Aspartate aminotransferase (AST) is an enzyme belonging to the class of

transferases. It is commonly referred to as a transaminase and is involved in the

transfer of an amino group between aspartate and keto acids. The older
terminology, serum glutamic-oxaloacetic transaminase (SGOT, or GOT), may also
be used. Pyridoxal phosphate functions as a coenzyme.
The transamination reaction is important in intermediary metabolism because of
its function in the synthesis and degradation of amino acids. The ketoacids
formed by the reaction are ultimately oxidized by the tricarboxylic acid cycle to
provide a source of energy.
Tissue Source
AST is widely distributed in human tissue. The highest concentrations are found
in cardiac tissue, liver, and skeletal muscle, with smaller amounts found in the
kidney, pancreas, and erythrocytes.
Diagnostic Significance
The clinical use of AST is limited mainly to the evaluation of hepatocellular
disorders and skeletal muscle involvement. In AMI, AST levels begin to rise within
6 to 8 hours, peak at 24 hours, and generally return to normal within 5 days.
However, because of the wide tissue distribution, AST levels are not useful in the
diagnosis of AMI.
AST elevations are frequently seen in pulmonary embolism. Following congestive
heart failure, AST levels also may be increased, probably reflecting liver
involvement as a result of inadequate blood supply to that organ. AST levels are
highest in acute hepatocellular disorders. In viral hepatitis, levels may reach 100
times ULN. In cirrhosis, only moderate levelsapproximately four times ULN
are detected (see Chapter 24). Skeletal muscle disorders, such as the muscular
dystrophies, and inflammatory conditions also cause increases in AST levels (48
ULN).
AST exists as two isoenzyme fractions located in the cell cytoplasm and
mitochondria. The intracellular concentration of AST may be 7,000 times higher
than the extracellular concentration. The cytoplasmic isoenzyme is the
predominant form occurring in serum. In disorders producing cellular necrosis,
the mitochondrial form may be significantly increased. Isoenzyme analysis of AST
is not routinely performed in the clinical laboratory.
Assay for Enzyme Activity
Assay methods for AST are generally based on the principle of the Karmen
method, which incorporates a coupled enzymatic reaction using malate
dehydrogenase (MD) as the indicator reaction and monitors the change in
absorbance at 340 nm continuously as NADH is oxidized to NAD (Eq. 12-11). The
optimal pH is 7.3 to 7.8.
Source of Error
Hemolysis should be avoided because it can dramatically increase serum AST
concentration. AST activity is stable in serum for 3 to 4 days at refrigerated
temperatures.

Reference Range
AST, 5 to 30 U/L (37C)

Alanine Aminotransferase
Alanine aminotransferase (ALT) is a transferase with enzymatic activity similar to
that of AST. Specifically, it catalyzes the transfer of an amino group from alanine
to - ketoglutarate with the formation of glutamate and pyruvate. The older
terminology was serum glutamic-pyruvic transaminase (SGPT, or GPT). Equation
12-12 indicates the transferase reaction. Pyridoxal phosphate acts as the
coenzyme.
Tissue Source
ALT is distributed in many tissues, with comparatively high concentrations in the
liver. It is considered the more liver-specific enzyme of the transferases.
Diagnostic Significance
Clinical applications of ALT assays are confined mainly to 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 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).
Cardiac tissue contains a small amount of ALT activity, but the serum level
usually remains normal in AMI unless subsequent liver damage has occurred. ALT
levels have historically been compared with levels of AST to help determine the
source of an elevated AST level and to detect liver involvement concurrent with
myocardial injury.
Assay for Enzyme Activity
The typical assay procedure for ALT consists of a coupled enzymatic reaction
using LDH as the indicator enzyme, which catalyzes the reduction of pyruvate to
lactate with the simultaneous oxidation of NADH. The change in absorbance at
340 nm measured continuously is directly proportional to ALT activity. The
reaction proceeds according to Equation 12-13. The optimal pH is 7.3 to 7.8.
Source of Error
ALT is stable for 3 to 4 days at 4C. It is relatively unaffected by hemolysis.
Reference Range
ALT, 637 U/L (37C)

Alkaline Phosphatase
Alkaline phosphatase (ALP) belongs to a group of enzymes that catalyze the
hydrolysis of various phosphomonoesters at an alkaline pH. Consequently, ALP is
a nonspecific enzyme capable of reacting with many different substrates.
Specifically, ALP functions to liberate inorganic phosphate from an organic
phosphate ester with the concomitant production of an alcohol.
The optimal pH for the reaction is 9.0 to 10.0, but optimal pH varies with the
substrate used. The enzyme requires Mg2 as an activator.
Tissue Source
ALP activity is present on cell surfaces in most human tissue. The highest
concentrations are found in the intestine, liver, bone, spleen, placenta, and
kidney. In the liver, the enzyme is located on both sinusoidal and bile canalicular
membranes; activity in bone is confined to the osteoblasts, those cells involved
in the production of bone matrix. The specific location of the enzyme within this
tissue accounts for the more predominant elevations in certain disorders.
Diagnostic Significance
Elevations of ALP are of most diagnostic significance in the evaluation of
hepatobiliary and bone disorders. In hepatobiliary disorders, elevations are more
predominant in obstructive conditions than in hepatocellular disorders; in bone
disorders, elevations are observed when there is involvement of osteoblasts.
In biliary tract obstruction, ALP levels range from 3 to 10 times ULN. Increases
are primarily a result of increased synthesis of the enzyme induced by
cholestasis. In contrast, hepatocellular disorders, such as hepatitis and cirrhosis,
show only slight increases, usually less than three times ULN. Because of the
degree of overlap of ALP elevations that occurs in the various liver disorders, a
single elevated ALP level is difficult to interpret. It assumes more diagnostic
significance when evaluated along with other tests of hepatic function.
Elevated ALP levels may be observed in various bone disorders. Perhaps the
highest elevations of ALP activity occur in Pagets disease (osteitis deformans).
Other bone disorders include osteomalacia, rickets, hyperparathyroidism, and
osteogenic sarcoma. In addition, increased levels are observed in healing bone
fractures and during periods of physiologic bone growth.
In normal pregnancy, increased ALP activity, averaging approximately 11 2 times
ULN, can be detected between weeks 16 and 20. ALP activity increases and
persists until the onset of labor. Activity then returns to normal within 3 to 6
days.24 Elevations also may be seen in complications of pregnancy such as
hypertension, preeclampsia, and eclampsia, as well as in threatened abortion.
ALP levels are significantly decreased in the inherited condition of
hypophosphatasia. Subnormal activity is a result of the absence of the bone
isoenzyme and results in inadequate bone calcification.
ALP exists as a number of isoenzymes, which have been studied by a variety of
techniques. The major isoenzymes, which are found in the serum and have been
most extensively studied, are those derived from the liver, bone, intestine, and
placenta.

Electrophoresis is considered the most useful single technique for ALP isoenzyme
analysis. However, because there may still be some degree of overlap between
the fractions, electrophoresis in combination with another separation technique
may provide the most reliable information. A direct immunochemical method for
the measurement of bone-related ALP is now available; this has made ALP
electrophoresis unnecessary in most cases.
The liver fraction migrates the fastest, followed by bone, placental, and intestinal
fractions. Because of the similarity between liver and bone phosphatases, there
often is not a clear separation between them. Quantitation with use of a
densitometer is sometimes difficult because of the overlap between the two
peaks. The liver isoenzyme can actually be divided into two fractionsthe major
liver band and a smaller fraction called fast liver, or 1 liver, which migrates
anodal to the major band and corresponds to the 1 fraction of protein
electrophoresis. When total ALP levels are increased, the major liver fraction is
the most frequently elevated. Many hepatobiliary conditions cause elevations of
this fraction, usually early in the course of the disease. The fast-liver fraction has
been reported in metastatic carcinoma of the liver, as well as in other
hepatobiliary diseases. Its presence is regarded as a valuable indicator of
obstructive liver disease. However, it is occasionally present in the absence of
any detectable disease state.
The bone isoenzyme increases due to osteoblastic activity and is normally
elevated in children during periods of growth and in adults older than age 50. In
these cases, an elevated ALP level may be difficult to interpret.
The presence of intestinal ALP isoenzyme in serum depends on the blood group
and secretor status of the individual. Individuals who have B or O blood group
and are secretors are more likely to have this fraction. Apparently, intestinal ALP
is bound by erythrocytes of group A. Furthermore, in these individuals, increases
in intestinal ALP occur after consumption of a fatty meal. Intestinal ALP may
increase in several disorders, such as diseases of the digestive tract and
cirrhosis. Increased levels are also found in patients undergoing chronic
hemodialysis.
Difference in heat stability is the basis of a second approach used to identify the
isoenzyme source of an elevated ALP. Typically, ALP activity is measured before
and after heating the serum at 56C for 10 minutes. If the residual activity after
heating is less than 20% of the total activity before heating, then the ALP
elevation is assumed to be a result of bone phosphatase. If greater than 20% of
the activity remains, the elevation is probably a result of liver phosphatase.
These results are based on the finding that placental ALP is the most heat stable
of the four major fractions, followed by intestinal, liver, and bone fractions in
decreasing order of heat stability. Placental ALP will resist heat denaturation at
65C for 30 minutes.
Heat inactivation is an imprecise method for differentiation because inactivation
depends on many factors, such as correct temperature control, timing, and
analytic methods sensitive enough to detect small amounts of residual ALP
activity. In addition, there is some degree of overlap between heat inactivation of
liver and bone fractions in both liver and bone diseases.

A third approach to identification of ALP isoenzymes is based on selective

chemical inhibition. Phenylalanine is one of several inhibitors that have been
used. Phenylalanine inhibits intestinal and placental ALP to a much greater
extent than liver and bone ALP. With phenylalanine use, however, it is impossible
to differentiate placental from intestinal ALP or liver from bone ALP.
In addition to the four major ALP isoenzyme fractions, certain abnormal fractions
are associated with neoplasms. The most frequently seen are the Regan and
Nagao isoenzymes. They have been referred to as carcinoplacental alkaline
phosphatases because of their similarities to the placental isoenzyme. The
frequency of occurrence ranges from 3% to 15% in cancer patients. The Regan
isoenzyme has been characterized as an example of an ectopic production of an
enzyme by malignant tissue. It has been detected in various carcinomas, such as
lung, breast, ovarian, and colon, with the highest incidences in ovarian and
gynecologic cancers. Because of its low incidence in cancer patients, diagnosis of
malignancy is rarely based on its presence. It is, however, useful in monitoring
the effects of therapy because it will disappear on successful treatment.
The Regan isoenzyme migrates to the same position as the bone fraction and is
the most heat stable of all ALP isoenzymes, resisting denaturation at 65C for 30
minutes. Its activity is inhibited by phenylalanine.
The Nagao isoenzyme may be considered a variant of the Regan isoenzyme. Its
electrophoretic, heat-stability, and phenylalanine-inhibition properties are
identical to those of the Regan fraction. However, Nagao also can be inhibited by
L-leucine. Its presence has been detected in metastatic carcinoma of pleural
surfaces and in adenocarcinoma of the pancreas and bile duct.
Assay for Enzyme Activity
Because of the relative nonspecificity of ALP with regard to substrates, a variety
of methodologies for its analysis have been proposed and are still in use today.
The major differences between these relate to the concentration and types of
substrate and buffer used and the pH of the reaction. A continuous-monitoring
technique based on a method devised by Bowers and McComb allows calculation
of ALP activity based on the molar absorptivity of p-nitrophenol.
Source of Error
Hemolysis may cause slight elevations because ALP is approximately six times
more concentrated in erythrocytes than in serum. ALP assays should be run as
soon as possible after collection. Activity in serum increases approximately 3% to
10% on standing at 25C or 4C for several hours. Diet may induce elevations in
ALP activity of blood group B and O individuals who are secretors. Values may be
25% higher following ingestion of a high-fat meal.
Reference Range
ALP, 30 to 90 U/L (30C)