I.

LIVER FUNCTION TESTS

LIVER
= major synthetic & metabolic organ of the body responsible for many major metabolic functions in the body
= the ONLY ORGAN that has the ability to regenerate of the organ
= the great physiologic significance of the portal flow to the liver is that ALL NUTRIENTS ARISING FROM THE
DIGESTION OF FOOD IN THE GIT, except fats, pass through the liver before entering the general circulation
for transmission to the rest of the body
= the secretion arising from the hepatic cells is called BILE
= the bile canaliculi carry away the secretions & excretions from the hepatocytes & bile canaliculi combine to form
bile ducts that carry the bile secretions into the small intestine

ANATOMY
= consists of two main lobes that together weigh from 1400-1600 g in the normal adult
= has an abundant blood supply, receiving approximately 15 ml/min from two major vessels:
 the hepatic artery
 the portal vein
= LOBULE
 forms the structural unit of the comprises cords of liver cells (hepatocytes) radiating from a central vein
 between the cords of liver cells are vascular spaces, called sinusoids, that are lined by endothelial cells
and Kupffer cells

PHYSIOLOGY
@ Functions of the Liver:

A. Metabolic Functions
a. CHO metabolism
i. Glycogenesis and glycogenolysis
ii. Converts lactate back to glucose
iii. Gluconeogenesis
iv. Maintains a relatively constant plasma glucose concentration

b. CHON metabolism
i. Most plasma proteins and blood coagulation proteins with the exception of the gamma globulins
ii. Converts proteins back to their constituent amino acids

c. Lipid metabolism
i. The liver is a major site of metabolism for cholesterol, apoproteins, and all liporotiens except
chylomicrons
ii. Synthesizes the endogenous lipids and lipoproteins necessary to transport these lipids to tissues
iii. Fatty acids are broken down into series of acetyl CoA units
iv. Hepatocytes catabolize cholesterol into bile acids & their conjugates
v. In liver disease, there is altered lipid or lipoprotein metabolism.
vi. Alcohol is a potent stimulus for hepatic synthesis of triglycerides and apoprotein AI and AII
vii. In NASH (Non-alcoholic steatohepatitis), there is increase triglycerides.
viii. In cirrhosis, lipids and lipoproteins are decreased.
ix. Patients with liver obstruction have abnormal lipoprotein (Lipoprotein X).

d. Ammonia
i. Enzyme ornithine carbomoyltransferease (OCT) is the rate-limiting factor in the urea cycle.
ii. Increase ammonia can result from congenital deficiency of OCT or other urea cycle enzymes;
Reye’s Syndrome or acute hepatic failure where there is severe hepatic dysfunction.
iii. Increased ammonia levels cause accumulation of glutamine in CNS and decreased inhibitor
neurotransmitter γ-aminobutyric acid or GABA, which can cause cerebral dysfunction.
iv. Enzymatic Assays involves Glutamate Dehydrogenase which catalyses the conversion of α-
ketoglutarate and ammonia to form glutamate with oxidation of NADPH to NADP (indicator).
v. Dry Slide Method in alkaline pH buffer can also be used, where NH 4- is converted to NH3
gas observable with indicator-bromphenol blue.

e. Drug Metabolism
i. Drug metabolism involves two phases
a. Phase 1 reaction
 b. Phase 2 reaction
ii. After metabolism, drug is cleared from the system.
iii. Certain drugs are used in the laboratory to evaluate drug metabolism of the liver, such as:
a. Aminopyrine labelled with radioactive Carbon (14C or 13C).
b. Caffeine.
c. Lidocaine

f. Bile Acids
i. Bile acids are derived from cholesterol. These compounds (bile acids) facilitate the absorption of fats
in the intestines.
ii. Primary bile acids are the cholic and chenodeoxycholic acids.
iii. Secondary bile acids arelithocholic, deoxycholic, and ursodeoxycholic acid.
iv. In conditions of impaired hepatic function, total bile acids are increased, but with normal ratio of
primary to secondary bile acids.
v. In cirrhosis, there is a reduced ratio of primary to secondary bile acids.
vi. Cholestatic jaundice causes increased ratio of primary to secondary bile acids because secondary
bile acids are not produced.
vii. Laboratory methods for determination - HPLC

g. Other Syntheses / Transformation

i. Transaminations

B. Synthetic Functions of the Liver

1. Protein Synthesis
a. Almost all proteins are synthesized by the liver except immunoglobulins and von Willebrand’s factor
(vWF).
b. Albumin. Albumin is the major protein synthesized by the liver. Synthesis is affected by :
i. low plasma oncotic pressure, and
ii. cytokines
c. α1-antitrypsin is also synthesized in the liver.
d. Ceruloplasmin (ferroxidase) is also produced in the liver. Ceruloplasmin is a copper containing
protein which is usually decreased in Wilson ’s disease.
e. Plasma clotting factors. Most plasma clotting factors are synthesized by the liver, except for vWF.
For detecting liver-associated coagulation abnormality, the most frequent laboratory test done is
prothrombin time (PT.
f. In severe liver disease, lipids are decreased.
g. In acute hepatic failure, hypoglycaemia is present

C. Excretory Function Test for the Liver

1. Bromsulphalein (BSP) is the most important excretory function test employing an artificial agent.
2. Major excretory function of the liver is the formation and excretion of bile acids
3. Two serum specimens are used
a. fasting serum sample serves as serum reference blank
b. serum collected 45 minutes after IV injection of BSP frequently 5 mL/kg

D. Conjugation-Detoxification Functions of the Liver

1. The liver can convert high concentrations of toxic substances in the circulation into non-toxic substances.
2. Bilirubin produced by catabolism of hemoglobin is converted to a water-soluble form by the liver so it can
be excreted
3. Medications are transformed to less toxic and more readily excreted derivatives

E. Storage Function
1. Primary storage site for glycogen
2. Iron, all fat-soluble vitamins and several water-soluble vitamins; and amino acids and some lipids are
stored in the hepatocytes.

F. Hematologic / Circulatory Functions

1. Certain protein clotting or coagulation factors (fibrinogen, prothrombin, & clotting factors V, VII, IX, X) are
synthesized solely by hepatic cells
2. Regulates blood volume by serving as a blood storage area
G. Protective / Immunologic Functions
1. Phagocytic action and detoxification reactions

BILIRUBIN METABOLISM
@ Metabolic functions of the liver, primarily involve bilirubin metabolism. BILIRUBIN, the principal pigment in
bile, is derived from the breakdown fo haemoglobin when aged rbcs are phagocytised by the RES, primarily in the
spleen, liver, and bone marrow. Bilirubin is a major metabolite of heme. Heme is found in haemoglobin,
myoglobin, and cytochromes.
1. Production of bilirubin is about 200 to 300 mg daily in normal individuals, 85% is derived from
turnover of senescent red blood cells.
2. Pathway for bilirubin production and clearance:
a. Methemoglobin from RBCs in the spleen is split into the heme fraction and free globin chains.
b. The porphyrin rings of heme are oxidized by microsomal heme oxygenase to biliverdin (which
are straight chain compounds) and iron (which is released).
c. Biliverdin is reduced by biliverdin reductase to unconjugated bilirubin / B1. Unconjugated bilirubin
is highly insoluble in water.
d. Free bilirubin and bilirubin carried by albumin, enter the space of Disse of the liver.
e. Bilirubin binds to Y and Z proteins, then to a ligandin. It then enters smooth endoplasmic
reticulum (SER) for conjugation.
f. In the SER, bilirubin is esterified to glucoronic acid by action of uridyldiphosphate-glucuronyl
transferase (UDPGT).
g. Diconjugates, monoconjugates, and triglucoronides of bilirubin are formed.
h. Conjugated bilirubin reaches canalicular membrane and then enters biliary system of the liver.
i. Final destination of conjugated bilirubin is the intestinal tract.
j. In the intestines, conjugated bilirubin is metabolized by bacteria into urobilinogen and converted
to stool pigment (stercobilin). Absence of stool pigment stercobilin causes clay-colored stools (an
early sign of impaired bilirubin metabolism)
k. Urobilinogen is water-soluble and is reabsorbed by enterohepatic circulation. Excess
urobilinogen is excreted in urine (urinary urobilinogen)
3. Clearance of unconjugated bilirubin is around 5 mg/kg/day; thus for a 75 kg male, about 4000
mg/day of unconjugated bilirubin is cleared.
4. Half-life of unconjugated bilirubin is very short.
5. Conjugated bilirubin has a half-life of < 24 hrs. When conjugated bilirubin is present in serum , It can
be bound to albumin; thus producing biliprotein or δ-bilirubin or delta bilirubin.
6. For bilirubin to be excreted by the liver, bilirubin must be in the conjugated form; that is, the water-
soluble diglucoronide.
7. Almost all the bilirubin formed is eliminated in the feces, and a small amount of the colorless product
urobilinogen is excreted in the urine.
8. When bilirubin concentration in the blood rises, the pigment begins to be deposited in the sclera of
the eyes and in the skin. The yellowish pigmentation in the skin or sclera is known as JAUNDICE or
ICTERUS.
9. Increase plasma levels of bilirubin are present in haemolytic anemia, decreased plasma clearance,
hepatitis, and cirrhosis.

Types of Bilirubin
1. Unconjugated bilirubin / Bilirubin 1 (other names)
a. Water insoluble / non-polar bilirubin
b. Indirect / indirect reacting bilirubin
c. Hemobilirubin
d. Free bilirubin / unbound bilirubin
e. Prehepatic bilirubin

2. Conjugated bilirubin / Bilirubin 2 (other names)

a. Water soluble / polar bilirubin
b. Direct / direct reacting bilirubin
c. Cholebilirubin / cholestatic bilirubin
d. One minute bilirubin
e. “Prompt” bilirubin
f. Post hepatic bilirubin
g. Obstructive bilirubin
h. Regurgitative bilirubin
CLINICAL SIGNIFICANCE OF ALTERED BILIRUBIN LEVELS
I. Bilirubin in serum can be elevated for a variety of reasons:
1. excessive load of bilirubin presented to liver
2. decreased conversion of unconjugated to conjugated forms of bilirubin
3. impairment of bilirubin excretion

II. Elevated unconjugated bilirubin (B1):

a. hemolytic anemias
b. newborns

III. Elevated conjugated bilirubin (B2):

a. bile duct obstruction
b. hepatitis
c. medications

IV. Elevated B1 & B2

V. Jaundice or Icterus
A. Jaundice is a yellow discoloration of the sclera, skin and mucous membrane caused by excessive amount
of bilirubin.
B. Hyperbilirubinemia is caused by
1. Increased breakdown of cells
2. Impaired liver cell uptake
3. Impaired conjugation
4. Impaired excretion
5. Obstruction to the flow of bile
C. The common types of jaundice/hyperbiilrubinemia are
1. Prehepatic jaundice / hemolytic jaundice
= excessive production of bilirubin due to excessive rbc destruction
Causes: a. Hemolytic anemias
b. Hemolytic disease of the newborn (HDN)
c. Parasitism (malaria)
d. Extensive hematoma
e. Hemolytic transfusion reactions

@ Disturbances in Excretion of Bilirubin (cholestasis)

= the elevated concentration of serum bilirubin consists of both the unconjugated & the conjugated,
because bilirubin can enter the hepatic cell & become esterified/conjugated
= the various types of disorders responsible for faulty or limited excretion of bilirubin by the liver may be
divided into 2 groups:

2. Hepatic jaundice / hepatocellular jaundice

= may result from impaired cellular uptake, defective conjugation, or abnormal secretion of
bilirubin by the liver cell
= implies damage to hepatocytes where both types of bilirubin is increased
= all intrahepatic problems must be treated by medical management; surgery would harm the
patient
Causes:
a. Retention jaundice or inability to conjugate as seen in:
i. Physiologic jaundice of the newborn
 the UDPG transferase enzyme system responsible for converting bilirubin into a
glucoronide ester is not fully developed at birth
 serum bilirubin may rise as high as 8 mg/dl (137 umol/L) in normal full-term
infants by the 3rd –6th day of life

ii. Hemolytic Disease of the Newborn (HDN) or Isoimmune Hemolytic Disease (IHD)
 caused by an Rh, ABO, or other blood system incompatibility between mother &
fetus (Rh- mother & Rh+ baby / type O mother & type A, B, or AB baby); the Rh
incompatibility is more severe than the ABO incompatibility
 the hemolytic process may continue for several weeks after birth bec many rbc
are coated with IgG Abs before delivery, so the hemolysis produces a large
amount of bilirubin for excretion by an immature liver; the load is too large to
 handle effectively, so the plasma bilirubin concentration rises rapidly & the
jaundice may be severe & may lead to mental retardation

iii. Gilbert’s Syndrome

 is a relatively common disorder characterized by impaired cellular uptake of
bilirubin
 the unconjugated bilirubin level is < 3 mg/100 mL
 Total bilirubin is about 2-3 mg/dl, seldom exceeding 5 mg/dl in patients with
Gilbert’s syndrome.
 Laboratory investigation includes obtaining bilirubin levels in fasting state and
with use of phenobarbital.

iv. Crigler-Najjar syndrome, Types I and II

 is a more serious disorder caused by a severe deficiency of uridyldiphosphate-
glucuronyl transferase (UDPGT)
 the syndrome is of two types:

“Crigler-Najjar Syndrome”
Type 1 Type 2
> No enzyme activity in the liver (since there is a > Less severe form since there is
complete absence of the enzyme in the liver less severe deficiency of the
> Is rare and is uniformly fatal enzyme
> No conjugated bilirubin is formed, and the bile is > 10 %Enzyme activity
colorless > Some conjugated bilirubin is formed
> Affected infants develop severe unconjugated
hyperbilirubinemia leading to kernicterus
(deposition of bilirubin in brain affecting
basal ganglia)

b. Hepatocyte injury (hepatocellular) as seen in:

i. viral hepatitis - at least 5 (HAV, HBV, HCV, HDV, HEV) different viruses are recognized
as specific agents for causing hepatitis
ii. cirrhosis and alcoholic hepatitis
iii. toxic liver injury
iv. parasitism (Fasciola hepatica)

c. Impaired excretion of products from the hepatocytes as seen:

i. Dubin-Johnson Syndrome
 a congenital disorder in the transport system for excreting the bilirubin diglucuronide
into the bile canaliculi due to blockade of the excretion of bilirubin into the canaliculi

ii. Rotor’s syndrome

 this condition is usually of viral origin; it is characterized by impaired excretion of
conjugated bilirubin.

iii. viral hepatitis

iv. cirrhosis

3. Post-hepatic / Obstructive jaundice

= failure of bile to flow due to obstruction of the biliary tree
= these disorders include all types of extrahepatic cholestasis or obstruction of the flow of bile
into the intestine
= obstruction caused by stones, benign tumors and strictures are definitely cured by surgery
Causes:
a. Gallstone or a tumor
b. Choledocholethiasis
c. Cholelithiasis
d. strictures and spasms of the common bile duct caused by bacteria
e. Pancreatic carcinoma
f. Parasitism (Ascariasis)
 Pre-hepatic / Hepatic / Post-hepatic /
Hemolytic Hepatocellula Obstructive
r
Direct bilirubin (B2) 15% or less More than 50% More than 50%
Indirect bilirubin (B1) 85% or more Less than 50% Less than 50%
Urine bilirubin Normal Increased Increased
Urine urobilinogen Increased Decreased Decreased to absent
Stool urobilinogen Increased Decreased Decreased to absent

VI. Disorders of bilirubin metabolism.

1. Congenital disorders:
a. Gilbert’s Syndrome
b. Crigler – Najjar Syndrome
c. Dubin-Johnson Syndrome
d. Rotor Syndrome
2. Acquired disorders:
a. Severe hemolysis
b. Septicemia
c. Total parenteral nutrition
d. Drugs such as androgens
e. Fasting

METHODOLOGIES OF BILIRUBIN ANALYSIS

SPECIMEN COLLECTION AND STORAGE:

 A fasting serum specimen, which is neither hemolyzed nor lipemic in nature is preferred.
 Samples for bilirubin analysis should be kept in dim light or in the dark and measured as soon as possible
(within 2-3 hours) after collection
 Serum may be stored in the dark in the refrigerator for up to 1 week and in the freezer for 3 months
without appreciable change in the bilirubin concentration

BACKGROUND:
 Laboratory test for bilirubin utilizes diazotized sulfanilic acid; the reaction product is measured at 540 nm.
Since unconjugated bilirubin reacts slowly, accelerants are added like caffeine or methanol.
 The conjugated bilirubin (water soluble and containing 1 or 2 attached glucuronic acid molecules) reacts
directly with the color reagent or the fraction that produced a color in aqueous solution is referred to as
DIRECT BILIRUBIN
 Unconjugated bilirubin (water-insoluble and nonconvalently attached to albumin) does not react with the
color reagent until the bilirubin is first dissociated from the protein, or the fraction that produced a color
only after alcohol was added is referred to as INDIRECT BILIRUBIN

GENERAL REACTION: Van den Bergh Reaction

Principle: Involves the diazotization of bilirubin with diazotized sulfanilic acid to form pink to purple
azobilirubin which is measured spectrophotometrically.

1. Evelyn-Malloy Method
Principle: Bilirubin is coupled with diazotized sulfanilic acid (Ehrlich ’s Diazo reagent) forming a pink to
purple azobilirubin.

The bilirubin that has reacted with the Diazo reagent after standing for 1 minute is the prompt or
conjugated bilirubin/direct bilirubin. Fifteen minutes after the addition of methanol (coupling
accelerator) TOTAL BILIRUBIN is measured. The difference between the total and the direct is
the INDIRECT BILIRUBIN.
Ehrlich’s reagent: Diazo A = 0.1% sulfanilic acid + HCl
Diazo B = 0.5% sodium nitrite
Diazo Blank = 1.5% HCl

2. Jendrassik-Grof Method
Principle: Plasma or serum is added to a solution of sodium acetate (buffer) and caffeine-sodium benzoate
(coupling accelerator), which is then added to diazotized sulfanilic acid to form purple azobilirubin.
The sodium acetate buffers the pH of the diazotization reaction, whereas the caffeine-sodium
 benzoate accelerates the coupling of bilirubin with diazotized sulfanilic acid. The diazotization is
terminated by the addition of ascorbic acid, which destroys the excess diazo reagent. With the
use of a strong alkaline tartrate solution, the purple azobilirubin is then converted to a blue
azobilirubin and the intensity of the color is read at 600 nm,

 Advantages of Jendrassik-Grof method over Evelyn-Malloy:

a. Is insensitive to sample pH changes
b. Is insensitive to a 50-fold variation in protein concentration of the sample
c. Has adequate optical sensitivity, even for low bilirubin concentrations
d. Has minimal turbidity and a relatively constant serum blank
e. Is not affected by hemoglobin up to 750 mg/dL

3. Icterus Index = this is a measure of the amount or degree of icteresia or yellowishness of serum or plasma in
cases of jaundice

Principle: Serum or plasma is diluted with NSS (normal saline solution ) sodium citrate solution until the
color of the specimen matches with that of a reference standard. The standard used is a 0.01%
potassium dichromate. The number of times the serum must be diluted is called the ICTERUS
INDEX

4. Other methods of determination:

a. high-pressure liquid chromatography (HPLC)
b. enzymatic methods
c. direct spectrophotometry - is a satisfactory method for evaluation of jaundice in newborns

REFERENCE RANGES FOR BILIRUBIN:

Conjugated / Direct bilirubin = 0-0.2 mg/dL (0-3 umol/L)
Unconjugated / Indirect bilirubin = 0.2-0.8 mg/dL (3-14 umol/L)
Total bilirubin = 0.2-1.0 mg/dL (3-17 umol/L)

REFERENCE RANGES FOR INFANT TOTAL BILIRUBIN:

Infants Premature, Total Full Term, Total
Conjugated / Direct bilirubin = 24 hours 1-6 mg/dL 2-6 mg/dL
Unconjugated / Indirect bilirubin = 48 hours 6-8 mg/dL 6-7 mg/dL
Total bilirubin = 3-5 days 10-12 mg/dL 4-6 mg/dL

TESTS FOR DISCLOSING HEPATIC DYSFUNCTION / ASSESSMENT OF LIVER FUNCTION

= the reasons for requesting liver function tests are threefold:
a. for diagnosis
b. for differentiation
c. for prognosis

1. Serum Bilirubin
= precautions:
a. when performing bilirubin tests on patients with hemolytic disease of the newborn, a control serum or
standard with a concentration close to 20 mg/dL (340 umol/L) should be used because this is near
the critical concentration at which decisions are made concerning exchange transfusions
b. bilirubin is altered by ultraviolet light in such a way that it is no longer able to form azobilirubin ;
serum samples and standards must be protected from direct sunlight or ultraviolet light
= increased concentration: total bilirubin = chronic hemolytic disease
hepatocellular disease
cholestasis
esterified bilirubin = hepatocellular disease
cholestasis
nonesterified bilirubin = hemolytic disease (HDN)
hepatocellular disease
cholestasis
 = decreased concentration: no clinical significance
2. Urine Bilirubin
= when present, urinary bilirubin indicates some pathologic condition of the liver or biliary system
= method of determination:
a. by tablet – Ictotest tablets
- (+), if bilirubin is present, a blue to purple color forms in the mat with in 30 sec
- (-), red or orange color, meaning a urinary compound other than bilirubin has been
converted to an azo dye
b. by Dipstick – multitest dipsticks

@ Urobilinogen
= most quantitative methods for urobilinogen are based on the reaction of this substance with p-dimethyl-
aminobenzaldehyde to form a red color
= Increased urinary urobilinogen: hemolytic disease
defective liver-cell function
= absence urinary and fecal urobilinogen: complete biliary obstruction
Hepatocellular disease
= decreased fecal urobilinogen: hepatocellular disease

3. Urine Urobilinogen
= method of determination:
a. dipstick = impregnated with p-dimethyl aminobenzaldehyde and an acid buffer turns red in the
presence of urobilinogen; comparison to a color chart if (+) / (-)
b. spectrophotometer
= Principle: Urobilinogen reacts with p-dimethyl aminobenzaldehyde (Ehrlich ’s reagent) to form
a red color, which is then measured spectrophotometrically. Ascorbic acid is added as a
reducing agent to maintain urobilinogen in the reduced state. The used of saturated
sodium acetate stops the reaction and minimizes the combination of other chromogens
with the Ehrlich’s reagent
= Specimen: A FRESH 2-hour urine collection and be kept cool and protected from light
= Comments and Sources of Error:
a. results reported in Ehrlich units rather than in milligram
b. bilirubin will form a green color (removed)
c. fresh urine is necessary, and test must be performed without and the
spectrophotometric
readings should be made within 5 minutes after color production
= Reference Range: urine urobilinogen: 0.1 – 1.0 Ehrlich units / 2 hr or
0.54.0 Ehrlich units/day (0.86.0 mmol/day)
(1 Ehrlich unit = 1 mg of urobilinogen)
= increased excretion: hemolytic disease
hepatocellular liver disease
congestive heart failure

4. Fecal Urobilinogen
= the original source of the brownish fecal pigment is the bilirubin that undergoes bacterial action in the
gut
= determination is by visual inspection; stools become pale or clay-colored with decreasing amounts of
pigment
= semiquantitative determination involves same principle described in urine urobiliniogen; it is carried out
in an aqueous extract of fresh feces, and any urobilin present is reduced to urobilinogen by treatment
with alkaline ferrous hydroxide before Ehrlich’s reagent is added
= reference range: 75-275 Ehrlich units / 100g of fresh feces or
75-400 Ehrlich units per 24-hour specimen
= decreased: hepatocellular disease
obstructions of the biliary tree

5. Serum Bile Acids Measurement

= these involve extraction with organic solvents, partition chromatography, gas chromatography – mass
spectroscopy, spectrophotometry, ultraviolet light absorption, fluorescence, radioimmunoassay, and
enzyme immunoassay methods
 = increased: liver disease

6. Tests Based on Abnormalities of Serum Proteins / Tests Measuring Hepatic Synthetic Ability
= protein analyses that provide useful information concerning liver disease:
a. albumin
b. -globulin
c. -globulins
d. clotting factors
e. -fetoprotein
i. AFP is one of the major plasma proteins synthesized during fetal life.
ii. It is structurally similar to albumin
iii. AFP levels fall throughout gestation, from 10,000 ng/ml at birth to 10 ng/ml at one year of age.
iv. In disease, such as acute hepatic injury, AFP is increased 10-20 times the upper limit.
v. In chronic viral hepatitis, there is minimal increase, slightly over 10% increase.
vi. In chronic liver disease (as fibrosis progresses), AFP exceeds 2-3 times the upper limit.
vii. Laboratory testing for AFP is done for screening and diagnosis of hepatocellular carcinoma
and for monitoring the course of disease in hepatocellular carcinoma and hepatoblastoma.

7. Tests of Liver Injury

A. Tests for Plasma Enzyme Levels
1. Those enzymes that have been used often are the following:
a. aspartate aminotransferase (AST) or serum glutamic-oxaloacetic transaminase (SGOT)
b. alanine aminotransferase (ALT) or serum glutamic-pyruvic transaminase (SGPT)
c. alkaline phosphatise (ALP)
d. γ-glutamyltransferase (GGT)

2. Cellular locations of enzymes

Cellular location of Enzymes Remarks

the Enzymes
Cytoplasmic Enzymes LDH These enzymes are released in membrane injury
AST
ALT
ASTc
Mitochondrial Enzymes Mitochondrial These enzymes are released with mitochondrial damage
isoenzyme of AST
Canalicular Enzymes ALP These enzymes are increased in obstructive processes
GGT

3. Mechanisms of Enzyme Release

a. Enzyme release is mainly due to irreversible cell membrane damage causing leakage to
cytoplasmic enzymes.
b. In cases of alcohol abuse, alcohol rapidly causes release of mitochondrial AST and expression of
the enzyme on cell surface.
c. Some medications (ethanol, phenytoin) can induce microsomal enzyme synthesis such as GGT,
and to lesser extent ALP.

4. Half-life of enzymes:
Cytoplasmic AST 17+ 3 hours
 Cytoplasmic ALT 42 + 11 hours
Mitochondrial AST 87 hours
LDH (LDH 4 & 5) 4 – 6 hours

5. Enzymes are evaluated by differing hepatic activity level and half-life. For instance:
a. In acute hapatocellular injury
* AST increases first, followed by ALT initially, because there is higher activity of AST in
hepatocytes
* Later on, within 24 to 48 hrs of ongoing damage; ALT will be higher than AST because ALT
has longer half-life than AST
* LDH transiently increased (although to a lesser degree than AST and ALT) and return to
normal by time clinical manifestation of the disease is observed.
b. In acute alcoholic liver damage: increase in AST persists
c. In chronic hepatocyte infection: ALT is more commonly elevated than AST; as fibrosis progresses,
ALT transiently declines, so that AST to ALT ratio increases. In cirrhosis, AST higher than ALT.

7. Some enzymes primarily reflect canalicular injury. GGT is slightly more sensitive than ALP for
canalicular injury, although less specific because it is increased in 80-95% of patients with liver
disease regardless of etiology. GGT is higher in obstructive disease and with space occupying lesions
of the liver.
.

8. Half-life of canalicular enzymes: ALP 3 days

GG 10 days
T

B. Selected Enzyme Tests

a. Alkaline Phosphatase (ALP)
= is widely distributed in many tissues including the osteoblasts (the bone-building cells), the
cells lining the sinusoids, and bile canaliculi in the liver
= NV: 20-105 U/L
= increased: hepatocellular disease
** cholestasis
carcinoma
amebic abscess
amyloidosis
granulomatous lesions
tuberculosis of the liver
** bile duct obstruction
pregnancy

b. Aminotransferases / transaminases (AST/GOT and ALT/GPT)

= NV: AST = 6-25 U/L
ALT = 3-30 U/L
= increased: severe viral hepatitis
acute toxicity with drug that severely damages liver tissue
= moderate elevation: cholestasis, cirrhosis
hepatic tumors
infections
infectious mononucleosis (IM)
= the rise in viral hepatitis begins early in the disease, frequently before jaundice is visible and
reaches a maximum during the acute stage of the disease when the destruction of hepatic cells
is at height
= the peak rise in aminotransferase after acute chemical toxicity, such as ingestion of carbon
tetrachloride in a suicide attempt may occur within 24 hr and decline rapidly by the second day

C. Less Common Enzyme Tests for Special Situations

a. -Glutamyltransferase (GGT)
= GGT transfers the -glutamyl portion of a peptide to another peptide, amino acid, or water
= located primarily on cell membranes and may assist in amino acid transport into cells
= present in relatively high concentration in kidney, pancreas, liver, and prostate
= NV: 3-35 U/L for men
 3-30 U/L for women
= increased: hepatocellular and obstructive liver diseases
= GGT is considered the enzyme of choice in investigating possible alcoholism

b. 5’-Nucleotidase (5NT)
= is a confirmatory test:
liver disease bone disease
 ALP increased increased
 5NT increased normal or slightly increased
= specifically hydrolyzes the phosphate from 5’-adenosine mononucleotide (AMP)
= present as a microsomal enzyme in liver
= NV: 1-7 U/L
= increased: hepatobiliary disease / hepatic disorders

c. Lactate dehydrogenase (LDH

= jaundice: elevated LD-5
= acute viral hepatitis and cirrhosis: moderate elevations of total serum LD
= biliary tract disease: slight elevations of total serum LD
= metastatic carcinoma of the liver: high serum levels of LD

d. Cholinesterase = decreased in liver diseases

HEPATIC ENZYMES
Enzymes
Alkaline phosphatise (ALP)
Aminotransferases
Gamma-glutamyltransferase
Cholinesterase
Lactate dehydrogenase (LD)
Clinical Utility in Liver Disorders
Elevated primarily in obstructive processes
Elevated in variety of liver diseases; LAT is more sensitive indicator
Some increase in liver diseases; sensitive indicator of ethanol intake
Normally quite high; values decrease in liver disorders
Elevated in wide variety of situations; clinical utility low in absence of isoenzyme
studies

8. Test for Autoimmune liver disease

a. PBC (primary biliary cirrhosis) is the most common autoimmune liver disease.
b. PSC (primary sclerosing cholangitis) is another autoimmune disease associated with liver dysfunction.

9. Markers of Hepatic Fibrogenesis

a. The process of fibrosis in the liver involves both production of collagen and breakdown of normal matrix
proteins.
b. Serum markers:
i. circulating fragments of collagen and procollagen
ii. enzymes involved in collagen synthesis (prolyl oxidase, lysl oxidase)
iii. matrix glycoproteins (laminin, fibronectin, elastin)
iv. proteoglycans (involved in matrix formation such as hyaluronate)
v. matrix breakdown products (metalloproteins)
vi. ALT and prothrombin time (but slight involvement only)

10. Dye Tests of Excretory Function

= indocyanine green (ICG)
= performed in patient who is not jaundiced but liver disease is suspected

11. Hippuric Acid Synthesis Test

= Principle: Benzoic acid which is given orally or intravenously in the form of sodium benzoate is
conjugated with glycine to form hippuric acid which is readily excreted by the kidneys.
= Procedure:
o 6 gms sodium benzoate dissolved in about 250 mL water
o Collect all urine specimen passed during the next 4 hours together with the urine obtained by
emptying the bladder at the end of the period
 o In the analysis of hippuric acid, the urine is saturated with sodium chloride, acidified with
concentrated sulfuric acid and cooled to precipitate out the hippuric acid that is only slightly
soluble
o The precipitated hippuric acid is centrifuged, washed and titrated with 0.1 NaOH, using
phenolphthalein as an indicator
= Reference Range: 3.0-3.5 gm excreted in 4 hours

12. Vitamin K Response Test

= Interpretation:
o In bleeding patients with post-hepatic jaundice (due to biliary obstruction), the prothrombin
time (Protime) is within normal limits after the parenteral administration of Vitamin K
o In bleeding patients with hepatic jaundice (due to severe destruction of liver cells), the
Prothrombin time (Protime) is prolonged even after the parenteral administratin of Vitamin K

13. Ammonia Determination / Tests Measuring Nitrogen Metabolism

AMMONIA AND THE LIVER

Source of Plasma Ammonia
= most ammonia in the circulation derives from 2 sources:
 breakdown of amino acids obtained from dietary protein in the intestine
 hydrolysis of urea, an end product of metabolism
= in the severe liver disease, toxic ammonia accumulate and reaches the systemic circulation which is
then converted to glutamine in the brain; increased synthesis of glutamine will compromise the Kreb ’s
cycle leading to coma due to lack of ATP for the brain

a. Analysis of Plasma Ammonia

= methods for determination:
o Enzymatic Assays involves Glutamate Dehydrogenase which catalyses the conversion of α-
ketoglutarate and ammonia to form glutamate with oxidation of NADPH to NADP (indicator).
o Dry Slide Method in alkaline pH buffer can also be used, where NH 4- is converted to NH3
gas observable with indicator-bromphenol blue.

b. Clinical Situations
= increased: hepatic encephalopathy
renal failure
pulmonary problems
Reye’ s syndrome
 it’s a form of hepatic destruction that usually occurs fllowing recovery form a
viral infection, such as varicella or influenza
 it has been related to aspirin therapy
 hallmark of the disorder is an increased plasma ammonia level;
 liver functions are always abnormal, but the bilirubin level is not usually
elevated

HEPATIC DYSFUNCTION / DISORDERS OF THE LIVER

1. Hepatitis

i. VIRAL HEPATITIS – at least 5 (HAV, HBV, HCV, HDV, HEV)different viruses are recognized as specific
agents for causing hepatitis

HEPATITIS NON-A, NON-B

Characteristics Hepatitis A Hepatitis B Hepatitis C Hepatitis Hepatitis
D E
Nucleotide RNA DNA RNA RNA RNA
Other names Infectious hepatitis /
Short-incubation hepatitis
Principal Mode Enteric / Fecal-oral Parenteral, Parenteral, Parenteral, Enteric /
of Transmission (contaminated food & Sexual, Sexual Fecal-oral
water) perinatal
Incubation Period 2-6 weeks 8-26 weeks 2-15 weeks ------- 3-6 weeks
Severity Mild Severe Moderate to Very Mild
severe severe
Carrier state none sometimes usually frequently None
Vaccine yes yes no yes No
Chronic Infection no yes yes yes ????
Serologic Dx yes yes yes yes yes
Available
* Hepatitis D occurs only in combination with hepatitis B; it requires the viral coat of hepatitis B

ii. ACUTE HEPATITIS

1. Hallmark of acute hepatic injury is elevation of aminotransferase levels which may be more than 8
times, up to 40 times reference limits; with nor or minimal elevation of ALP.
2. Most common form of damage to hepatocytes are primarily related to immune response (viral or
drug induced injury). In viral or drug induced toxicity, there is:
a. Gradual rise of cytoplasmic enzymes (AST and ALT)
b. Increase in bilirubin
c. Normal or slightly elevated ALP
d. Increased GGT
e. Bilirubin <15 mg/dl, with 50-80% of which is direct (conjugated and delta bilirubin)
f. Normal or increased prothrombin time >3 seconds upper limit of the normal
3. Less common cause for acute hepatic injury is due to direct damage. In toxic and ischemic hepatic
injury:
a. AST and ALT are markedly increase, >100 times upper reference limits.
b. Bilirubin rises minimally
c. Prothrombin time is >4 seconds
4. In cases of alcoholic liver injury:
a. AST and ALT are elevated minimally
b. AST increased 8 times upper limit
c. ALT increased 4 time upper limit
5. Patients with massive hepatic injury (massive hepatic necrosis) have:
a. High AST and ALT levels
b. Bilirubin is increase (predominantly unconjugated bilirubin)
c. Prothrombin time is increased
d. Ammonia level is abnormal (wherein signs of hepatic encephalopathy develop)
6. There are several causes of acute hepatocellular injury.
a. Viral infection
b. Alcoholic hepatitis
c. Toxic and ischemic hepatocellular injuries
7. There are markers of severity of injury such as prothrombin time, which is the best predictor of
prognosis in acute hepatocellular injury. Marked increase in PT > 4 seconds above reference limit
means increased risk of hepatic failure.
8. Dramatic symptoms of Acute Liver Disease and their Causes:

Symptom Cause
Clay colored stool Decreased biliary clearance of bilirubin into the intestine
Dark-colored Water soluble conjugated bilirubin in plasma appearing in urine (bilirubinuria)
urine
Jaundice (yellow Acute hepatitis or biliary tract obstruction
Discoloration of
eyes and skin)

III. CHRONIC HEPATITIS

1. Chronic hepatitis is a major predisposing factor for cirrhosis and hepatocellular carcinoma (two
leading cause of death for liver diseases)
2. Causes of Chronic Hepatitis
a. Chronic viral infections such as HBV and HCV can cause chronic hepatitis
 b. A number of other disorders can lead to chronic liver injury such as:
i. Hemochromatosis

ii. Wilson’s disease

* The gene involved in copper handling is impaired, leading to defective incorporation of copper
to ceruloplasmin
* Final result is excessive copper deposition in tissues
* Clinical manifestations include psychiatric or neurologic finding or liver disease
* Most widely used test is ceruloplasmin, which in this case is low
* Other findings: serum copper is consequently reduced, and urinary copper is increased

iii. Another cause of chronic hepatitis is autoimmune hepatitis

iv. Other causes of chronic hepatitis are:

* drug-directed toxicity such as from sulphonamides
* abnormal drug metabolism such as isoniazid
* idiosyncratic, hypersentivity reactions
* NASH (non-alcoholic steatohepatitis)
* fatty liver
* deficiency of α1 anti-trypsin (risk factor)

c. Laboratory recognition of chronic hepatocellular injury includes:

i. increased cytoplasmic enzymes
ii. ALT increase to about 1 to 4 times upper limit
iii. AST is increased to a lesser extent

2. Alcoholic Liver Disease – acetaldehyde, the metabolite formed from ethanol catabolism
- injury is from fatty infiltration to fibrosis to cirrhosis and death

3. Hepatic Drug Toxicity – some drugs (acetaminophen, isoniazid, tetracyclines, chlorpromazine, thiazides,
prophylthiouracil, etc.); tranquilizers such as phenothiazines, certain antibiotics, antineoplastic agents
and anti-inflammatory drugs are toxic to hepatic cells (liver injury) and induce various injuries that may
range from cholestasis to cell injury of organelles and even cause cell necrosis

4. Cholestasis – is the obstruction or stagnation of biliary flow that causes disturbances in bilirubin excretion
- cholesterol is the principal constituent in 80% of gallstones
- cholestatic or Obstructive Jaundice is another form of acute liver disease
A. Obstruction of drainage through the biliary tree is a common cause of acute developing jaundice
(cholestatic jaundice). Obstruction may either be of the common bile doctor both hepatic ducts.
B. Laboratory features of cholestasis include:
i. Increases in canalicular enzymes (ALP and GGT) up to 10 to 20 times reference limits or higher
ii. Increase in conjugated bilirubin
iii. Increase of cholesterol
iv. Increase bile acids
C. Causes of Cholestasis
i. Defects in the energy dependent excretion of compounds from hepatocytes into canaliculi cause
increased conjugated bilirubin or bilirubinostasis
ii. Damage to hepatocytes causes functional or mechanical obstruction of canaliculi
iii. Drug reactions is most commonly seen with cholestatic hepatitis
iv. Α-1-antitrypsin deficiency causes cholestasis among infant
v. Mechanical obstruction of extrahepatic bile ducts because of tumors near the ampulla or Vater or
due to gallstones
vi. Others that cause damage to intrahepatic bile ducts; Primary Biliary Cirrhosis, Primary Sclerosing
Cholangitis , Sarcoidosis
D. Differential Diagnosis of Cholestasis

Condition Jaundice Cytoplasmic Enzymes Canalicular Enzymes

Hepatocellular present Moderately to markedly Normal or minimally increased
injury elevated
 Obstructive present Normal or minimally increased Moderately to markedly
jaundice elevated

5. Hepatic Encephalopathy (hepatic coma) –

- ammonia per se is a toxic material that causes an encephalopathy or coma if its concentration in the
brain and CSF becomes sufficiently high especially in cases of fulminant hepatitis or in cirrhosis of the
liver

6. Jaundice
- except in infants, hyperbilirubinemia is generally well tolerated and does not produce serious clinical
adverse effects; however, in infants, hyperbilirubinemia (levels exceeding 15-20 mg/dL) may be
associated with kernicterus, a serious disorder of the central nervous system resulting from increased
bilirubin levels
- hypercarotenemia, a disorder caused by the excessive ingestion of vitamin A

7. Cirrhosis
- one way to classify cirrhosis is by the appearance of the liver, that is by the size of the nodules; this
conditions are referred to as macronodular and micronodular cirrhosis, although mixed forms occur
- the leading cause of cirrhosis is alcohol abuse, which leads to a micronodular type of cirrhosis
- other cause of cirrhosis include hemochromatosis, postnecrotic cirrhosis (which occurs as a late
consequence of hepatitis), and primary biliary cirrhosis (which is an autoimmune disorder)
- portal hypertension results when blood flow through the portal vein is obstructed by the cirrhotic liver;
this may result to splenomegaly and esophageal varices which may rupture and lead to fatal hemorrhage
- the synthetic ability of the liver is reduced, causing hyperalbuminemia and deficiency of the clotting
factors which may lead to hemorrhage
- ascitic fluid may accumulate in the abdomen

@ Development of cirrhosis:
a. Early stage: compensated cirrhosis where impair of hepatic function is minimal
b. As fibrosis progresses the resistance to portal venous flow is increased causing portal hypertension
c. Later on, there arises impaired hepatic synthetic and metabolic functions
d. In late stages, complications such as hepatic encephalopathy, hypoxemia, severe ascites, and
hepatorenal syndrome arises

@ Cirrhosis can be recognized in the laboratory by the following:

a. ALT is decreased, while AST remains the same; AST to ALT ratio is > 1.
b. Prothrombin time is prolonged
c. Liver production of protein is low, with markedly decreased albumin
d. There is a polyclonal increase of IgG and IgA and bridging of β and γ regions in serum protein
electrophoresis
e. Cholesterol is at baseline then becomes absolutely decreased
f. BUN is also decreased
g. Total bilirubin is increased; with direct bilirubin accounting for < 50%. (jaundice develops)
h. Ammonia level is chronically increased
i. Platelet count is mildly to moderately decreased

8. Tumor – the primary malignant tumors of the liver, known as hepatocellular carcinoma (HCC),
hepatocarcinoma, or hepatoma
- screening includes evaluating AFP levels (α-fetoprotein) with or without ultrasound of the liver
- malignant tumor in the liver is a serious finding with poor prognosis

9. Space-Occupying lesions
- For local cholestasis, increase in canalicular enzymes are primarily reflected by increase in GGT
- ALP isoenzyme are bound to fragments of canalicular membrane and are also increased in this
condition
- Lipoprotein-X is present.
- Direct bilirubin is also increased
 - Prothrombin time is normal

REFERENCE INTERVALS FOR LIVER FUNCTION TESTS

Laboratory Tests Reference Interval

Albumin 3.8 -5.0 g/dL (38 – 50 g/L
Ammonia < 120 ug/dL (67 umol/L)
Bile acids 0.3 – 3.0 mg/dL (3.0 – 30 mg/L)
Bilirubin
Direct < 0.3 mg/dL (< 5 umol/L)
Indirect 0.1 – 1.0 mg/dL (2 – 17 umol/L)
Total O.1 – 1.2 mg/dL (2 – 21 umol/L)
Ceruloplasmin 23 – 50 mg/dL (230 – 500 mg/L)
Prothrombin Time 11 – 15 seconds
Liver Enzymes c/o next topic

CHANGES IN SERUM PARAMETERS IN LIVER DISEASE

Change in Liver Disease Other Possible Cause

Bilirubin Hemolysis
Hereditary erythrocyte enzymopathies
Alkaline Phosphatase (ALP-increase) Bone cancer
Pregnancy
Muscle damage
Cancer
Pancreatitis
Renal disease
AST (increase) Myocardial Infarction
Skeletal muscle damage
Cholinesterase (decrease) Inhibition by pesticides
Lactate dehydrogenase (LD- Myocardial infarction
increase) Skeletal muscle damage
Renal disease
Cancer
 II. CLINICAL ENZYMOLOGY

1. ENZYME
1.1 Definition
A. Enzymes are protein catalyst that hasten a chemical reaction without themselves being consumed or
undergoing a chemical change.

1.2 Characteristic of Enzymes

A. Enzymes have high specificity for their substrate
B. As catalysts, enzymes lower the energy of activation of chemical reactions.
C. They are present in trace amounts in ECF and are subject to saturation
D. Enzymes tertiary structure is important to its normal function, a change in temperature, ionic strength,
and pH may alter enzymes tertiary structure, hence, its ability to serve as a catalyst.
E. They are found in high concentration within the cells

1.3 Nomenclature
I. According to the name of the substrate with the addition of the suffix “ase”.

II. According to the type of reaction they catalyzed.

Examples:
A. transfer of amino group from stubstrate to another = transferase
B. transfer to phosphate group from a high energy phosphate compound to its substrate =
kinase
C. effect of hydrolysis on phosphate esters = phosphatise
D. removal of hydrogen atoms from its substrate = dehydrogenase

III. According to the numerical designation given by the Enzyme Commission (EC)
A. It is standardized by (EC) Enzyme Commission of the International Union of Biochemistry.
Example:
i. the IUB nomenclature for Acid Phosphatase (ACP), for example, is EC 3.1.2.3;
ii. for Creatine Kinase (CK) = EC 2.7.3.2.
iii. EC 1.1.1.27 = for lactate dehydrogenase
iv. EC 3.2.1.1 = for amylase
v. EC 2.6.1.2 = for alanine aminotransferase

B. The numerical designation for each isoenzyme consists of four separate numbers separated by
periods. EC stands for Enzyme Commission. The first number defines the class (one to six
reactions) to which the enzyme belongs, whereas the next two numbers indicate the subclass and
sub-subclass to which the enzyme is assigned. A specific serial number is the last number given to
each enzyme in its sub-subclass.

C. The isoenzyme migrating farthest towards the anode in electrophoretic migration is designated
isoenzyme one (1).

1.4 Classification
I. Enzymes are classified as
 A. Oxidoreductases
Examples:
i. oxidase = cytochrome oxidase
ii. dehydrogenase = lactate dehydrogenase (LDH)
= malate dehydrogenase (MDH)
= isocitrate dehydrogenase (ICD)
= glucose-6-phosphate dehydrogenase (G-6-PD)

B. Transferases
= Examples:
> aspartate aminotransferase (AST) or glutamate oxaloacetate transferase (GOT)
> alanine aminotransferase (ALT) or glutamate pyruvate transaminase (GPT)
> creatine kinase (CK) or creatine phosphokinase (CPK)
> gamma glutamyl transferase (GGT)
> ornithine carbamyl transferase (OCT)

C. Hydrolases
= Examples: i. esterases: ALP, ACP, cholinesterase (CHS), lipase LPS)
ii. peptidases: leucine aminopeptidase (LAP ), trypsin (PTS), pepsin
(PPS)
iii. glycosidase: amylase (AMS), galactosidase

D. Lyases
= Examples: i. aldolases = most common
ii. glutamate decarboxylase
iii. pyruvate decarboxylase
iv. tryptophan decarboxylase

E. Isomerases
= Examples: i. glucose phosphate isomerase
ii. ribose phosphate isomerase

F. Ligases
= Examples: i. aminoacyl-tRNA synthetase

1.5 Factors Affecting Plasma Enzyme Activities

 Mechanisms by which plasma levels of enzymes may be increased
A. Increased plasma enzymes is death of the enzyme-containing cell.
B. Increased membrane permeability
C. Increased rates of intracellular synthesis and the subsequent diffusion of these secreted enzymes
into the circulation.
D. Enzymes are also released in the process of normal cell turnover
E. Increased synthesis of enzymes can also increase plasma enzyme levels.
F. Drugs that stimulate microsomal enzymes,
G. Ethanol ingestion causes expression of mitochondrial isoenzyme of AST on hepatocyte surface.
H. Food ingestion leads to release of intestinal ALP into lymphatic fluid then into the plasma.
I. Canalicular obstruction leads to increased concentration of bile salts which may released
fragments of membrane with liver enzymes
J. Other factors include ischemia
K. Decreased clearance from circulation also increases plasma enzyme levels.

1.6 Parts of an Enzyme

ENZYME

activation

HOLOENZYME
(apoenzyme + cofactor)
 APOENZYME + COFACTOR
(protein moiety) (nonprotein moiety)

Activators Coenzyme
> inorganic > organic

A. Holoenzyme – an active substance formed by combination of a coenzyme (cofactor) and apoenzyme

B. Apoenzyme – is the protein portion of the holoenzyme that is subject to denaturation,
- catalytically inactive protein when cofactor is removed; they are heat labile and
dialyzable
C. Cofactors – are the nonprotein portions of an enzyme that are essential by an enzyme before any
catalytic activities or enzymatic activity can be manifested
- if the cofactor is a / an:

1. inorganic ion or metal ion (Activators)

– are inorganic substances, usually metal ions, which modify reactions catalyzed
- the metal ion may serve as:
a. bridge between an enzyme and substrate
b. primary catalytic center
c. stabilizing agent in the conformation for catalytic activity
- examples: AMS = Cl-, Br- LPS = Ca++
ALP = Zn++ CPK = Mg++
2. organic molecule (Coenzyme)
– is the nonprotein dialyzable portion of an enzyme and is essential for catalytic activity
- examples:
i. NAD / NADH
[R-H2 (reduced form) + NAD+ ↔ R (oxidized form) + NADH + H+]

ii. NADP / NADPH = the same reaction with NAD / NADH; differ in the phosphate group
attached to the molecule

iii. Pyridoxal-5-phosphate [P5P.

= serves as the intermediary for the transfer by accepting the amino group (NH2)
from amino acid to a keto acid and vice versa
[amino acid + PP → keto acid + PP - NH2]

 TERMS ASSOCIATED WITH ENZYMES:

A. isoenzymes

B. proenzymes

C. substrates

D. isoforms

1.7 Enzyme Kinetics

A. Enzymes catalyze reversible conversion of substrate (S) to reaction product (P). This is referred as
enzyme kinetics.
K1 K3
Efree + S E-S E free + P
K2 K4

A. Michaelis-Menten Equation
Where: v = velocity / rate of enzyme activity
Vmax = maximal rate of reaction when the enzyme is saturated
Km = (Michaelis-Menten constant) the substrate concentration that produces ½ of
the maximal velocity
 It is often assumed that the reverse reaction does not occur, since enzymes catalyzed reactions
proceed only under thermodynamically favorable conditions (where P energy level is less than that of
S).
Examples:
1. Some enzymes in the glycolytic pathway are also involved in gluconeogenesis
2. CK acts to store energy as creatine phosphate during inactive periods, while converting
creatine phosphate to ATP whe needed for muscle contraction
3. In vitro, both the “forward” and “reverse” reactions have been employed for the
measurement of LD, by changing the pH and reaction conditions.

B. Enzyme Action
= the first step in the action of an enzyme is the formation of an enzyme-substrate complex
= the process is done in a precise lock-and-key fashion
= after the enzymatic action (rupture of a particular bond in the substrate) has occurred, the
products of the reaction are no longer bound and the enzyme is recycled and is free to bind to
another substrate molecule and repeat its action

C. Michaelis-Menten curve shows the relationship of the reaction velocity to the substrate
concentration.
1. First Order Kinetics
i. The reaction rate varies directly with the substrate ’s concentration with a fixed amount of
enzyme.
ii. At low S concentration, there is a direct relationship between S concentration and reaction
velocity.

o As S concentration increases, the rate of increase gradually diminishes until all E exists as E-S
complex (Saturation kinetics); at this point, the rate becomes maximum (Vmax)

2. Zero Order Kinetics

i. The velocity of the reaction is not affected by the addition of more substrate at a certain point.
ii. When maximum velocity (Vmax)is reached, the rate of increase in velocity is zero. (zero
order reaction)
iii. At the point of Vmax, the rate of reaction is independent of the S concentration (Zero-Order
Kinetics with respect to S concentration) and directly proportional to the concentration of the
enzyme.

1.8 Enzyme Inhibitors

A. Major Types:
1. Irreversible Inhibitors – there is a permanent attachment of the inhibitor to the enzyme
2. Reversible Inhibitors - 3 Patterns of enzyme inhibition may be:
i. Competitive inhibition, where the inhibitor competes with substrate for binding to active,
catalytic site
= with large amounts of substrate, the inhibitor is unable to compete effectively, and thus
Vmax remains the same
= Example: succinylcholine, which competes with acetylcholine for binding sites on
cholinesterase

ii. Non-competitive inhibition, where the inhibitor binds to site on the enzyme, which is different
from catalytic site. In the process, it removes cofactors of the enzyme; these lower
affinity of the enzyme for the substrate and also lowers the Vmax
= Example: citrate, which complexes Mg and Zn, necessary cofactors for ALP activity

iii. Uncompetitive inhibition, are rare inhibitors where the inhibitor binds to E-S complex,
preventing its dissociation; Vmax in the process is reduced
= Example: inhibition of placental and intestinal ALP by L-phenylalanine

1.9 Tissue Specificity of Enzymes

1.8 Tissue specificity of enzymes may be divided into
A. High Specificity
 i. ACP in RBC and prostate
ii. ALT in liver
iii. AMS in pancreas and salivary glands
iv. LPS in pancreas
B. Moderate Specificity
i. AST in liver, heart, skeletal muscles
ii. CK in heart, skeletal muscles, brain
iii. ALP in liver, bone, kidney
C. Low Specificity
i. LD in all tissues

1.10 Assay of Enzymes

A. Units for Expressing Enzyme Activity
i. International Units (IU)
= 1 IU = 1 umol / min

ii. Katal Unit (KU

= 1 KU = 1 mole/sec
= 1 IU equals 16.7 nkat.(nanokatals)

B. Enzyme Measurements
i. In enzyme level measurements, measuring small increase in product is much easier than
measuring small decrease in large amounts of substrate.
ii. Enzyme reaction may be coupled with another reaction where an indicator substance uses the
product.

C. Factors Affecting the Velocity of Enzyme Reactions

1. Enzyme Concentration
i. As enzyme level increases, the reaction rate also increases. Again the rate levels off when
the level of enzyme gets to the point where the substrate concentration is no longer saturating.
ii. In this situation, enzyme level can be estimated by another assay using a smaller volume of
body fluid or diluting the sample and reassaying it.

2. Substrate Concentration
i. If enzyme concentration present is higher, the rate of reaction is determined by the
concentration of the substrate.
ii. As substrate level increases, enzyme reaction rate also increases until such point where
further increase in substrate produces no more enhancement of the enzyme reaction rate
iii. When measuring enzyme concentration present in a body fluid, the substrate is in excess.

3. Temperature
i. To ensure accuracy, the temperature of the reaction mixture must not deviate +0.1°C —0.1°C
from the optimal temperature.
ii. In general, every 10°C increase in temperature causes doubling of activity.
iii. The use of higher temperature gives faster reaction rate, improving sensitivity, an advantage
when the enzyme activities are low.
iv. At very high temperatures, enzyme dissociates and becomes inactive
v. Some enzymes, remains stable at extremely high temperatures
vi. Lower temperature increase linear limit of assay, requiring fewer dilutions
vii. 37°C appears to be the best single choice of reaction temperature, followed by 30°C
viii. All enzyme activity ceases when the protein is completely denatured
ix. Low temperature also decrease enzyme activity, and be completely inactive at temperature
0C and below
x. Any enzymes in tissues or extracts may be preserved for months by storing at –20 or -70 C
xi. Low temperature or freezing does not usually destroy enzymes, except
 CK, freezing at -20°C destroys its activity, whereas freezing at -70°C does not
 LD, losses its activity when stored at 4°C

4. Hydrogen Concentration (pH)

i. At optimal pH, enzymes possess maximal activity.
iv. Enzyme activity is in its maximum rate when reaction is done at an optimum pH
 Example: ALP has maximum activity at pH between 9 and 10
vi. pH 7.0 is the optimum pH for most intracellular enzymes.
vii. Extremes of pH may cause hydrolysis of enzyme loosing its structural integrity and decreasing
its activity

5. Coenzyme Concentration
i. Many enzyme require a coenzyme (ex NAD/NADH, most common) of some sort for the
reaction to proceed, but must be present at the proper concentration.
6. Inhibitor Concentration

7. Activator (ions) Concentration

i. Roles: > Helps bind the substrate to the active site by forming ionic bridges
> Helps orient the substrate at the proper point of attachment and in correct
configuration to the protein

8. Buffer

3. Salt and protein concentration

i. Salt concentration – of ionic strength is too high, enzyme activity drops
ii. Increase in protein concentration, increases enzyme activity.
iii. Enzymes lose activity rapidly in protein-free solutions either by denaturation or by adsorption to
the walls of the container

4. Anticoagulants
i. Heparinized samples are BEST (equivalent to serum) but may inhibit amylase (AMS) and ALT.
ii. Citrate inhibits CK and ALP.
iii. EDTA should never be used
iv. Fluoride

D. Antibody-based techniques for measuring enzymes

1. Such techniques include immunoassay.
a. Radioimmunoassay (RIA)
b. Immunoradiometric Assay (IRMA)
c. Homogenous Enzyme Immunoassay
d. Enzyme-Linked Immunoassay (ELISA)
e. Fluorescence Immunoassays
f. Chemiluminescence and Biolominescence

E. Pitfalls in Enzyme Assays

1. Hemolysis causing falsely high serum values.
2. Serum rather than plasma is the preferred specimen
3. Lactescence, or milky serum, may result in variable absorbance readings spectrophotometric
assays
4. Storage for enzymes in biological fluids:
a. most enzymes are stable at 6°C for at least 24 hours
b. some are stable at room temperature for lesser periods
c. for prolonged storage, -20°C or lower must be used
d. for LD4 and LD5, room temperature only
e. heat liability must be considered with respect to each enzyme to be assayed as well as to
other enzyme components, especially coenzymes and substrate
5. Accuracy in timing each assay and use of meticulously clean glassware are essential
6. It is vital that the pH of the reaction must be maintained constant.
7. Causes of increased serum enzyme levels:
a. impaired removal of enzymes form the plasma
b. in pathologic conditions involving tissue necrosis and degeneration
c. increased permeability of cell membrane
d. physiological or pathological increase in the number of cells or increase in the rate of
production of cells
8. Causes of decreased enzyme levels:
a. increased removal of enzymes from the plasma
b. decreased synthesis due to organ impairment, injury or removal
c. malnutrition leading to decreased enzyme production
 F. Quality Control Program for Clinical Enzyme Assays
1. Adherence to sero-order kinetics
2. Proportionality studies with increments of samples.
3. Use of pooled serum or stable reference materials (lyophilized) as control solutions
4. Replicate measurements to evaluate precision of assay

Enzymes as Reagents or Labels:

a. hexokinase & glucose-6-phosphate dehydrogenase = measurement of CK activity
b. glucose oxidase = for serum glucose determination
c. uricase = in the determination of uric acid

Major Enzymes of Clinical Significance

ENZYME CLINICAL SUBSTRATE

SIGNIFICANCE
Acid Phosphatase (ACP) – Prostatic Carcinoma Many phosphate esters (p-nitrophenylPO4, G6P,
[hydrolase] phenyl-PO4, ά-glycero-PO4, phenolphthalein-PO4,
thymolphthalein PO4, naphthol-PO4)
Alanine Amino-transferase Hepatic Disorder L-alanine + 2-oxoglutarate
(ALT) – [tranferase]
Alkaline Phosphatase (ALP) Hepatic Disorder Same as ACP
[hydrolase] Bone Disorder
Amylase (AMS) Acute Pancreatitis
Aspartate Amino-transferase Myocardial Infarction L-aspartate + 2-oxoglutarate
(AST) – [transferase] Hepatic Disorder
Skeletal Muscle
Disorder
Creatine Kinase (CK) – Myocardial Infarction Creatine + ATP or creatine PO4 + ADP
[transferase] Skeletal Muscle
Disorder
γ-Glutamyltransferase (GGT) Hepatic Disorder Transfer a γ-glutamyl group from many “donor”
[transferase] peptides to an “acceptor” peptide
Glucose-6-Phosphate Drug-induced
Dehydrogenase haemolytic
(G-6-PD) anemia
Lactate Dehydrogenase (LD) Myocardial Infarction Pyruvate and other deto acids + NAD
[oxido-reductase] Hepatic Disorder Lactate and other α-hydroxyl acids + NAD
Carcinoma
Lipase (LPS) Acute Pancreatitis
Pseudocholinesterase (PChE) Organophosphate Many aliphatic esters of choline
[hydrolase] poisoning
Genetic variants

ENZYMES OF CLINICAL SIGNIFICANCE

I. Phosphatases

1. Acid Phosphatase (EC 3.1.2.3) or ACP

A. Present in the prostate gland, erythrocytes, platelets, liver, spleen, milk and bone marrow. To
differentiate the prostatic form from the non-specific form like RBC acid phosphatase, inhibitors are
added.
 > L-tartrate
> formaldehyde and cupric ions
B. Subtrates used in prostatic determination:
Beta-glycerophosphate
Thymolphthalein
Phosphate
Substrates for non-specific form determination:
Phenylphosphate
p-nitrophenylphosphate
C. Isoenzymes:
1. Band 1.
2. Band 2 and 4.
3. Band 3
4. Band 5
E. Optimum pH: 4.9-5.0
F. Reference Range: Prostatic ACP: males = 0.2 – 3.5 IU/L or 0 – 3.5 ng/mL
females = 0.0 – 0.8 IU/L
Total ACP: males = 2.5 – 11.7 IU/L
females = 0.2-3.5 IU/L
G. ACP elevations
1. Tartrate-inhibited ACP (Prostatic isoenzyme)
a. Increased ACP may be due to prostatic cancer, although ACP is inferior to PSA (Prostate
Specific Antigen) in early cancer.
b. Increased in patients with benign prostatic hyperplasia and prostatic infarction.
c. Urinary tract obstruction, carcinoid tumors of the rectum, and prostatic massage
d. Medico-legal cases: ACP also has implications in suspected rape.
2. Tartrate-resistant ACP (bone isoenzyme) is usually elevated in:
a. active osteoclast-mediated bone resorption
b. Gaucher’s disease
c. hairy cell leukemia
3. Moderate elevation of total ACP:
a. female breast CA
b. Paget’s disease
c. hyperparathyroidism
4. Considerations in ACP Assays:
a. Due to release of ACP from platelets during clotting, serum ACP is slightly higher than plasma.
b. Sample: Citrate plasma
c. Fluoride inhibits ACP
d. Heparin and oxalate produce decreased activity.

H. Measurement
ACP
p-nitrophenolphosphate p-nitrophenol + P 1

1. Total ACP
a. Total ACP is measured by its ability to cleave phosphate groups at an acid pH. Substrate used in
the assay is p-nitrophenolphosphate. End colored product is measured.
b. Reaction may be measured before and after addition of tartrate (0.02 mol/L); activity before the
addition of tartrate will measure total ACP while activity after the addition of tartrate will measure
prostatic ACP.
2. Prostatic ACP
a. Prostatic isoenzymes level may be determined after addition of tartrate since it is inhibited by
tartrate.
Activity before addition of tartrate (total ACP) -- (minus)
Activity after addition of tartrate → (equals) Prostatic ACP
b. Thymolphthalein monophosphate is the preferred substrate cleaved by the prostatic
isoenzyme to thymol.

2. Alkaline Phosphatase (EC 3.1.3.1) or ALP

A. Isolated from various tissues like liver, bone, spleen, kidney and intestines.
B. Tissue Distribution
 1. Chromosome 1 = codes for the most abundant plasma ALP isoforms
2. Chromosome 2 (has 2 genes) = a. Placental and intestinal origin
b. Germ-cell or placental-like isoenzyme
C. Several isoenzymes exist which include:
1. placental isoenzyme
2. intestinal isoenzyme
3. liver isoenzyme
4. bone isoenzyme
D. Half-life / Rate of clearance of ALP isoenzymes:
1. Intestine = minutes
2. Bone = 1 day
3. Liver = 3 days
4. Placenta = 7 days
E. Sample Collection
1. Serum or heparinised plasma
2. Long standing clotted blood may loss CO2, hence increases ALP values by 20-30%.
3. Physical activity does not affect ALP level.
F. Measurement
1. Assays to measure ALP activity use p-nitrophenyl phosphate substrate at an alkaline pH.
2. Activators for this enzyme are zinc, magnesium, and other cations.
3. Chelators can falsely lower activity owing to chelation of magnesium ions.
4. Activity of enzyme increases slightly on storage due to loss of inhibitors.
5. ALP is relatively stable at 4°C for up to one week.
6. Optimum pH: 8.6 – 10 (average = 9)

G. Separation of ALP isoenzymes can be done by the following methods:

1. Inhibition with phenylalanine.
2. Heat fractionation.
3. Electrophoretic fractionation
4. Use of certain enzymes or lectins.
5. High resolution electrophoresis
6. Immunoassays
H. Reference Range: 30-90 U/L (30°C)
I. ALP Elevations
1. “Physiologically” elevated ALP
a. in growing children and in pregnant women in the third trimester.
2. Elevated liver / biliary ALP
b. Hepatic causes including obstructive cholestasis (biliary tract obstruction), jaundice from
hepatic injury, passive congestion of the liver
3. Elevated bone ALP
a. Paget’s disease, osteosarcoma, tumor metastatasize to bone, rickets, osteomalacia,
hyperparathyroidism
4. Elevated intestinal ALP
a. Diabetes, renal failure, cirrhosis. Intrahepatic jaundice, intestinal tract disorders
5. Germ cell tumors (such as seminoma, dysgerminoma) causes elevated placenta-like isoenzymes; as
well as serous carcinoma of the ovary, nonseminomatous germ cell tumors, endometrial carcinoma,
and leukemia.
6. An increase in 1% hour in ALP levels is seen in frozen and thawed serum and 10% increase after 96
hours. This is due to the breaking down of ALP-lipoprotein complex.
7. Liver and bone diseases
J. Decreased ALP
1. After blood transfusions or cardiopulmonary bypass
2. Hypophosphatasia
3. Zinc deficienc.
4. Hypophosphatemia and malnutrition / malnourished patients
K. ALP isoenzymes in cancer patients
1. Regan isoenzyme is found in patients with a particular type of lung cancer
2. Nagao isoenzyme is found in patients with adenocarcinoma
3. Kasahara isoenzyme is found in patients with hepatoma

II. Aminotransferases
 A. Aminotransferases are of two types:

1. Aspartate Aminotransferase (EC 2.6.1.1) or AST / Serum Glutamate Oxaloacetate

Transaminase (SGOT)
 Tissue content of AST in decreasing concentrations:
a. Heart f. pancreas
b. Liver g. spleen
c. Skeletal muscle h. lung
d. Kidney i. serum
e. Brain
 Optimum pH: 7.4
 Reference Range: 5-30 U/L (37°C)
 Clinical Significance:
a. In MI, AST levels are usually 4-10 times the upper limit of normal.
b. AST levels are elevated and used in the diagnosis of chronic alcohol abuse and drug
hepatoxicity
c. Increase AST levels are also seen in pulmonary infarction, pericarditis, acute hepatitis,
skeletal muscle disorders, etc.
d. Decreased AST levels are seen in pregnanct women.

2. Alanine Aminotransferase (EC 2.6.1.2) or ALT / Serum Glutamate Pyruvate Transaminase

(SGPT)
 ALT is found primarily in the liver but also in the kidneys, heart, skeletal muscle and pancreas.
 Optimum pH: 7.4
 Reference Range: 6-37 U/L (37°C)
 Clinical Significance:
a. acute or chronic viral hepatitis
b. acute myocardial infarction and other hepatocellular diseases
B. Both of these cytoplasmic enzymes uses pyridoxal-5’ -phosphate or P-5’-P as a necessary cofactor.
C. The half-life of AST is 17 + 5 hours while ALT has a half-life of 47 + 10 hours.
D. Specimen
1. AST is stable in serum at refrigerator temperature for up to three weeks, indefinitely if frozen. ALT
has the same stability but markedly decreases with freezing.
2. Specimen for AST and ALT are stable in whole blood for up to 12 or 24 hours, but increase with time
due to release from red blood cells.
E. Measurement
ALT
L-alanine + alpha ketoglutarate pyruvate + glutamate

AST
L-aspartate + 2-oxoglutarate oxaloacetate + glutamate
1. Coupled enzymatic reactions, using NADH as the final reaction product.
2. Reagents with NH4+ will give falsely increase ALT and AST owing to the conversion of NADH to NAD
by the ammonium ion.
3. International Federation of Clinical Chemistry (IFCC) recommended that methods have added
P-5’-P in the reagents.
F. Aminotransferase levels are altered in:
a. Hepatocyte injury
b. Muscle injury
c. Kidney infarcts
d. Renal failure

III. Angiotensin-Converting Enzyme (EC 3.4.15.1) or ACE

A. ACE is responsible for conversion of angiotensin I to angiotensin II and inactivation of bradykinin,
enkephalin, tachykinins.
B. Enzyme structure is described as a single polypeptide chain with zinc at the active center
C. Most activity is present in the lungs
D. Measurement
1. Activity is measured by ACE’s ability to cleave synthetic peptides, releasing hippuric acid, or other
indicator molecules.
3. There is a modification for CSF ACE assay because of low activity of isoenzyme in CSF.
 E. Disease Correlation
1. Most common reason is for diagnosing and monitoring sarcoidosis.
2. Increased ACE activity:
a. multiple sclerosis f. Alcoholic hepatitis
b. addison’s disease g. peptic ulcer
c. hyperthyroidism h. Bacterial & pneumocystis pneumonitis
d. diabetes mellitus i. HIV infection
e. nephritic syndrome
3. Decreased ACE activity:
a. chronic renal failure d. anorexia nervosa
b. malignancies e. hypothyroidism
c. chronic liver disease
IV. Cholinesterase (EC 3.1.1.7) and Pseudocholinesterase (EC 3.1.1.8)
A. True cholinesterase
1. It has high activity in CNS, red blood cells, lung and spleen.
C. Pseudocholinesterase or acylcholine acylhydrolase
1. It is important in cleavage of succinylcholine (muscle relaxant used during surgery)
2. It is primarily produced in the liver, but is also synthesized by myocardium and pancreas.
D. Measurement
1. True cholinesterase uses acethylcholine while pseudocholinesterase uses butyrylthiocholine as a
substrate.
2. The released thiocholine reacts with Ellman’s reagent (dithiobisnitobenzoic acid DTNB), releasing 5-
mercapto-2-nitro benzoic acid, which is measured photometrically.
E. Clinical Significance
1. Enzyme measurement is done:
i. to monitor those exposed to cholinesterase inhibitors
ii. as a liver function test.
iii. for diagnosis of genetic variants.

V. Creatine Kinase (EC 2.7.3.2) or CK / Creatine Phosphokinase or CPK

A. CK is an enzyme that catalyzes the reversible transfer of phosphate from adenosine triphosphate (ATP)
to creatine CK
(Rxn 1: creatine phosphate + ADP creatine + ATP )
HK
(Rxn 2: ATP + glucose ADP + glucose-6-phosphate)

(Rxn 3: Glucose-6-Phosphate + NADP+ 6-phosphogluconate + NADPH

B. Is present in high concentration in skeletal muscle, cardiac muscle, thyroid, prostate, and brain
C. Creatine kinase requires magnesium as a cofactor.
D. Isoenzymes of creatine kinase are:
1. CK1 or CK-BB = BB isoenzyme is normal serum is undetectable by electrophoretic mtds, but may
increase in women immediately post partum & in pxs w/ cardiovascular accidents
(stroke), acute renal disease, adenocarcinomas of the prostate or other tissues, severe
hypoxia (lack of oxygen), & brain injury
2. CK2 or CK-MB (normal heart contains 15% to 20% of CK-MB; in skeletal muscle, CK-MB
comprises 0% to 1% of total CK in type 1 fibers, and 2% to 6% of total CK in type 2 fibers)
3. CK3 or CK-MM
4. Macro-CK (an oligomer present in mitochondria and is seldom released into circulation)
E. Optimum pH:
1. Forward reaction = pH 9.0
2. Reverse / Backward reaction = pH 6.8
F. Considerations in CK Assays:
1. CK is light sensitive and anticoagulants like oxalates and fluorides inhibit its action.
2. CK in serum is very unstable and rapidly loss during storage.
3. Laked blood is not used
4. Exercise and intramuscular injections cause CK elevations.
G. Measurement
1. Electrophoresis is the method of choice.
2. Immunoinhibition assays for CK-MB
3. Mass immunoassay is the most commonly used method for measuring CK-MB.
 4. Column Chromatography
H. Reference Range:
 Total CK:
Male = 15-160 U/L (37°C)
Female = 15-130 U/L (37°C)
 CK-MB: < 6% total CK / CK-MB = 2-30 ug/L

I. Clinical significance:
1. Increases in CK-MB may be due to:
a. Cardiac or skeletal muscle damage
b. Chronic myopathies
c. Chronic renal failure
d. Acute respiratory exertion due to lung diseases
2. Damage to skeletal muscle
3. CK-BB is increased in:
a. Brain damage
b. Smooth muscle injury (intestinal ischemia)
c. Malignancies (prostate cancer, small cell carcinoma of the lung, and intestinal malignancies)
d. Acute cerebrosvascular disease

4. CK levels are also increased in:

a. Progresssive muscle dystrophy
b. Polymyositis
c. Acute psychosis
d. Alcoholic myopathy
e. Hypothyroidism
f. Trichinosis and dermatomyositis

5. Macro-CK2 is present in:

a. Malignancies
b. Myocardial infarction
c. MI (less than 5%) of the cases

VI. γ-Glutamyl Transferase or Gamma-Glutamyl Transferase (EC 2.3.2.1) or GGT

A. GGT are plasma membrane bound on cells that have high secretory or absorptive
B. GGT elevations
1. Liver damage
2. Smoking.
3. Medications increase GGT levels up to 5 times normal.
C. GGT decrease
1. Pregnancy.
2. Oral contraceptives reduce GGT by 20%
D. Half-life of GGT is about 7-10 days. In alcoholic liver disease, half-life increases to 28 days.
E. Measurement
1. GGT activity is measured by cleavage of chromogen o-carboxy, p-nitroaniline from glutamate-
modified form of the compound.
2. Szasz Assay
F. Uses of GGT
1. Evaluation of liver injury
2. Test for alcoholic abuse
3. Urine GGT: High amount of GGT is found in urine, since the kidneys contain large amount of GGT for
the physiological readsorption of amino acids. Pathological increased in urine GGT may be due to
long intake of aminoglycoside antibiotics which may damage the kidneys.

VII. Lactate Dehydrogenase (EC 1.1.1.27) or LD or LDH

A. Tissue source: found in all tissues and blood cells
is present in high concentration in liver, cardiac muscle, kidney muscle, erythrocytes
B. LD is a tetramer of two subunits, H (for heart) and M (for muscle).
1. LD1 (HHHH; H4)
2. LD2 (HHHM; H3M)
 3. LD3 (HHMM; H2M2)
4. LD4 (HMMM; HM3)
5. LD5 (MMMM; M4)
C. In normal serum LD isoenzymes activity is in decreasing order LD2>LD1>LD3>LD4>LD5
D. Distribution of LD isoenzymes:
1. LD1 and LD2 = found mostly in the myocardium and erythrocytes, also found in the renal cortex
2. LD3 = found in a number of tissues predominantly in the white blood cells and brain
3. LD4 and LD5 = found mostly in the liver and skeletal muscle, also in the ileum and skin
E. Measurement
LDH
Lactate + NADH+ Pyruvate + NADH+ + H+

1. This can be done either by the forward (L-lactate to pyruvate) or the reverse (pyruvate to lactate) reaction.
1. Dry slide method for LD utilizes the reverse reaction
2. Inhibition methods for LD1 are also available. Results are reported in ratio of LD 1 to total LD.
Hydroxybutyrate is cleaved by LD1.
F. Specimen
1. Serum should not be frozen for assay of LD isoenzymes
2. LD is not stable at 4°C storage due to cold lability of LD5.
3. Within three days of storage, total LD decreases by about 20%.
4. Samples may be stored up to 24 hours at room temperature.
5. Hemolysis must be avoided.
G. “Hydroxybutyrate dehydrogenase” was employed as a diagnostic test for myocardial infarction, but this is
no longer the case.
H. Reference Range: 100-225 U/L (37°C)
I. Clinical significance:
1. In myocardial infarction, LD1 is higher than LD2 thus called “flipped “ LD pattern.
(Normal: LD2 > LD1 while in MI: LD1 > LD2)
2. Marked elevations of LD levels (> 5 to 10 times) are seen in:
a. Megalobalstic anemia
b. Pulmonary infarction
c. Hemolytic anemia
d. Advanced malignancies
e. Sepsis
f. Cardiopulmonary arrest
3. Moderate elevations are seen in:
a. Pneumocystis carinii pneumonia
4. Other causes of increased levels:
a. Germ cell tumors (seminoma or dysgerminoma)
b. Haemolytic anemia, megaloblastic anemia, and renal cortical diseases
c. Skeletal muscle injury or patients with ischemic or toxic hepatic injury (increases in LD 4 and LD5)
5. In tumors of WBCs (leukemia, lymphoma, multiple myeloma) LD3 and LD4 are increased, while LD1
and LD2 are decreased.
J. Isomorphic pattern for LD is often referred to as when total LD is increased but isoenzyme levels are
normal; and a “ tombstone” pattern occurs when the relative amount of each isoenzyme is roughly the
same. This isomorphic pattern is typical for patients with diffuse tissue damage, accompanied by shock
and hypoxemia.

VII. Leucine Aminopeptidase (EC 3.4.11.1) or LAP

A. LAP isoenzymes
1. Liver isoenzyme
2. Placental isoenzyme
B. Measurement
1. Starch gel electrophoresis
C. Clinical significance
1. LAP is increased in liver injury but NOT in bone disease
2. LAP is as sensitive as ALP and 5’-NT in detecting obstructive liver diseases
3. LAP is elevated in patients with SLE or systemic lupus erythematosus.
4. A number of other malignancies that causes increase in LAP enzyme are breast, endometrial, and
ovarian carcinomas, germ cell tumors of the ovary and testis.
IX. 5’ -Nucleotidase (EC 3.1.3.5) or 5’-NT
A. 5’-NT is a metalloprotein also known as 5’-ribonucleoside phosphohydrolase or NTP.
B. Is predominantly derived from the liver.
C. Zinc is an integral part of 5’-NT
D. Measurement
1. Measurement of this enzyme utilizes the specific substrate adenine 5’-monophosphate (5’-AMP),
which is converted to adenosine and Pi:
5’-AMP → adenosine + Pi
The either the product Pi or adenosine can be measured accordingly:
a. Pi
b. Adenosine
i. Titrimetric method
ii. Coupled Reaction

2. Chelating agents interfere in 5’-NT measurement.

3. In measurement of 5’-NT, the assay uses large amounts of other non-nucleoside substrates to
“competitively inhibit” ALP, since other phosphatase such as ALP interfere with reaction.
D. Increased 5’ -NT:
1. acute hepatitis
2. ovarian carcinoma
3. rheumatoid arthritis

X. Pancreatic Enzymes
1. Amylase or α-1,4-Glucan-4-Glucanohydrolase (EC 3.2.1.1) or AMS
A. Isoenzymes of amylase
1. pancreatic isoenzyme (P-form)
2. salivary gland isoenzymes (S-form)
B. Electrophoresis .
1. Three fast moving toward the cathode (S1, S2 and S3)
2. Three slow moving (P1, P2 and P3)
C. P3 was identified only in patients with acute or chronic pancreatitis and in those with renal insufficiency
D. Optimum pH: 6.9 – 7.0
E. Measurement: Methods for measuring amylase are divided into:
1. Starch substrate-based methods
a. Saccharogenic – measures the amount of reducing sugars produced by the hydrolysis of starch
by the usual glucose methods or measures the appearance of the product
b. Amyloclastic – amylase activity is evaluated by following the decrease in substrate concentration
or disappearance of starch substrate
c. Chronometric – measures the time required for amylase to hydrolyzed completely all the starch
present in a reaction mixture. End point is reach when there is absence of any substrate
capable of forming the blue starch iodine color
d. Amylometric – measures the amount of starch hydrolyzed in a fixed period of time using the
intensity of the blue starch iodine color.
e. Chromogenic – measures the increasing color from production of product couples with a
chromogenic dye
f. Continuous monitoring – coupling of several enzyme systems to monitor amylase activity

2. Defined-substrate methods (preferred)

 They contain well defined substrates rather than heterogenous polymers.
 Beckman assay: Maltotetrose is hydrolyzed by amylase into two maltose units. The reaction is
coupled with maltose phosphorylase, which converts glucose-1-phosphate to gluce-6-phosphate.
G-6-P is oxidized by glucose-6-phosphate dehyrdrogenase to dlucolactone-6-phosphate wherein
NADP is converted to NADPH (indicator measured at 340 nm)
 Other methods use nitrophenyl saccharide substrates
G. Amylase is the only protein that can be cleared by the kidneys.
1. In pancreatitis, the clearance of amylase is increased. The ratio of amylase clearance to
creatinine clearance is usually elevated in acute pancreatitis.
2. In renal failure, serum levels of amylase are elevated as a result of decreased renal clearance.
3. In macroamylasemia, amylase is bound to immunoglobulin, and the complex is too large to be
filtered by the glomeruli.
 H. Increased amylase levels are seen in:
1. Pancreatitis
2. Salivary gland lesions
3. perforated peptic ulcer
4. appendicitis
5. ruptured ectopic pregnancy
6. dissecting aortic aneurysm
7. biliary tract disease
I. Lipemic specimens cause a reduction in amylase activity.

2. Lipase or Triacylglycerol Acylhydrolase (EC 3.1.1.3) or LPS

A. Present mainly in the pancreas and in other sources like the stomach and intestines.
B. Optimum pH: 7.8-8.0
C. Reference Range:0 – 1.0 U/mL

D. Measurement
1. Titrimetric method
2. Turbidimetric method
E. Increased lipase levels are seen in:
 Pancreatic disorders (acute or chronic pancreatitis)
 Pancreatic duct obstruction and tumors of the pancreas.
F. Considerations in LPS Assays:
1. Lipemic specimen cause a reduction in lapse activity.
2. Use of olive oil as substrate precludes the measurement of non-specific esterases like the “clearing
factor”
3. Opiates and morphines increase LPS activity due to their spastic effects on the duodenal
musculature and sphincter of Oddi.

XI. Fructose Diphosphate Aldolase (ALD)

A. Three isoenzymes:
1. Aldolase A
2. Aldolase B
3. Aldolase C
B. Optimum pH 6.8-7.2
C. Measurement:
1. Immunoassay – enzyme immunoassay
D. Considerations of ALD assays:
1. Laking should be avoided since the red cells contain 10 times as much ALD as serum.
2. The ALD activity of serum and plasma are identical, with oxalate, citrate, fluoride, heparin and EDTA
as anticoagulants
E. Clinical significance:
1. Severe elevations in: Muscle degeneration & Viral hepatitis
2. Moderate elevations in:
a. Gangrene d. Metastatic liver carcinoma
b. Megaloblastic anemia e. Psychosis
c. Granulocytic leukemia f. trichinosis

XII. Glucose-6-Phosphate Dehydrogenase

A. G-6-PD is an oxidoreductase that catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconate
or the corresponding lactone.
G-6-PD
Glucose-6-phosphate + NADP+ glucose-6-phosphogluconate + NADPH + H +

B. Tissue source: adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland and erythrocytes;
little activity is found in serum
C. Function or role of G-6-PD:
1. Its role focuses in erythrocyte. It functions to maintain NADPH in reduced form. An adequate
concentration of NADPH is required to regenerate sulhydryl-containing proteins, such as glutathione
 in the reduced form. A deficiency of G-6-PD results in an inadequate supply of NADPH and the
inability to maintain reduced glutathione levels. When erythrocytes are exposed to oxidizing agents,
hemolysis occurs because of oxidation of haemoglobin an damage of the cell membrane.
D. Measurement
G-6-PD
Glucose-6-phosphate + NADP+ glucose-6-phosphogluconate + NADPH + H +

1. A red cell hemolysate is used to assay for deficiency of the ALD enzyme.
2. Serum is used for evaluation of enzyme elevations
E. Reference Range:: G-6-PD = 10-15 Ug/Hgb
F. Clinical significance:
1. Increased levels of G-6-PH: Myocardial infarction & Megaloblastic anemias
CLINICAL USAGE OF ENZYMES

Diagnosis of Myocardial Infarction (MI)

= MI is diagnosed by the presence of 2 out of 3 features:
1. Chest pain (“prolonged” chest pain)
2. ECG changes
3. Rise and fall of cardiac enzymes

1. Creatine Kinase (CK)

= the serum CK activity begins to rise 3-6 hrs (4-8 hrs according to Bishop) after a MI
& reaches a maximum around 24 hr; the maximum rise in serum for a patient is usually about 5-8
times the upper limit of normal ( X ULN) / (50-100 times ULN according to Bishop), but in severe
cases, the increase may reach 10-20 X ULN; the CK activity in serum may return to normal levels in
3 or 4 days
= INCREASE ACTIVITY:
 Damage heart tissue – CK is the first enzyme to appear in serum in higher conc after MI & is probable
(CK-MB) the first to return to normal levels if no further coronary damage occurs
- serum CK may be increased in some cases of coronary insufficiency w/o MI

2. Creatine Kinase Isoenzyme (CK-MB / CK2)

= NV: CK-MB = 2-30 ug/L
= INCREASED ACTIVITY:
 following MI, the CK-MB levels begin to rise within 4-8 hours, peak at 12-24 hours; this time frame
must be considered when interpreting CK-MB levels
 CK-MB activity has been observed in other cardiac disorders, therefore
 the maximum rise may range from 10 U to as high as 400 U
 MB isoenzyme is always elevated w/in 48 hr of an MI, but it can also be elevated w/ coronary
insufficiency

3. Lactate Dehydrogenase (LD)

= principal clinical uses of LD test are the ff:
a. in MI – serum LD activity increases after MI, levels begin to rise within 12-24 hours, reach peak levels
within 48-72 hours; but the rise occurs later than that for CK & is of less intensity; it may remain
elevated for 7-10 days, long after the CK level has returned to normal
- CK-MB isoenzyme when taken together the LD isoenzmes, the finding of a flipped ratio (LD1 >
LD2) plus an elevated MB isoenzyme is a positive indication of an MI

4. Lactate Dehydrogenase Isoenzymes

= CLINICAL APPLICATIONS:
b. in MI - LD1 is higher than LD2 thus called “flipped “ LD pattern.

@ AMI / MI: * LD1 > LD2 referred to “flipped” LD pattern (when normally LD2 > LD1);
characteristically appears 12-24 hours and within 48 hours
* LD1/LD2 ratio > 1 (within 48 hours) when normally, LD1/LD2 ratio = < 1 or 0.5-0.75

- the flipped ratio may occur after a renal infarct & in hemolytic situations
- the combination of an elevated CK-MB isoenzymes & a flipped LD isoenzymes ratio in a px
suspected of having had an MI makes the dx certain; this combination never occurs in
coronary insufficiency w/o an MI
 a. in liver dse
- the LD-4 and LD-5 isoenzymes are found primarily in liver and skeletal muscle tissue, with LD-5
being the predominant fraction in these tissues; LD-5 levels have greatest clinical significance
in the detection of hepatic disorders, particularly intrahepatic disorders;
- LD-6 is alcohol dehydrogenase; LD-6 has been present in patients with arteriosclerotic
cardiovascular failure; it is suggested, that LD-6 may reflect liver injury secondary to severe
circulatory insufficiency
b. in pulmonary infarction – prominent increased in LD3

= migration: LD1, LD2, LD3, LD4, LD5

= serum concentration or tissue distribution: LD2, LD1, LD3, LD4, LD5
= bacterial meningitis: LD5, LD4, LD3, LD2, LD1
= LD1 – MI; LD3 – pulmonary embolism; LD5 – liver diseases
= in AMI/MI:
o CK & CK-MB = peaks within 24 hours
o AST = follows CK & CK-MB, peaks within 48 hours
o LD, specifically LD1 = peaks within 72 hours (elevated levels persist longer as 10-14 days)

 Differential Diagnosis between Myocardial Infarction (MI) and Pulmonary Infarction (PI):
CK2/CK-MB AST LD
MI: ↑ ↑ ↑
PI: N N ↑ (due to LD3)

5. Serum Aspartate Aminotransferase (AST) / Glutamine-oxaloacetic transaminase (GOT)

= in AMI, AST levels begin 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, AST levels are not useful in the
diagnosis of AMI

6. Nonenzyme Proteins / Proteins Markers:

A. Myoglobin
= is the earliest marker to become abnormal after MI
= can be detected within 2 hours of AMI (increased), serves as screening test for MI [when
striated muscle is damaged, myoglobin is released, elevating the blood levels; in an AMI, this
increase is seen within 1-3 hours of onset and reaches peak concentration in 5-12 hours; if a
repeated myoglobin level doubles within 1-2 hours after the initial value, it is highly diagnostic
of an AMI; and blood levels return to normal in 18-30 hours after the AMI
= it has a reasonable sensitivity of 95-100% but poor specificity for diagnosis of MI
= methods of measurement: latex agglutination, ELISA, immunonephelometry,
fluoroimmunoassays, immunochromatography (qualitative spot
test)

A. Troponin (troponin T) = is a complex of three proteins that bind to the thin filaments of striated muscle
(cardiac and skeletal) but are not present in smooth muscle
= the complex consists:
1. Troponin T (TnT)
= parallels with CK-MB but the increase is greater (up to 80% upper limit) than CK-MB
and declines after 10 days even after MI (cardiac troponin T levels in serum begin to rise
within 3-4 hours following the onset of myocardial damage, peak in 10-24 hours, and
remain elevated for 10-14 days following AMI
= high levels of cardiac troponin T correlates with worsening prognosis

2. Troponin I (TnI)
= cTnI, like cTnT, does not normally circulate in the blood and it is 13 times more
abundant in the myocardium than CK-MB on a weight basis,
= cTnI is a very sensitive indicator of even minor amount of cardiac necrosis (following
an AMI, cTnI levels begin to rise in 3-6 hours, reach peak concentration in 14-20 hours,
and return to normal in 5-10 days
= method of measurement: specimen can be serum or heparinized plasma
> ELISA or immunoenzymometric assay; immunochromatographic dry strip
assays
 3. Troponin C (TnC)

= troponin is replacing CK-MB as diagnostic test of choice for MI; troponin is now known as the “ gold
standard” for diagnosis of MI
= specimen of choice for troponin assay is heparinized samples

2. New Tests for Myocardial Injury

a. These include markers for activation of the coagulation system such as P-selectin (indicative of platelet
activation), fibrin monomers, and thrombus precursor proteins (thrombus formation is a critical
element in the development of unstable angina and MI)
b. Glycogen phosphorylase BB isoenzymes
c. Fatty acid-binding protein

Diagnosis of Muscle Disorders

1. creatine kinase (CK) – total CK and CK-MM
2. aspartate aminotransferase (AST)
3. aldolase (ALD)

Diagnosis of Skeletal Muscle Injury

1. Biochemical Markers of Cardiac Injury
A. CK
i. Direct trauma causes mild elevations of CK (5-6 times normal)
ii. Chronic skeletal muscle damage cause more persistent elevations of CK
iii. CSN disorders (central vascular accident, seizures, nerve degeneration and CNS shock)
iv. Neurologic malignant syndrome

B. CA-III (CA isoenzyme III) is a protein marker found in skeletal but NOT in cardiac muscle. If both
myoglobin and CA-III are increased, skeletal muscle injury is the cause of marker released.

2. Severe acute muscle damage or rhabdomyolysis

A. This may be caused by: drugs (ethanol and cocaine) crush injuries
Viral infections ischemia of lower extremeties
Extreme exertion inflammatory myopathies
B. There is evidently higher total CK (CK > 20 times normal), and appearance of myoglobinuria in this type
of muscle injury.
B. Myoglobin has a shorter half-life, making CK measurements more reliable.
C. In “ cell lysis syndrome”, other cell contents – potassium, phosphate, and nucleotides (converted to uric
acid) are present.

Markers of cardiac muscle injury existing together with skeletal muscle injury
1. Markers present in both skeletal and cardiac muscle (total CK, AST, and myoglobin) will be increased
2. Troponin is the diagnostic test of choice for recognition of cardiac injury (when skeletal muscle damage is
known or suspected)

Diagnosis of Hepatic Disease

1. Serum Alanine Aminotransferase (ALT) / Glutamic-pyruvic transaminase (GPT)
= present in moderately high conc in liver, but is low in cardiac & skeletal muscles & other tissues
= is more specific transaminase for the liver, & is elevated in the presence of hepatocellular necrosis
= measurement can be helpful in distinguishing hepatocellular jaundice from obstructive jaundice, as ALT
shows marked elevations in hepatocellular jaundice & only mild elevations in obstructive jaundice
= blood specimen should be drawn & handled carefully to avoid hemolysis bec it falsely elevates the ALT
= serum ALT is relatively unstable & should be kept at 4C for short periods or stored frozen at -20C or
lower for longer periods
= INCREASED ACTIVITY: various intracellular hepatic disorders
= cardiac tissue contains a small amount of ALT activity, but the serum level usually remains normal in
 AMI unless subsequent liver damage has occurred

2. Serum Aspartate Aminotransferase (AST) / Glutamine-oxaloacetic transaminase (GOT)

= found in every tissue & rbcs & is in high concentration in cardiac muscle & liver
= most helpful for the dx & monitoring of hepatocellular dse
= blood specimens should be drawn in a manner that avoids hemolysis to avoid false increase of AST
= INCREASED ACTIVITY: intracellular liver damage (hepatitis, cirrhosis, hepatotoxins)
after MI, in trauma to or dses affecting skeletal muscle
after a renal infarct, in various hemolytic conditions
congestive heart failure bec of hepatic ischemia & anoxia produced

3. Gamma Glutamyltransferase (GGT)

= is sensitive to alcohol that chronic alcoholic persons often have elevated GGT, regardless of liver
damage
= the most clinically useful application of GGT is dx of diseases of the liver & biliary tract
= only moderately increased in hepatocellular liver dse, but is markedly increased in obstructive liver dse
= helps to determine whether an elevated ALP is coming from bone or liver
= Reference method of measurement: SZASZ method

4. Ornithine Carbamoyltransferase (OCT)

= elevations are quite marked in hepatocellular disease
= a genetic deficiency of this enzyme causes severe & often fatal hyperammonemia in the newborn
period

5. Alkaline Phosphatase
= is most useful in dx of cholestasis or obstructive liver disease

6. 5’-Nucleotidase (5NT)
= useful in interpreting an elevated ALP result of unknown origin
= elevated in liver disorders, but not in bone diseases

Diagnosis of Bone Disease

1. Alkaline Phosphatase (ALP)
= present in high conc in bone (osteoblasts, the cells of growing bone), intestinal mucosa, & renal tubule
cells & in lower conc in the liver (highest in the biliary tree), leukocytes & placenta

2. Alkaline Phosphatase Isoenzymes (Bone isoenzyme)

= INCREASED ACTIVITY:
 in all bone disorders (Paget’s dse / osteitis deformans, osteoblastic tumors w/ metastasis)
 hyperparathyroidism (when mobilization of Ca & P from bone exists)
 rickets & osteomalacia
 in liver dse (esp disorders of the hepatic biliary tree)
 transient but extreme elevations in children (between ages of 2 mons - 2 yrs
= DECREASED ACTIVITY:
 hypophosphatasia, a rare congenital defect in dwarfs resulting from depressed osteoblastic activity;
also known as phosphoethanolaminuria, named for the presence of phosphoethanolamine in the
urine (pxs show premature tooth loss & rickets-like bone lesions)
 hypothyroidism
 pernicious anemia

Diagnosis of Acute Pancreatitis

1. Serum Amylase (AMS)

= INCREASED ACTIVITY:
 acute pancreatitis (AMS level is 4-6 times the upper reference limit, obstruction of the pancreatic
ducts and parotid (salivary) gland [whether by stone, inflammation, or compression of the common
bile duct by a cancerous growth of the head of the pancreas] = reaches its maximum level in about 24
hr, w/ a return to normal in 2 or 3 days
= DECREASED ACTIVITY:
  acute or chronic hepatocellular damage

2. Serum Amylase Isoenzyme

= pxs with acute pancreatitis usually exhibit a prominent P3 isoenzyme; also elevated in pxs w/ severe
kidney dse, presumably bec of impaired renal clearance of the isoenzyme

3. Urine Amylase
= bec AMS has a molecular wt of lest than 50,000 daltons, it is readily excreted by the kidney
= the urinary excretion of AMS is high in px w/ acute pancreatitis
= the urinary AMS may be elevated for 7-10 days, whereas the serum AMS returns to normal in 2 or 3
days after an attack
= Amylase Creatinine Clearance Ratio (ACCR)
Urine sample (U/L) X serum creatinine (mg/dL)
ACCR (%) = X 100
Serum amylase (U/L) X urine creatinine (mg/dL)
= INCREASED EXCRETION:
o acute pancreatitis
= DECREASED EXCRETION:
o acute or chronic renal dse
o severe damage to hepatic cells

4. Serum Lipase
= INCREASED CONC:
 pancreatic disorders (acute or chronic pancreatitis
 elevated in pancreatic duct obstruction and tumors of the pancreas.

= lipase is a specific marker for pancreatic disease and becomes elevated within the first 12 hours of
the onset of pancreatitis and remains elevated for 7 days
= in contrast to AMS levels, LPS levels are normal in conditions of salivary gland involvement; therefore,
LPS levels are useful in differentiating serum AMS elevations as a results of pancreatic versus salivary
involvement; of the three lipase isoenzymes, LPS is thought to be the most clinically specific and
sensitive

Diagnosis of Metastasizing Cancer of the Prostate

1. Acid Phosphatase (ACP)
= bec rbcs & blood platelets also contain an ACP, the ACP derived from these sources during the clotting
of the blood specimen must be distinguished from the ACP coming from the prostate
= present in significant amount in semen therefore, used forensically in the diagnosis of rape
= INCREASED CONC:
o metastasizing carcinoma of the prostate
o Gaucher’s dse or some bone dses
= DECREASED CONC:
o no physiologic significance

2. ACP Isoenzymes
a. Prostatic phosphatase isoenzyme (PAP) – used to detect a prostatic carcinoma before it is palpable
(detectable by touch in a rectal examination)
- 1000-fold more sensitive
b. Prostate Specific Antigen (PSA)
- the specificity of this protein for prostate makes it an excellent tumor marker
- PSA is tissue specific, but it is not cancer specific, as it is found in benign, normal & malignant
prostases
= useful as a screening tool in the older male population, esp when symptoms (frequent urination) occur

Investigation of Genetic Disorders

1. Pseudocholinesterase
= some people have a genetic deficiency of this enzyme, & when injected w/ succinylcholine, they fail to
 inactivate the drug & may be subjected to its action for as long as 2-3 hr instead of the usual 2 min; the
respiratory muscles may be so relaxed by the drug that breathing is inadequate & the px ’s life is
endangered

2. Glucose-6-phosphate Dehydrogenase (GPD)

= is needed for the oxidation of glucose-6-phosphate as the first step in the pentose phosphate pathway
= GPD is present in many types of cells, but it is particularly impt in the rbc bec the NADPH generated is
a necessary ingredient for other enzyme systems in the erythrocyte to prevent the accumulation of
methemoglobin
= a genetic deficiency of GPD or have 30% less of the normal amount of the enzyme are susceptible to
acute hemolysis of the rbc if exposed to certain drugs, such as antimalaria drugs (primaquine), some
sulfonamides, quinine, & others

3. Galactose-1-phosphate Uridyltransferase
= dse galactosemia is caused by hereditary deficiency of the enzyme galactose-1-phosphate uridyl
transferase, w/c is necessary for the conversion of ingested galactose to glucose; this conversion
must take place before galactose can be used for energy
= in the absence of the enzyme, continued ingestion of galactose causes a buildup of galactose-1-
phosphate in cells; this buildup produces the typical symptoms of galactosemia: cataract formation,
enlarged liver & spleen, & mental retardation
 V. ACID-BASE BALANCE AND BLOOD GASES
I. INTRODUCTION
@ CHEMICAL ASPECTS OF ACIDS AND BASES
A. Acid and Base
1. Acid is a substance capable of dissociating to yield hydrogen ion.
2. Base is a substance that can accept hydrogen ion.
B. Buffers
1. Buffers are chemical systems that resist pH changes.
2. They are typically composed of a mixture of a weak acid and a salt of its conjugate base.
3. To be effective, there must be approximately equal amounts of both buffer acid and buffer base.
4. pH range is within + around the pKa of the buffer acid
5. The ability to resist pH changes is also controlled by the total amount of buffer acid and base
6. Carbonic acid and bicarbonate are the most important buffer system in the body.
7. Specific Henderson-Hasselbalch equation is derived by substitution of K’a value of carbonic acid and
using the solubility of carbon dioxide in water, that is 0.03 times the pCO 2.
pH = 6.1 + log (HCO3- /0.03 X pCO2)

II. DEFINITION OF BASIC TERMINOLOGY:

1. Acid – is a substance that can yield a hydrogen ion (H +) or hydronium ion when dissolved in water

2. Base – is a substance that can yield hydroxyl ions (OH-)

3. pK – defined as the negative log of the ionization constant, also the pH in which the protonated and
unprotonated forms are present in equal concentrations

4. Buffer – the combination of a weak acid or weak base and its salt

5. Bicarbonate (HCO3-) – the second largest fraction of the anions in the plasma

6. Carbonic acid (H2CO3) – this fraction of blood, plasma, or serum includes the undissociated carbonic acid
and the physically dissolved anhydrous CO2
- since CO2 concentration is higher than HCO 3-, the symbol cdCO2 (concentration of
dissolved CO2) is frequently used which is measure from pCO2 multiplied by the
solubility coefficient of CO2

7. Partial pressure – the partial pressure of a gas in a liquid is the partial pressure of that gas with which the
liquid is in equilibrium

8. Partial Pressure of CO2 (pCO2) – the pressure or tension exerted by CO2 gas dissolved in blood
- it is an index of efficiency of gas exchange in the lungs and not a measure of CO 2
concentration in the blood

9. Carbon Dioxide Combining Power – the value of the CO 2 combining power is an index of the amount of
CO2 that can be bound by serum, plasma, or whole blood as HCO 3- at a pCO2 of 40 mm
Hg at 25°C
 10. Total Carbon Dioxide Concentration (ctCO2) – it refers to the total concentration of CO2 in the blood
consisting of ionized and unionized fraction and physically dissolved CO 2

11. Standard Bicarbonate Concentration – the bicarbonate ion concentration in the blood that has been
equilibrated with CO2 at 40 mm Hg at 37°C

12. Partial Pressure of Oxygen (pO2) – the pressure or tension exerted by oxygen gas dissolved in arterial
blood which reflects the availability of the gas in the blood but not its content

13. Oxygen Saturation (O2 Sat) – refers to the amount of oxygen carried by the haemoglobin

14. pH – the negative logarithm of hydrogen ion activity

15. Buffer Base – the sum of the concentration of bicarbonate and the net protein anion or concentration of
titrable base when titrating to the apparent isoelectric pH of the protein at a pCO 2 of zero.

16. Base Excess – the concentration of the titrable base minus the concentration of titrable acid when titrating
the blood or plasma with a strong acid or base to a plasma pH of 7.4 at a pCO 2 of 40 mm
Hg at 37°C

17. Base Deficit – refers to a negative base excess, or decrease in blood buffering capacity

18. Alkali Reserve – the total amount of substance in the blood available for neutralizing acids.

III. IMPORTANCE OF pH
1. Sources of Protons
a. glucose metabolism

b. fatty acids / triglycerides metabolism

c. amino acid metabolism

d. oxidative phosphorylation, a process that utilizes oxygen & generates ATP


under normal circumstances, the supply of oxygen is adequate and no appreciable increase in H + occurs

in conditions of oxygen deficiency (anoxia), acidosis develops owing to the body ’s inability to transfer all
H+ to O2 to form water

thus several buffer systems preserve the internal environment by reducing the concentration of H +

2. Relationship of H+ to pH: inversely proportional

H+ pH therefore: ↑ H+ = ↓ pH = acidic
45 7.35 ↓ H+ = ↑ pH = alkaline
40 7.40
35 7.45
30 7.50

IV. PHYSIOLOGY OF ACID-BASE BALANCE / MECHANISMS THAT MAINTAIN BOTH ECF AND ICF pH
1. BUFFER SYSTEMS IN THE BODY / BLOOD BUFFERING MECHANISM (c/o Kaplan)
@ BUFFER = is a mixture of a weak acid and its salt with the capability of combining with protons or
releasing protons in response to external shifts in pH
= its purpose is to furnish (or neutralize) protons in solution to maintain a defined blood pH
= the buffer mixture consists of an equilibrium between a protonated species & its
nonprotonated counterpart:
HA  H+ + A-
o HA – stands for the undissociated componenet of the buffer system
o H+ - the dissociated protons
o A- - the anion component of the buffer
 = as protons are added to the system (decrease in pH), the equilibrium shifts (to the left) to form more
HA, consuming the extra protons [↑ H+ (←) = acidic = ↓ pH] (1/α)
= if the proton level decreases (increase in pH), the equilibrium shifts to the right, releasing protons into
solution [↓ H+ (←) = alkaline = ↑ pH]
= the ff are the (#s 1 & 2 predominant) buffer systems to maintain blood pH:

BLOOD BUFFERS (in descending order of buffering capacity):

A. PLASMA BICARBONATE BUFFER SYSTEM / BICARBONATE-CARBONIC ACID BUFFER SYSTEM
CA CA
H2O + CO2 H2CO3 H+ + HCO3- (can be conveniently expressed as the derived
Hendersen-Hasselbalch Equation:)
(HCO3-) bicarbonate
pH = pKa + log
(H2CO3) carbonic acid

pH = 6.1 (constant) + 1.3 (usual result)

pH = 7.4 (thus normal range is 7.35-7.45)

c. MAINTAINING EQUILIBRIUM:
1. Shift to the Right
= results from an increase in CO2, the equilibrium shifts to the right, producing more hydrogen
ions (H+) and bicarbonate anions (HCO3-)
2. Shift to the Left
= The reaction can shift to the left under two (2) different sets of circumstances:
i. an increase in protons (H+) or
ii. a decrease in CO2 (through respiration )
d. H2CO3 H+ + HCO3- is the most important buffer system because of the high concentration of
-
HCO3 and the readiness with which H2CO3 may be increased through diminished lung activity or
decreased by blowing off CO2 through increased pulmonary ventilation
e. The carbonic acid (H2CO3) is simply an intermediary in the equilibrium; it decomposes either to form
water and carbon dioxide or protons and bicarbonate, depending on the concentration of other
chemicals in the system
f. The equation above can also be expressed as:

(total CO2) - 0.03 )(pCO2)

pH = pKa + log
0.03 pCO2
Where: 0.03, is the solubility coefficient of CO2 gas in a normal plasma at 37°C.
Hence: H2CO3 = 0.03 x pCO2
HCO3- = (total CO2) - 0.03 (pCO2)

It is easier to think of acid-base balance in terms of H + concentration (normally at 40 mmol/L). A

conversion from pH to [H+] is made using the Henderson equation to evaluate acid-base perturbations.
(pCO2) mm Hg
[H+] nmol/L = 24
[HCO3-] mmol/L

Reference value for H+: 45 – 35 nmol/L (pH = 7.35-7.45)

B. ERYTHROCYTE HEMOGLOBIN / OXYHEMOGLOBIN BUFFER SYSTEM

a. Protons and O2 interchange on hemoglobin as a result of pH changes in the cells and the
circulation. As the O2 content increases, a proton is released and an O 2 molecule attaches to the
heme portion of the protein.
b. Hemoglobin, the protein present in high concentration in rbc (erythrocytes), binds oxygen in the
lungs and releases it in tissues
c. Protons and oxygen molecules interchanges on hemoglobin as a result of pH changes in the cells
and the circulation (when Hgb is protonated, it does not bind oxygen; as the oxygen content
increases, a proton is released and an oxygen molecule attaches to the heme portion of the
protein)
d. If we start in the lungs, protonate hemoglobin (HHb) entering the bloodstream; the high oxygen
 content in the lung (from respiration) adds oxygen to homoglobin with the loss of a proton
e. The oxyhemoglobin (HbO2) travels through the arterial circulation and reaches the cells; cellular
metabolism has generated protons, which attach to hemoglobin and cause oxygen to dissociate
and enter the cell
f. The deoxyhemoglobin (protonated Hgb) then carries the protons back to the lung through the
venous circulation encounters oxygen, and the process begins again
g. A major role of hemoglobin in pH maintenance is to remove protons from cellular sites and carry
them to the lung where they are utilized by the bicarbonate buffer system

C. PLASMA PROTEINS
a. Acts as buffers because both their free carboxyl and amino groups are able to bind H +.

D. PHOSPHATE BUFFER SYSTEM

H+ + HPO4- H2PO-4 or H3PO4
a. In the plasma at pH 7.4, 80% of the phosphate is in the form of HPO 4-; but in the acid urine, the
bulk of it exists as H2PO-4
b. At a pH 7.4, the ratio of HPO4- / H2PO-4 is: 4:1

2. ORGANS INVOLVED ON ACID-BASE BALANCE

I. RESPIRATORY REGULATORY MECHANISM
a. The lung’s pH regulatory mechanism is through the transport of H + by hemoglobin and the elimination
or disposal of carbonic acid in the form of exhaled CO 2.
 Hemoglobin entering the lung contains protons which are displaced as oxygen binds to this
protein
 The oxyhemoglobin moves through the arterial circulation to the cell
 Metabolism within the cell has produced carbon dioxide, which combines with water to form
carbonic acid; dissociation of carbonic acid yields hydrogen ions and bicarbonate anions
Krebs Cycle: CO2 + H2O + ATP
CA CA
H2O + CO2 H2CO3 H+ + HCO3-
 The hydrogen ions combine with oxyhemoglobin, displacing the oxygen and making it available to
the cells for further metabolism
 Both bicarbonate ions and protonated hemoglobin travel through the venous circulation back to
the lung
 In the lung, oxygen from respiration displaces protons, reforming oxyhemoglobin
 The displace protons combine with bicarbonate to form carbonic acid, which quickly dissociates
to carbon dioxide and water.
 The carbon dioxide is then removed from the lung by exhalation; the previously formed protons
are now part of water molecules.
b. Carbonic acid is the chief acid product of metabolism. This is neutralized by sodium bicarbonate. The
ratio of NaHCO3 to H2CO3 in the blood is 20:1
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

@ PULMONARY FUNCTION
A. FACTORS REGULATING THE PULMONARY FUNCTIONS
1. RESPIRATORY RATE
a. Increase in carbon dioxide decreases cellular pH and stimulates the respiratory center, while fall
in carbon dioxide has the opposite effect.
2. ALVEOLAR GAS EXCHANGE
a. Obstructive lung disease, such as, chronic bronchitis and asthma, cause trapping of air in the
alveoli preventing oxygenation as well as carbon dioxide exchange.
b. Disorders that impair pulmonary perfusion
c. Diseases that cause loss of airspace
3. NORMAL ALVEOLAR MEMBRANE
a. Alveolar wall thickening initially results to impairment of oxygen diffusion than that of carbon
dioxide leading to low arterial oxygen and carbon dioxide. With more severe damage, the ability
to excrete carbon dioxide is also often lost.
 ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

II. RENAL REGULATORY MECHANISM

The kidney’s major role of excreting acids produced in the body’s metabolic processes and
controlling mainly the plasma bicarbonate level.
There are several processes to attain equilibrium by the neutralization of H +.
a. In both the proximal and distal tubules, CO2 and H2O combine to form H2CO3, which then dissociates
into H+ and HCO3-. The H+ are then excreted into the tubular filtrate when Na + are reabsorbed.
Charge balance is provided by the exchange H+ for Na+ and the reabsorption of HCO3- with Na+. The
excreted protons (H+) can combine with bicarbonate in the tubular filtrate to produce water and carbon
dioxide; a portion of the CO2 is reabsorbed, and water may be reabsorbed or excreted in the urine.
b. A second process to neutralize hydrogen ions takes place in distal tubule, as H + are secreted into the
tubular filtrate, they combine with phosphate anions and be carried out in the urine:
H+ + HPO42- H2PO4 or H3PO4
Hydrogen ion secretion is again balanced by sodium reabsorption and co-transport back into the
circulation with bicarbonate forming NaHCO 3.
c. Also, a number of amino acids are deaminated in the distal tubule, creating ammonia as one of the
products; when ammonia is secreted into the tubular filtrate, it combines with protons to form
ammonium ion, which is then excreted in the urine.

NH3 + H+ NH4+ (excreted in urine)

V. HENDERSON-HASSELBALCH EQUATION
CA CA
H2O + CO2 H2CO3 H+ + HCO3-
Can be conveniently expressed as:
Bicarbonate
pH =
Carbonic acid (dissolved CO2)
OR
HCO3_
pH =
pCO2
OR
Function of kidneys (metabolic)
pH =
Function of lungs (respiratory)

VI. OXYGEN TRANSPORT AND DELIVERY

A. Factors that Affect Ability of Hemoglobin to Bind and Release Oxygen Normally
1. Factors that shift oxygen dissociation curve to the right:
a. Increase in hydrogen (decrease in pH)
b. Increase carbon dioxide
c. Increase temperature
d. Increase 2,3 diphosphoglycerate

2. Factors that shift the oxygen dissociation curve to the left:

a. Decrease in hydrogen (increase in pH)
b. Decrease carbon dioxide
c. Decrease temperature
d. Decrease 2,3-DPG
e. Presence of high affinity hemoglobins (Hgb F and carboxyhemoglobin)
VII. ACID-BASE IMBALANCE AND THEIR COMPENSATORY MECHANISMS
Acidemia / Acidosis – is defined as a blood pH of less tha 7.35, [H+] > 45 mmol/L

Classification of Acidemia:
1. Respiratory Acidosis
Cause: excess CO2 (hypercapnia) accumulation in the body, due to hypoventilation.
Compensatory mechanism: Increasing the reabsorption of HCO3 by the kidneys. .

2. Metabolic Acidosis
Cause: excess accumulation of fixed acids (non-volatile acids, other than H 2CO3) resulting to a primary
decrease (alkali loss) in HCO3-.

Two types of metabolic acidemia:

a. Acidemia with an increased anion gap (> 17 mmol/L)
Causes:
 Uremia with retention of fixed acids
 Ketotic states
 Lactic acidosis
 Toxin ingestion
 Increased plasma proteins
b. Acidemia with a normal anion gap (< 17 mmol/L), or hyperchloremic metabolic acidosis
Causes:
 Gastrointestinal losses
 Renal tubular acidosis (RTA)
 Mineralocorticoid deficiency
Compensatory mechanism: hyperventilation

Alkalemia / Alkalosis – is defined as a blood pH greater than 7.45, [H+] < 35 mmol/L
Classification of Alkalemia
1. Respiratory Alkalosis
Cause: excess pCO2 loss in the blood resulting from hyperventilation
Compensatory mechanism: decreasing the reabsorption of HCO3- by the kidneys.

2. Metabolic Alkalosis
Cause: excess loss of fixed acids or a primary increase (alkali excess) in HCO3-
Alkali excess can occur in:
a. Excessive ingestion of basic substances (ex. antacids)
b. Disease states in which there intracellular accumulation of H + and/or excess excretion of H+
into the urine, such as:
 Mineralocorticoid excess syndrome
 Hypokalemia
Compensatory mechanism: hypoventilation, or decreasing respiratory rate causing an increase in
pCO2.

CONSEQUENCES OF ACID-BASE IMBALANCE

1. In alkalosis, tenany ensues due to hypocalcemia, which can lead to death because of respiratory muscle
spasm.
2. In acidosis, there is an inhibition of the neural mechanisms which will then lead to coma. A blood ph of
6.9 has been proven fatal.

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

COMPENSATION IN ACID-BASE DISORDERS

A. Metabolic Disorders
1. The primary problem in these disorders is abnormal metabolism leading to changes in plasma
bicarbonate
2. Compensation of metabolic acid-base disorders involves a change in respiratory excretion of carbon
dioxide.
 B. Respiratory Disorders
1. These disorders are due to changes in pulmonary excretion of carbon dioxide.
2. Compensation for respiratory acid-base disorders involves altered renal handling of bicarbonate.
3. Following is a table of acid-base disorders

DISORDER PRIMARY COMPENSATION

CHARACTERISTIC
Respiratory acidosis ↑ pCO2 ↑ HCO3-, ↓ Cl-
Respiratory alkalosis ↓ pCO2 ↓ HCO3-, ↑ Cl-
Metabolic acidosis ↓ HCO3- ↓ pCO2
Non-anion gap metabolic acidosis ↓ HCO3- ↓ pCO2, ↑ Cl-
Metabolic alkalosis ↑ HCO3- ↑ pCO2, ↓ Cl-

 In mixed acid-base disorders, however, blood pH is not an accurate determination of acid-base

status.
 Determination of bicarbonate, blood pCO2, serum chloride and anion gap gives more reliable result in
mixed acid-base disorders.
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

ANION GAP
In plasma, there is normally an exact balance of positively and negatively charged ions (electroneutality).
Major plasma cations: Na+ (140 mEq/L), K+ (4 mEq/L), Ca++, Mg++, and immunoglobulins
Major plasma anions: Cl- (100 mEq/L), HCO3- (24 mEq/L), phosphate, sulfate, amino acids, most
proteins

Normally, the total amount of each is approximately 150 – 155 mmol/L. The anion gap is then defined as:
Anion gap = Na+ -- (Cl- + HCO3-)
Normally, anion gap would be around 16 mEq/L, which actually compreised the other counter-ions that
neutralize sodium but are not measured in serum.

Interpretations:
1. Increase anion gap = typically due to replacement of HCO3- with the anion of an organic acid such as
(25.30 mEq/L) acetoacetic acid (in diabetic acidosis) or lactic acid (in sepsis or hypoperfusion)
= thus, the amount of anion gap is an indirect measure of the molar concentration of
acid added to the system
2. Low anion gap = signifies the presence of high levels of basic protein (often myeloma protein). Basic
(1-3 mEq/L) protein contains the “invisible” ammonium ion, the counter-ions for which are chloride
= persistently low anion gaps are serious signs of possible malignancy (ex. myeloma)

VIII. REFERENCE VALUES

* pH = 7.35 – 7.45

* pCO2 = 35 – 45 mm Hg
* pO2 = 80 – 110 mmol/L
HCO3- = 22 – 26 mmol/L
Total CO2 = 23 – 27 mmol/L
O2 saturation = > 95%
O2Hb = > 95%

(*) are the only ones being measured, the rest are calculated

IX. PARAMETERS OF INTEREST

1. Evaluate the pH: (normal pH = 7.35 – 7.45)
< 7.35 = acidosis (7.31-7.34)
> 7.45 = alkalosis (7.46-7.49)
2. Evaluate the ventilation (lungs): pCO2 = 35-45 mm Hg
< 35 = respiratory alkalosis
> 45 = respiratory acidosis

3. Evaluate the metabolic process (kidneys): HCO3- = 22-26 mEq/L

< 22 = metabolic acidosis
> 26 = metabolic alkalosis

4. Determine which is the primary (1°) and compensating disorder = pH

5. Determine the degree of compensation:

a. Non-compensatory / Uncompensatory
b. Partial compensation
c. Complete compensation / Full compensation

pH pCO2 HCO3-
Non-compensatory A A N
N A
Partial compensation A A A
Complete compensation Nearly N A A

6. Evaluate the degree of oxygenation

pO2 = 80 – 110 mm Hg (adequate oxygenation)
decreased pO2 = hypoxemia (decreased oxygenation in the blood)
 Mild = 61 – 79
 Moderate = 41 – 60
 Severe = 40 or less

Increased pO2 = hyperoxemia

7. Final Interpretation:
a. Degree of compensation
b. Primary disorder
c. Degree of oxygenation

8. Examples:

A) pH = 7.22 acidosis
pCO2 = 20 mm Hg respiratory alkalosis
HCO3 = 11 mEq/L metabolic acidosis
pO2 = 66 mm Hg moderate hypoxemia

Partially compensated metabolic acidosis with mild hypoxemia

B) pH = 7.48 alkalosis (but 7.48 is nearly normal)

pCO2 = 48 mm Hg respiratory acidosis
HCO3 = 30 mEq/L metabolic alkalosis
pO2 = 50 mm Hg moderate hypoxemia

Complete compensatory metabolic alkalosis with moderate hypoxemia