1. Plasma Enzyme tests in
diagnosis
Dr. Elham Sharif,PhD
Assistant professorof BiomedicalSciences
College of Health Sciences
Qatar University
Tel: 00974-4403-4788
Email: e.sharif@qu.edu.qa
2. Objectives
Dr Elham Sharif 3
• Identify/discuss the different factors affecting the rate of an enzymatic reaction.
(TL1)
• Identify/explain enzyme kinetics, including zero-order and first-order kinetics. (TL1)
• Identify/explain why the measurement of serum enzyme levels is clinically useful.
(TL1)
• Identify/discuss which enzymes are useful in the diagnosis of various disorders,
including cardiac, hepatic, bone, and muscle disorders, malignancies, and acute
pancreatitis.
• Identify/discuss the tissue sources, diagnostic significance, and assays, including
sources of error, for the following enzymes: CK, LD, AST, ALT, ALP, ACP, GGT,
amylase, lipase, cholinesterase, and G-6-PD. (TL1)
• Evaluate and assess laboratory data of patient serum enzyme levels in relation to
disease states. (TL3)
• Identify/discuss the role of enzymes in drug metabolism. (TL1)
• Calculate the clinical sensitivity, clinical specificity, positive and negative predictive
value of a given enzyme test. (TL2)
• Interpret the biochemical data used in the investigation and diagnosis of disease,
through interpreting problem based case studies. (TL3)
3. ENZYMES
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Measurements of the activity of enzymes in plasma are of value in the diagnosis
and management of a wide variety of diseases.
Most enzymes measured in plasma are primarily intracellular, being released into
the blood when there is damage to cell membranes,
but many enzymes, for example renin, complement factors and coagulation factors, are
actively secretedinto the blood, where they fulfill their physiological functions.
Small amounts of intracellular enzymes are present in the blood as a result of
normal cell turnover.
When damage to cells occurs, increased amounts of enzymes will be released
and their concentrations in the blood will rise.
However, such increases are not always due to tissue damage. Other possible
causes include:
increased cell turnover
cellular proliferation (e.g. neoplasia)
increased enzyme synthesis (enzyme induction)
obstruction to secretion
decreased clearance.
4. Disadvantages of enzyme assays
5
1. Lack of specificity for a particular tissue or cell type:
because many enzymes are common to more than one tissue.
Overcome by:
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), termed isoenzymes;
Individual isoforms are often characteristic of a particular tissue:
although they may have similar catalytic activities, they often differ in some
other measurable property, such as heat stability or sensitivity to inhibitor
After damage to a tissue, the activity of intracellular enzymes in the plasma rises
as they are released from the damaged cells, and then falls as the enzymes are
cleared.
It is thus important to consider the time at which the blood sample is taken in
relation to the damage.
If taken too soon, there may have been insufficient time for the enzyme to
reach the bloodstream,
and if too late, it may have been completely cleared.
Dr Elham Sharif
5. Enzyme composition
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• Enzymes are proteins,
• biological catalysts that appear in the serum in increasing
amounts after cellular injury or tissue damage.
• Isoenzyme:
o different forms of the same enzyme that exist in the body (differ in
quaternary structure).
• Active site or catalytic site:
o Enzymes have a region with specific structurethat binds the
substrateand facilitates conversion to product.
o It confer the specificity of an enzyme for its substrate and the
reaction it catalyses.
6. Reaction
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Note: Enzymes are susceptible to denaturation due to
changes in their physical surrounding.
The activity of an enzyme can be enhanced or inhibited by
conditions and factors:
1. Cofactor & Coenzyme
Many enzymes require a cofactor or coenzyme for full activity.
Cofactor:
is a small, inorganic compounds, such as metal ions
a non-protein compound that may be necessary for enzyme activity.
Activators:
non-organic enzyme activators such as Mg or CL.
Coenzyme:
organic compounds, Vitamins.
7. Terminology for enzymes & their
associated cofactors & coenzymes
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• Prosthetic groups:
o Cofactors or coenzymes that are tightly bound and difficult
to dissociate from the enzyme.
• Apoenzymes:
o enzymes lacking their prosthetic groups.
• Holoenzyme:
o Combined enzyme and prosthetic group.
• Metalloenzyme:
o enzymes that have a metal ion prosthetic group.
8. Enzyme kinetics & activity
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• Role of enzyme is to function as biological catalysts.
• Enzyme accelerates the rate at which chemical reaction take
place, without being consumed in the reaction process.
• Enzyme assays usually depend on the measurement of the
catalytic activity of the enzyme, rather than the concentration
of the enzyme protein itself.
• Since each enzyme molecule can catalyse the reaction of
many molecules of substrate, measurement of activity
provides great sensitivity.
• It is, however, important that the conditions of the assay are
optimized and standardized to give reliable and reproducible
results.
9. Factors That Influence Enzyme Reaction
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• 1. Substrate concentration:
A. At low concentrations of substrate, the rate of the enzyme catalyzed reaction
is directly proportional to substrate concentration (first order kinetics) (one
reactant).
A. When the substrate concentration is high enough to bind with all available
enzyme, the reaction velocity is at its maximum and when the product is
formed, the enzyme reacts with more substrate (zero order kinetics).
A. Assays to measure enzyme activity in serum are designed for zero order
kinetics, since the rate of the reaction should be dependent only on the conc.
of the enzyme present in the specimen being analyzed.
10. cont
Dr Elham Sharif 11
• 2. Enzyme concentration: as long as the substrate
concentration exceeds the enzyme concentration, the velocity
of the reaction is proportional to the enzyme concentration.
• 3. pH: this variable must be controlled very carefully because
extreme pHs can denature an enzyme, change its ionic state
and possibly its active site, and change its activity completely.
• 4. Temperature: increasing the temperature increases the rate
of a chemical reaction by increasing molecular energy.
Enzymes have optimal reaction temperatures which is usually
37ºC. (Note: that enzymes are inactivated at 55-60 °C).
11. Factors cont
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• 5. Inhibitors:
o A substance that interferes with an enzyme catalyzed reaction.
• Competitive inhibitor:
- Occurs when a substance (inhibitor) binds free enzyme at the active
site, compete with substrate for the active site & preventing it. This inhibition
is reversible.
E+S+IES+EIE+P
• Noncompetitive inhibitor:
o occur when an inhibitor combines with either the free enzyme or the
enzyme substrate complex.
- This inhibition may be reversible or irreversible. It may be irreversible
if it destroys part of the enzyme or makes the active site inaccessible.
• E+S+IES+EI+ESIE+P
• Uncompetitive inhibitor:
o Occurs when inhibitor binds to the enzyme substrate complex only, so
that increasing the substrateconcentration leads to more enzyme
substratecomplexes and more inhibition.
• E+S+IESI+ESE+P
12. Measuring Enzyme Activity
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• 1. Enzyme reactions are performed in zero-order kinetics,
with enough substrate in excess to ensure only 20 percent
is converted to product.
• 2. It is extremely important in enzyme reactions that the
pH, temperature, and additives (i.e., cofactors,
coenzymes, activators) remain constant.
• 3. There are two methods used to measure enzyme
reactions: endpoint and kinetic.
o a. Endpoint: these reactions combine reactants and at a fixed time (i.e., 5
minutes) the products are measured. Concentration of the enzyme is based on the
final absorbance reading.
o b. Kinetic: these reactions combine reactants, then measure the change in
absorbance at specific time intervals (i.e., 30, 60 seconds) over a specific time
interval. Concentration of the enzyme is based on the change in absorbance over
time.
13. E. Calculation of Enzyme Activity
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• 1. Enzymes are reported in activity units because they are
reported relative to their activity instead of their true
concentration. i.e IU
• 2. International unit (IU): the amount of enzyme that will
catalyze the reaction of one micromole of substrate per
minute under specified conditions of temperature, pH,
substrates, and activators.
14. ENZYME CLASSIFICATION
• 1. Oxidoreductases.
o Catalyze an oxidation-reduction reaction between two substrates
• 2. Transferases.
o Catalyze the transfer of a group other than hydrogen from one
substrateto another
• 3. Hydrolases.
o Catalyze hydrolysis of various bonds
• 4. Lyases.
o Catalyze removal of groups from substrates without hydrolysis; the
product contains double bonds
• 5. Isomerases.
o Catalyze the interconversion of geometric, optical, or positional
isomers
• 6. Ligases.
o Catalyze the joining of two substrate molecules, coupled with breaking of the
pyrophosphate bond in adenosine triphosphate (ATP) or a similar compound
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22. Alkaline phosphatase (ALP)
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• ALP is a general name for a group of phosphohydrolases that
demonstrate an alkaline pH optimum; about 9 to 10.5.
• Mg2+ is a cofactor
• Metalloenzyme with Zn2+
• In health: ALP in serum is from bone & liver origin.
• ALP facilitate bone formation & synthesized by osteoblasts.
• 5 Isoenzyme:
o Bone, liver, placental, kidney, spleen
o Specimen: fresh serum, or plasma with heparin as anticoagulant.
23. Source of Error
• Hemolysis may cause slight elevations because ALP is approx:
6x > concentrated in RBC than in serum.
• ALP assays should be run as soon as possible after collection.
• Activity in serum increases approximately 3% to 10% on
standing at 25°C or 4°C for several hours.
• Diet may induce elevations in ALP activity of blood group B
and O individuals who are secretors.
• Values may be 25% higher following ingestion of a high-fat
meal.
• The most heat labile fraction of alkaline phosphatase is
obtained from the bone
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24. ALP activity increase in
Physiological Pathological
• Rise in 20% of (blood O& B)
after meal, intestinalALP).
• Childhood, due to bone
growth, age specific RI.
• Healing of bone fracture
o mild increase in bone repair
and growth
• 3d trimester of pregnancy.
• > 40 years due to bone
turnover.
• Bone disease, with increase
osteoblastic activity:
o Highest elevations of ALP:
• > 10-25x URL Paget’s disease.
(bone disease).
• > 2-4 X URL Rickets
• Osteomalacia, bone tuomors
o ALP levels increase with healing bone
fractures.
o In Extra-hepatic obstruction of bile flow
(Cholelithiases (gallstone) or pancreatic
tumor, ALP levels are >3-10x URL.
o In hepatobiliary disorders, the increased
levels are due to obstructive disease
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26. 2. Aspartate aminotransferase (AST)
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• Highest concentrations: cardiac tissue, liver, and skeletal muscle,
RBCs.
• AST isoenzymes - cytoplasm, mitochondria
• AST is used to evaluate:
o Acute myocardial infarction (AMI),
o Hepatocellular disorders,
o Skeletal muscle disorders.
• AST is usually elevated in pulmonary embolisms, extremely
elevated in liver disease (100 times ULN in viral hepatitis and 4
times ULN in cirrhosis), skeletal muscle disorders, and
inflammatory conditions.
27. AST
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Very high levels, sometimes in excess of 100 × ULN, are seen
with severe tissue damage, such as in acute hepatitis, crush
injuries and tissue hypoxia.
More usually in hepatitis, the peak level is only 10-20 × ULN.
In myocardial infarction:
plasma AST begins to rise 12 h afters the infarct,
Peak 10× ULN at 24-36 h
decline over 2-3 days providing that there is no further cardiac
damage.
In most conditions in which AST is elevated, there is a concurrent,
though proportionally smaller, rise in ALT.
In hepatitis, however, plasma activities of ALT may exceed those
of AST.
29. Aspartate aminotransferase (AST) cont,
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• Assay
• Coenzyme pyridoxal-5’-phosphate (P-5’-P) (vitamin B6) for
both AST & ALT
30. Source of Error in AST assay
• Hemolysis should be avoided because it can dramatically
increase serum AST concentration.
• AST activity is stable in serum for 3 to 4 days at refrigerated
temperatures.
• Plasma collected in EDTA, oxalate or citrate.
• Best plasma or serum collected in heparin tube.
• Reference range: 5-30 IU/L at 37C
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32. 3. Alanine aminotransferase (ALT)
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• a. High concentrations in the liver.
• b. Diagnostic significance: liver disorders, hepatocellular disorders
(hepatitis, cirrhosis) are higher than intra or extrahepatic obstruction.
• AST and ALT: widely distributed, ALT more liver specific, AST more
muscle specific
ALT is stable for 3 to 4 days at 4°C.
It is relatively unaffected by hemolysis.
33. Gamma-glutamyltransferase GGT
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This enzyme is present in high concentrations in the liver, kidney, intestine and pancreas.
Increased levels in all hepatobiliarydiseases.
Measurement of its plasma activity provides a sensitive indicator of hepatobiliary
disease although it is of no value in distinguishing between cholestatic and
hepatocellular disease.
Higher levels observed in biliary tract obstruction.
In biliary obstruction, plasma GGT activity may increase before that of alkaline
phosphatase.
Sensitive indicator of alcoholism: enzyme elevations are from 2 to 3 times normal.
Plasma GGT is raised in the absence of liver disease in many patients taking the:
anticonvulsant drugs phenytoin and phenobarbital; rifampicin, used in the treatment of
tuberculosis, can have a similar effect. This is an example of enzyme induction.
The increased plasma GGT is not due to cell damage but to an increase in enzyme
production within cells with the result that an increased amount is released during normal
cell turnover.
Levels can be also increased in pancreatitis, diabetes mellitus, and AMI.
35. GGT
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Source of Error
• GGT activity is stable, with no loss of activity for 1 week
at 4°C.
• Hemolysis does not interfere with GGT levels because
the enzyme is lacking in erythrocytes.
36. Creatine kinase (CK) and CK isoenzymes
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37
• Phosphocreatine - high energy phosphate compound in muscle
• widely distributed - highest amount in muscle, heart, brain
• CK is regulated by excretion by the kidneys.
• CK concentration is influenced by sex, muscle mass, physical activity, and race.
o In serum, healthy individuals have CK-MM as the major isoenzyme and a
small amount of CK-MB (< 6% of total CK), whereas CK-BB is not
normally detectable.
o Normally higher in male than female.
o Increase with sport.
• For its activity requires Mg+2
• CK consists of two subunits: M for muscle and B for brain.
• Each CK isoenzyme is a dimer with three possible types:
o CK-1 (BB) Brain & prostate gland.
o CK-2 (MB) Cardiac muscle
o CK-3 (MM) skeletal muscle
updated
37. Dr Elham Sharif
38
• Total CK increased
-muscle trauma
-myocardial infarction
-muscular dystrophy
-cerebral ischemia
-head injury
-Reye’s syndrome
• CK-2 (MB) increase in myocardial infarction MI
o Following AMI, CK-MB levels rise within 4-6 hours, peak at 12-24
hours, and return to normal within 2-3 days.
o CK-MB can be measured either by measurement of enzyme activity in the
presence of an antibody that inhibits the M subunit,
o or by measurement of enzyme mass using an immunoassay.
• Analysis of isoenzymes – electrophoresis - detection by enzyme reaction
leading to NADPH and reduction of tetrazolium salt by NADPH to a colored
formazan.
updated
41. Test methodologies
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42
• 1) CK isoenzymes
o Measured by electrophoresis, ion-exchange chromatography, ELISA, and
RIA.
• 2) Oliver-Rosalki CK test methodology
• Hemolysis of serum samples may be a source of falsely elevated CK activity
• Serum should be stored in a dark place because CK is inactivated by light.
42. Acid phosphatase (ACP)
Dr Elham Sharif 43
• Tissue location: Highest concentration in prostate gland & seminal fluid, with
lesser amounts in bone (osteoclasts), liver, spleen, erythrocytes, platelets.
• a group of phosphatases with an optimum pH of 7
• up to 50% of serum activity in males due to prostatic ACP.
• Increase in ACP in benign hypertrophy of the prostate
• Mild increase in some bone cancers.
• ALSO Increased in Paget disease, breast cancer with bone metastases,
Gaucher disease, platelet damage, idiopathic thrombocytopenic purpura.
• immunoassay methods, specific for prostatic ACP.
• Clinical significance
o 1) Diagnosing prostate cancer.
o 2) More common in men over 50 years of age
o 3) chemical determination maybe helpful in establishing the presence of
seminal fluid.
• ACP is useful in forensic cases involving rape because vaginaL
washings containing seminal fluid would exhibit ACP activity.
UPDATE
43. ACP cont.
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Specimens to be used in acid phosphatase assays should be:
• Placed on ice as soon as they are obtained
• acidified if stored
• Analyzed as soon as possible
44. Source of Error
• Serum should be separated from the red cells as soon as the blood has
clotted to prevent leakage of erythrocyte and platelet ACP.
o Hemolysis should be avoided because of contamination from erythrocyte
ACP.
• Serum activity decreases within 1 to 2 hours if the sample is left at RT without
the addition of a preservative.
o Decreased activity is a result of a loss of carbon dioxide from the serum,
with a resultant increase in pH.
• If not assayed immediately, serum should be frozen or acidified to a pH lower
than 6.5.
o With acidification, ACP is stable for 2 days at room temperature.
• RIA procedures for measurement of prostatic ACP require non-acidified serum
samples. Activity is stable for 2 days at 4°C.
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45. Prostate specific antigen PSA
• PSA is a serine protease released by the prostate
• more specific and sensitive than ACP
• mild increase seen in hypertrophy of the prostate.
• Increase in prostate cancer.
• PSA measured by specific immunoassay techniques
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46. Amylase AMS
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47
• Serum amylase produced by pancreas and salivary glands, small intestine,
fallopian tubes.
• Clinical significance
o 1) Diagnosis: acute pancreatits.
• Occur in 2-12 hours after the onset of pain, with peak values in 24 hours,
and return to normal in 3-4 days.
o 2) Increased: mumps, perforated peptic ulcer, intestinal obstruction,
cholecystitis, ruptured ectopic pregnancy, mesenteric infection, and acute
appendicitis.
o 3) Amylase has isoenzymes originating from the pancreas (p type) and the
salivary glands (S type), and these tissue-specific isoenzymes can be
distinguished by means of electrophoresis or the use of inhibitors.
• Optimum pH 7.0
• Metalloenzyme - requires Ca+2
• c. Assay
o 1) Amyloclastic: measures the decrease in the starch substrate.
o 2) Saccharogenic: measures the increase of the product produced (maltose).
o 3) Chromogenic: measures the increase of the product that is coupled with a
chromogenic dye.
o 4) Enzymatic: combines several enzyme assays to monitor activity.
o Reference range: serum: 95-290 IU/L; urine: 35-400 IU/h.
47. Amylase
Dr Elham Sharif
48
• Plasma amylase activity is usually increased, often to 5× or even to >10
× ULN, in acute pancreatitis.
• It is used in the diagnosis of patients presenting with an acute
abdomenal pain, and the other causes of an increase in plasma amylase
activity.
• Diagnosisof acute pancreatitis
o serum levels increase 2 - 12 hr after onset
o peak levels 12 - 72 hr
o return to normal 3 - 4 days
o 4 - 6 x increase in blood
o greater increase in urine
o 20 % of all cases are normal
• typically normal serum amylase levels in chronic pancreatitis
48. Source of Error in amylase assay
• AMS in serum and urine is stable.
• Little loss of activity occurs at RT for 1 week or at 4°C for 2
months.
• Because plasma triglycerides suppress or inhibit serum AMS
activity, AMS values may be normal in acute pancreatitis with
hyperlipemia.
• The administration of morphine and other opiates for pain relief
before blood sampling will lead to falsely elevated serum AMS
levels.
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49. Lipase LPS
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• Tissue location: Found in pancreas, with lesser amounts in gastric mucosa,
intestinal mucosa, adipose tissue
• FUNCTION: Hydrolyses dietary triglycerides.
• Clinical significance:
• Increased: Perforated peptic ulcer, duodenal ulcers, intestinal obstruction,
cholecystitis
• Confirms diagnosis of acute pancreatitis.
• Clinical use - diagnosis of acute pancreatitis
o 2 - 50x increase in serum
o increased levels in serum 4 - 8 hr after onset
o peak at 24 hr
o normal levels 8 - 14 days
o levels parallel amylase, but start earlier and last longer more
specific than amylase
50. Measurement of lipase activity
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51
1. Titrimetric methods
•Enzyme added to standard oil suspension, olive oil or triolein
•Enzyme activity releases fatty acids
•Titrate fatty acids with standard alkali
2. Turbidimetric methods
•Measure light scatter of standard oil suspension, olive oil or triolein
•Enzyme added to oil suspension
•Monitor enzyme activity by following the clarification of the sample.
51. Source of Error
• LPS is stable in serum, with negligible loss in activity
at RT for 1 week or for 3 weeks at 4°C.
• Hemolysis should be avoided because hemoglobin
inhibits the activity of serum LPS, causing falsely low
values.
• Reference range: Up to 38 U/L at 37°C
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52. Cholinesterase
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• This enzyme is secreted by the liver into the blood-stream and low
plasma activities occur in chronic hepatic dysfunction.
o It is, however, rarely measured for this reason.
• Plasma cholinesterase activity also falls in organophosphate poisoning.
• Low activities occur physiologically during pregnancy.
• Group of two enzymes: acetycholinesterase (ACHE) and
pseudocholinesterase(SChE).
• Clinical significance
• 1) Pseudocholinesterase: indicator of complications in hepatocellular
disease and of prognosis in cancer.
• 2) Decreased SChE: fertilizer and insecticide poisonings.
53. Tissues containing the cholinesterase
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AchE: mediates neurotransmission
Nervous system
RBCs
Lungs
Spleen
SChE:
Serum
Heart
Liver
Pancrease
White matter of the brain.
55. Glucose-6-phosphate Dehydrogenase (G-6-PD)
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• Tissue location: Found in erythrocytes, adrenal glands, thymus,
lymph nodes, spleen
o Very little G-6-PD in healthy individual’s serum.
• Clinical significance
• 1) Deficiency: RBC hemolysis
• 2) Increased: megaloblastic anemia's and AMI.
56. Glucose-6-phosphate Dehydrogenase (G-6-PD),
Cont’d
1) Decreased: Primary importance of G6PD is in cases of
deficiency, inherited as a sex-linked trait (X-chromosome).
a. In G6DP deficiency, a drug-induced hemolytic anemia
occurs when an individual is administered antimalarial
drugs or primaquine.
b.Hemolysis may also be caused by infections and after
ingestion of fava beans.
2) Increased: Megaloblastic anemias and AMI.
• Test methodology
• 1) G6PD deficiency requires the analysis of a red blood cell
hemolysate.
• 2) Analysis of G6PD elevations requires a serum sample.
• 3) Reference range (RBC): 8-14 U/g Hgb
uodated
57Dr Elham Sharif
57. Lactate dehydrogenase (LD/LDH)
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• Cytoplasmic enzyme present in all cells
• This enzyme exists in body tissues as a tetramer. Two monomers, H and M,
can combine in various proportions with the result that five isoenzymes of LD
are known.
• LD Isoenzymes
o LD-1 HHHH H4 myocardium & RBCs
o LD-2 HHHM H3M1 myocardium & RBCs
o LD-3 HHMM H2M2 Lungs & spleen
o LD-4 HMMM H1M3 Lungs & spleen
o LD-5 MMMM M4 Liver & skeletal muscle
• Increases in plasma LD activity are seen in a wide variety of conditions
including acute damage to the liver, skeletal muscle and kidneys, and also in
megaloblastic and haemolytic anaemias.
• In patients with lymphoma, a high plasma LD activity indicatesa poor
prognosis.
• LDH is also known as α-hydroxybutyrate dehydrogenase (HBD). HBD/LD1
increases later after myocardial infarction
62. Lactate dehydrogenase (LD)
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• a. Clinical significance
• 1) Elevated: hepatic, cardiac, or skeletal muscle disease
• 2) Highest elevation: pernicious anemia
• 2) In AMI, LD levels rise within 8-12 hours, peak at 24-48 hours, and
return to normal in 7-10 days. Although LD and LD isoenzymes are not
used to diagnoseAMI, knowledge of their pattern may be useful
when assessing concurrent liver damage.
• Reference range: 100-225 IU/L
N
updated
63. Lactate dehydrogenase (LD) cont’d
Source of Error in LDH assay
• RBC contain an LDH conc. approx. 100-150 times that found in
serum.
o Therefore, any degree of hemolysis should render a sample
unacceptable for analysis.
• LDH activity is unstable in serum regardless of the temperature
at which it is stored.
• If the sample cannot be analyzed immediately, it should be
stored at 25°C and analyzed within 48 hours.
• LDH-5 is the most labile isoenzyme.
• LD-4 and LD-5 labile at 4°C
• Loss of activity occurs more quickly at 4°C than at 25°C
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