Clinical enzymology

University of Babylon

College of pharmacy

Second semester - biochemistry

3rd class 2014 – 2015

By

Dr. Abdulhussien M. K. Aljebory

Enzyme activityEnzyme assays usually depend on the measurement the

catalytic activity of the enzyme, rather than the concentration of the enzyme protein itself.

• One international unit is the amount of

enzyme that will convert one micromole

of substrate per minute per litre of

sample and is abbreviated as U/L.

• Katal (catalytic activity) is defined as

the number of mole of substrate

transformed per second per litre of

sample.

Disadvantages of enzyme assaysA major disadvantage in the use of enzymes for the

diagnosis of tissue damage is their lack of specificity to a particular tissue or cell type. Many enzymes are common to more than one tissue.

• This problem may be obviated to some extent in two ways:

• first, different tissues may contain (and thus release when they are damaged) two or more enzymes in different proportions

• Second, some enzymes exist in different forms (isoforms)

• Individual isoforms are often characteristic of a particular tissue: although they may have similar catalytic activities

Enzymes as Cardiac Markers

Creatine kinase (CK or CPK)

Aspartate amino transferase (AST)

Lactate dehydrogenase (LDH or LD)

Creatine KinaseA cytoplasmic and mitochondriac enzyme

Total CK is %40 sensitive and %80 specific

CK-1(BB) : Brain

CK-2(MB) :Myocardium

CK-3(MM) :Skeletal muscle ,heart

CK-3 has 3 isoforms :CK-31, CK-32 and CK-33 (posttranslational products)

CK-2 has 2 isoforms

Half life :CK-31 > CK-32>CK-33, CK-21>CK-22

• CK-MB (CK-2) has the most specificity for cardiac muscle (>%85)

• CK-MB (CK-2) accounts for only 3-20 % of total CK activity in heart

• Normal skeletal muscle contains 1 % CK-2

CK-2 is increased in severe skeletal muscle injury (trauma/surgery ),chronic muscle disease (muscular dystrophy, polymyositis ) and extreme exercise.

Reason:Regeneration process of muscle (reexpression of CK-MB genes )

In AMI,CK-MB increases at least 4-6 hr.after onset of chest pain,makesa peak at 12-24 hr. and returns to baseline level in 2-3 days (t 1/2 is 10-12 hr )

Factors that might affect the classic pattern

1-Size of infarction

2-CK-2 composition in myocardium

3-Concomitant skelatal muscle injury and reperfusion

Differentation of increased CK-2 due to heart or muscle monoclonic anti CK-2 antibody based assays

%relative index (%RI)= CK-2 activity /total CK X %100

helpful in the detection of AMI

• CK-22: the predominant isoform in blood collected 2-6 hr. after AMI .It makes a peak ≈10-18 hr.,decreases rapidly 12-24 hr concomitantly as CK-21 gradually increases.

Following trombolitic therapy in AMI patients, greater than two fold increases of CK-2 occur within 90 min. of reperfusion

• The rate of which CK-2 isoform is released from the myocardium to circulation appears to be the more useful index of coronary reperfusion

• CK22 ∕ CK-21 ratio→best discriminant within 1 hr. after treatment

Lactate Dehydrogenase (LDH)

A cytoplasmic enzyme found in skeletal, muscle, liver, heart, kidney and

red blood cells

• 5 izoenzymes, composed of 4 subunit peptides of 2 distinct types:M

(muscle ), H (heart)

• LDH isoenzyme determination increases specificity for cardiac tissue.

– LD1 (HHHH):moves fastest towards anode

– LD5 (MMMM):moves closest towards cathode

– LD2 (HHHM )

– LD3 (HHMM )

– LD4 (HMMM )

– LD1 in heart,kidney(cortex), red blood cells

– LD5 in liver,skeletal muscle

In healthy adults:

14-26 % LD1 (slow removal )

• 29-39% LD2

• 20-26% LD3

• 8-16 % LD4

• 6-16 % LD5 (rapid disappearance )

• Not a tissue specific enzyme

• In AMI total LD -elevation at 12-18 hr– peak at 48-72 hr

– return to baseline level after 6-10 days

LD 1 (heart)

– elevation at 10-12 hr

– clinical specificity 85-90 in patients suspected of AMI

– peak at 72-144 hr

– return to normal >10 days

• Total LD patern ≈LD1 pattern (contrast with total CK and CK-2 pattern)

• Because of its prolonged half life, LD-1 is a clinically sensitive (%90)

marker for infarction when measured after 24 hr.

• LD1∕LD2 >1,0 →FLIPPED PATTERN (clinical sensitivity

%75 , clinical specificity 85-90 in patients suspected of AMI)

• if CK-2 values are diagnostic for AMI, cancel LD isoenzyme test request,

because LD isoenzyme results don’t increase the clinical specificity of

diagnostic CK-2 results for AMI

• As with CK-2 in skeletal muscle, the heart

specific LD-1 isoenzyme in skeletal

muscle can increase twofold during a 9-wk

exercise training, with paralel decreases in

LD-5

• LD and isoenzymes play no role in

unstable and stable angina.

Aspartate aminotransferase (AST )

Widely distributed in many tissues

• Highest concentrations are found in cardiac tissue, liver and skeletal muscle.

• Clinical utility in hepatocellular disorders and skeletal involvement.

• Unuseful in diagnosis of AMI

• Beginsto rise within 6-8 hr.

• Makes a peak at 24 hr.

• Generally returns to normal within 5 days.

Functions of liver

① Excretory function: bile pigments, bile salts and cholesterol are excreted in bile into intestine.

② Metabolic function: liver actively participates in carbohydrate, lipid, protein, mineral and vitamin metabolisms.

③ Hematological function: liver is also produces clotting factors like factor V, VII. Fibrinogen involved in blood coagulation is also synthesized in liver. It synthesize plasma proteins and destruction of erythrocytes.

④ Storage functions: glycogen, vitamins A, D and B12,and trace

element iron are stored in liver.

⑤ Protective functions and detoxification: Ammonia is detoxified to

urea. kupffer cells of liver perform phagocytosis to eliminate foreign

compounds. Liver is responsible for the metabolism of xenobiotic.

Classification of liver functions test

Classified based on the major functions of liver:

① Excretion: Measurement of bile pigments, bile salts.

② Serum enzymes: Transaminase (ALT, AST), alkaline

phosphate(ALP), 5’-nucleotidase, LDH isoenzyme.

③ Synthetic function: Prothrombin time, serum albumin.

④ Metabolic capacity: Galactose tolerance and

antipyrine clearance

⑤ Detoxification :

Serum enzymesA large number of enzyme estimations are available which are used to

ascertain liver function. They are be divided into two groups:

• I: most commonly and routinely done in the laboratory.

• serum transaminase(ALT/AST)

• serum alkaline phosphate(ALP)

• II: not routinely done in the laboratory.

• ALT or sGPT (serum glutamate pyruvate transaminase)

AST or sGOT (serum glutamate oxaloacetate transaminase)

organ GOT GPT

heart 156000 7100

liver 142000 44000

skeletal 99000 4800

kidney 91000 19000

organ GOT GPT

pancrease

spleen

lung

serum

28000 2000

14000 1200

10000 700

20 16

GPT:

Normal range: 2-59 U/L

GOT:

Normal range: 10-34 U/L

Therefore, when the liver is injured, GPT is released into the

bloodstream.

Elevated levels of GPT may indicate :

alcoholic liver disease

cancer of the liver

cholestasis or congestion of the bile ducts

cirrhosis or scarring of the liver with loss of

function

death of liver tissue

Hepatitis or inflammation of the liver

noncancerous tumor of the liver

use of medicines or drugs toxic to the liver

GOT

GOT also reflects damage to the hepatic cells and

is less specific for liver disease. It can also be

released with heart, muscle and brain disorders.

Therefore, this test may be ordered to help

diagnose various heart, muscle or brain disorders,

such as a myocardial infarct (heart attack).

Elevated levels of GOT may indicate :

acute hemolytic anemia, acute pancreatitis or inflammation of the pancreas. acute renal failure or loss of kidney function. cirrhosis of the liver. Hepatitis heart attack primary muscle disease recent surgery severe burns muscle injury

Although GOT is not a specific for liver as the GPT, ratios between GPT and GOT are

useful to physicians in assessing the etiology of liver enzyme abnormalities.

◆ normally: GPT is normal, GOT is normal, GPT/GOT is about 1.15.

◆ Virus hepatitis: GPT↑, GOT is normal ,GPT/GOT＞1,even more than 2.5；

◆ chronic hepatitis : GPT↑ ,GOT ↑GPT/GOT is about 1.

◆ Liver cancer, cirrhosis, Alcohol-induced hepatitis: GPT↑ ,GOT ↑ < 1, about 0.6~0.7.

◆Accute myocardial infarct :< 1

GPT and GOT is in the different distribution of the hepatocytes. GPT exists primarily in

the cytoplasm of liver cell. if there is a slight liver cell damage, GPT firstly leak into the

bloodstream, so that the serum GPT increased.

The GOT mainly in the "mitochondria“of liver cells, the mitochondria are "bubble" in the

liver cell cytoplasm. if there is a slight liver cell damage, GOT don`t leak into the

bloodstream.

When the GOT was significantly higher, mitochondria of liver cells are injuries.

Alkaline phosphatase (ALP)• Normal serum level of ALP is 40-125 U/L or 0.5-1.3

mmol/(hour L).• This enzyme present in high concentrations in the• liver• bone (osteoblasts)• placenta • intestinal epithelium.

• ALP is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. – most effective in an alkaline environment

– in humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta.

• Levels are significantly higher in children and pregnant women.

Higher levels of ALP than normal may indicate:

liver disease

bone disease

leukemia, a cancer of the blood and bone marrow

various hormone problems

pregnancy

Lower levels of ALP than normal may indicate:

anemia, or a low red blood cell count

malnutrition

various hormone problems

Mechanism of increase in ALP in liver disease:

• Increase in the activity of ALP in liver disease is not due to hepatic cell disruption , nor to a failure of clearance , but rather to increased synthesis of hepatic ALP .

• The stimulus for this increased synthesis in patients with liver disease has been attributed to bile duct obstruction by stone ,tumors , intrahepatically by infiltrative disorders or space-occupying lesions.

• It is used for many years in differential diagnosis of jaundice. it is increased in both infectious hepatitis (viral hepatitis) and posthepatic jaundice, but the rise is usually much greater in case of obstructive jaundice .

• dividing line which has been suggested is 35KA units/ml. a value higher than 35KA units/ml is strongly suggestive of diagnosis of obstructive jaundice, in which very high figures even up to 200KA units/ml or more may be found.

• Serum ALP is found to be normal in haemolytic jaundice.