

the increase in absorbance at 405 nm is directly proportional to ALP activity.
- pH of the reaction is 10.2.
2. Bessey-Lowry-Brock

Sources of Error: Hemolysis may cause slight elevation in ALP levels since ALP is six times more
concentrated in erythrocytes than in serum.

ALP assays should be run as soon as possible after collection as its activity in serum
increases 3 to 10% on standing at 25C or 4C for several hours.

Diet may induce elevations in ALP activity of people with blood type B or O and who are
secretors. Values may be 25% higher following ingestion of a high-fat meal.

Smoking and Anti-Epileptic Drugs cause falsely increased ALP values.

Chelators such as EDTA, Citrate and Oxalate cause falsely low ALP activity. (It inhibits
calcium and zinc which also helps in the action of ALP)

Increased levels of phosphate ion is capable of inhibiting ALP activity that has not yet
reacted in the reaction.

***In order to minimize phosphate interference, a buffer is added which will bind to phosphate and
prevents ALP inhibition.

Addition Henry’s Info on ALP:

Half-Life of ALP isoenzymes: Intestine = minutes, Bone = 1 day, Liver = 3 days, Placenta = 7 days

ACID PHOSPHATASE (EC 3.1.3.2)

Systematic Name and Class: Orthophosphoric Monoester Phosphohydrolase (Acid Optimum) / Class
3 (Hydrolase)

Function: It is a hydrolase which catalyzes the same type of reaction as ALP. Their only difference is
the pH of the reaction where the enzyme functions. With ACP, the optimal pH of the reaction is 5.0.

Chemical Reaction: It functions to liberate inorganic phosphate from an organic phosphomonoester

with the concomitant production of an alcohol.
Cofactor: Unknown but probably similar to ALP which is Magnesium (Mg 2+)

Tissue Source: ACP activity is found in the prostate, bone, liver, spleen, kidney, erythrocytes and
platelets.

The prostate is the richest source of ACP with many times the activity found in other tissues.

Clinical Significance: ACP measurement has been used as an aid in the detection of prostatic
carcinoma, particularly metastatic carcinoma of the prostate. ACP determinations are relatively
insensitive techniques which can only detect elevated ACP levels results from prostatic carcinoma
only when the tumor has already metastasized (low sensitivity).

One of the most specific substrates for prostatic ACP is thymolphthalein monophosphate.
Chemical inhibition methods used to differentiate the prostatic fraction of ACP most frequently use
tartrate as the inhibitor since the prostatic fraction of ACP is inhibited by tartrate.

In order to measure prostatic ACP, serum and substrate are incubated both with and without the
addition of L-tartrate. ACP activity remaining after inhibition with L-tartrate is subtracted from the total
ACP activity without inhibition: TOTAL ACP – ACP after tartrate inhibition = Prostatic ACP.
However, this reaction is not entirely specific for prostatic ACP as lysosomal ACP is also inhibited by
tartrate.

Prostatic ACP elevations also occur in other prostatic conditions such as prostate hyperplasia
and prostatic surgery.

Due to this limitation of ACP, newer markers such as prostate-specific antigen is used as a
diagnostic tool for prostatic carcinoma. Prostate-specific antigen (PSA) is particularly useful to monitor
the success of treatment however it is controversial as a screening test as it may also be elevated in
conditions other than prostatic carcinoma such as benign prostatic hypertrophy and prostatitis.

ACP assays have proved useful in forensic clinical chemistry in the investigation of rape. Vaginal
washings are examined for seminal fluid-ACP activity which can persist for up to 4 days. Elevated
seminal fluid-ACP activity is presumptive evidence of rape in such cases.

Serum ACP may be elevated in bone disease as bone ACP activity is often associated with
osteoclasts. ACP may also be elevated in platelet damage as some ACP activity is found in platelets.

Isoenzymes: The five important types found in the human body are lysosomal, prostatic,
erythrocyte, macrophage and osteoclastic ACP. ACP normally occurs in serum at low
concentrations.

The erythrocyte isoenzyme differs from other ACP isoenzymes in that it is inhibited by 2%
formaldehyde and 1 mM cupric sulphate solution which does not inhibit the other isoenzymes.
Additionally, the erythrocyte isoenzyme is not inhibited by 20 mM tartrate solution which does inhibit
the other isoenzymes.
 Erythrocyte ACP – inhibited by 2% formaldehyde and 1mM cupric sulphate solution.

- Not inhibited by tartrate. (Tartrate-resistant)

Pancreatic ACP and Other ACP isoenzymes – inhibited by tartrate solution.

Tartrate-resistant Acid Phosphatase (TRAP) or Erythrocyte ACP is present in certain

leukemias and some lymphomas, most notably in hairy cell leukemia. A particular isoform, TRAP-5b,
occurs predominantly in osteoclasts in bone marrow and is used as a marker for bone remodelling
and proposed as a marker for metastatic bone marrow cancer.

Bone ACP and bone ALP are used as tumor markers for bone cancers.

Prostatic ACP has a half-life of 1-3 hours.

Laboratory Assay: Use the same techniques as in ALP assays but are performed in acidic pH of 5.
(INCREASING ABSORBANCE)

The reaction product (p-nitrophenol) is colorless at the acidic pH of the reaction, but the addition
of alkali stops the reaction and transforms the products into chromogens which can now be
measured spectrophotometrically at 405 nm. (P-nitrophenol is yellow in the ALP assay but that is
because of the alkaline pH of the reaction used for ALP assays. P-nitrophenol can only exhibit its color
in an alkaline pH and will be colorless at an acidic pH which is why we add an alkali in ACP assays)

Immunochemical techniques for prostatic ACP used are radioimmunoassay (RIA),

counterimmunoelectrophoresis, immunoprecipitation and immunoenzymatic assay (Tandem E)
which includes incubation with antibody to prostatic ACP and nitrophenylphosphate

Nitrophenylphosphate = Nitrophenol (alcohol) + Phosphate

Thymolphthalein Monophosphate = Thymolphthalein (alcohol) + Phosphate

- For Quantitative Endpoint Reactions.

Alpha-naphthyl phosphate = Alpha-naphthol (alcohol) + Phosphate

- For Continuous-Monitoring Reactions.

***It is the alcohol that is detected in all assays for ALP and ACP because phosphate has too many
interferences.

Source of Error: Serum should be separated from RBCs as soon as the blood has clotted to
prevent leakage of erythrocyte and platelet ACP.

Serum activity decreases within 1-2 hours if the sample is left at room temperature without a
preservative. If not assayed immediately, serum should be frozen or acidified to a pH lower than 6.5.
With acidification, ACP is stable for 2 days at room temperature.

Hemolysis should be avoided because of contamination from erythrocyte ACP.

 High bilirubin causes falsely low values for TRAP activity but not for total ACP.

RIA procedures for prostatic ACP require non-acidified serum samples. Activity is stable for 2
days at 4C.

ALP ACP

(nitrophenylphosphate) (thymolphthalein/alpha-napthol)

False Decrease (Due to

False Increase (H2CO3 + O2 =
H2CO3 in blood which when
Stability in Room Temp. CO2 which is basic and
exposed to oxygen becomes
increases pH of the reaction)
CO2 which increases pH)

Hemolysis False Increase False Increase

Diet (In Blood type B or O)

Other Factors Causing False
Smoking
Increase
Anti-epileptic Drugs

EDTA, Citrate and Oxalate

Anticoagulants

Post-Blood Transfusions
High Bilirubin (False decrease
False Decrease
Post-Cardiopulmonary Bypass for TRAP but not for Total ACP)

Zinc Deficiency

Hypophosphatasia