BLOOD

Normal Values

Adult females 4–25 U

9–31 mU/mL

3.5–13 IU/L

3–33 U/L at 37°C

Adult males 7–40 U

12–38 mU/mL

4–23 IU/L

9–69 U/L at 37°C

Children

Cord blood

Premature infants

1–3 days

4–21 days

3–12 weeks

3–6 months, female

3–6 months, male

> 6 months, female

> 6 months, male

1–15 years

190–270 U/L at 37°C

< 140 U/L at 37°C

56–233U/L at 37°C

0–130U/L at 37°C

4–120U/L at 37°C

5–35U/L at 37°C

5–65U/L at 37°C

15–85 IU/L

5–55 IU/L

0–23 U/L at 37°C

Glutamyl Transferase (Carboxy Substrate Method)

(Courtesy: Tulip Group of Companies)

For the determination of γ-glutamyl transferase activity in

serum (For in vitro diagnostic use only).

Summary

γ-glutamyl transferase (GGT) is an enzyme found mainly

in serum from hepatic origin, though the highest levels are

in the kidneys. Elevated levels are found in hepatobiliary

Enzymology 537

and pancreatic diseases, chronic alcoholism, myocardial

infarction with secondary liver damage, and diabetics.

Principle

GGT catalyzes the transfer of amino group between L-γglutamyl-3-carboxy-4 nitroanilide and glycylglycine to form

L-γ-Glutamyl-glycylglycine and 5-amino-2-nitrobenzoate.

The rate of formation of 5-amino-2-nitrobenzoate

is measured as an increase in absorbance, which is

proportional to the GGT activity in the sample.

 GGT L-γ-Glutamyl-3-carboxy- L-γ Glutamylglycyl4-nitroanilide glycine

+ +

Glycylglycine 5-amino-2-nltrobenzoate

Normal Reference Values

Serum (Males) : 10–50 U/L at 37°C

(Females) : 7–35 U/L at 37°C.

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 10 × 2 mL 35 × 2 mL

L1 : Buffer reagent 25 mL 80 mL

T1 : Substrate tablets 10 Nos 35 Nos

Storage/stability

Contents are table at 2–8oC till the expiry mentioned on

the labels.

Reagent Preparation

Working reagent: Dissolve 1 substrate tablet in 2.2 mL of

buffer reagent. This working reagent is stable for at least

15 days when stored at 2–8°C.

Sample material

Serum. Free from hemolysis. GGT is reported to be stable

in serum for 3 days at 2–8°C.

Procedure

Wavelength/filter : 405 nm

Temperature : 37°C / 30°C / 25°C

Light path : 1 cm

Pipette into a clean dry test tube labeled as test (T).

Addition

Sequence

(T)

(mL)

Working reagent 1.0

Incubate at the assay temperature for 1 minute and add

Sample 0.1

Mix well and read the initial absorbance A0 after one

minute and repeat the absorbance reading after every 1, 2,

and 3 minutes. Calculate the mean absorbance change per

minute (∆A/min).

Calculations

GGT activity in U/L = ∆A/min × 1158.

Temperature Conversion Factors

Assay

Temperature

Desired

25°C

Reporting

30°C

Temperature

37°C

25°C 1.00 1.37 1.79

30°C 0.73 1.00 1.30

37°C 0.56 0.77 1.00

Linearity

The procedure is linear up to 700 U/L at 37°C. If the absorbance change (∆A/min) exceeds 0.250, use only the value

of the first 2 minutes to calculate the result, or dilute the

sample 1 + 9 with normal saline (NaCI 0.9%) and repeat

the assay (Results × 10).

Note

Samples having a very high activity show a very high initial

absorbance. If this is suspected then dilute the sample and

repeat the assay.

System Parameters

Reaction : Kinetic Interval : 30

Wavelength : 405 nm Sample volume : 0.10 mL

Zero setting : Distilled water Reagent volume : 1.00 mL

Incubation

Temperature

: 37°C Standard :

Incubation

time

: — Factor : 1158

Delay time : 30 sec React slope : Increasing

Read time : 120 sec Linearity : 700 U/L

No. of read : 4 Units : U/L

Clinical Relevance

1. Increased GGTP levels are associated with:

a. Cholecystitis

b. Cholelithiasis

c. Cancer metastasis to the liver

d. Cirrhosis of the liver

e. Acute pancreatitis

f. Cancer of the bile duct

g. Alcoholism

h. Barbiturate use

538 Concise Book of Medical Laboratory Technology: Methods and Interpretations i. Lipoid nephrosis

j. Obstruction of biliary tract

k. Hepatotoxic drugs for treatment of cancer increase

levels more than the cancer itself.

2. In myocardial infarction, GGTP is usually normal.

However, if there is an increase, it occurs about

the fourth day after myocardial infarction and

probably implies liver damage secondary to cardiac

insufficiency.

3. Values are not enhanced in:

a. Bone disorders

b. Pregnancy

c. Skeletal muscle disease

d. Neonatal hepatitis

e. Renal failure.

LACTIC DEHYDROGENASE

Lactic dehydrogenase (LDH) is a hydrogen transfer enzyme

that catalyzes the following reaction:

 LD

Lactic acid + NAD Pyruvic acid + NADH

The reaction is reversible but the conditions for the

reverse reaction are different than those for the forward

(e.g. the pH for the forward reaction is 8.8 to 9.8 and for the

reverse reaction is 7.4 to 7.8).

LD activity can be determined colorimetrically using 2,

4-dinitrophenyl-hydrazine (2, 4-DNPH) as the chromogen

in alkaline medium. It is a discrete or two-point method.

The alternative method of kinetic measurement or

continuous monitoring enzyme assay is definitely superior

to the colorimetric method.

LD activity is present in almost all the tissues of the

body yet its increased activity in serum reflects several

pathologic states. The five isoenzymes of LD (1 to 5) can

be separated by electrophoresis. Increased activity of

LD-1 is related to myocardial infarction while that of LD-5

is interpreted to be due to liver disorder. LD-1 and LD-5

can also be separated by thermal treatment. If serum is

heated to 65oC for 30 minutes, the thermolabile LD-5

is destroyed. Thus, the difference between the total LD

activity of a non-heated serum specimen and the activity

of the thermostable isoenzyme (LD-1) gives the measure

of LD-5 activity.

Clinical Significance

Serum LD activity is related to myocardial infarction, liver

diseases, pernicious anemia, megaloblastic anemia, renal

diseases, malignant diseases, and progressive muscular

dystrophy.

Normal Values

SI Units

Wroblewski method

30°C

150–450 U/L 72–217 U/L

Adult

< Age 60 45–90 U/L 45–90U/L

> Age 60 55–102 U/L 55–102 U/L

Newborn 160–500 U/L 160–500 U/L

Neonate 300–1500 U/L 300–1500 U/L

Infant 100–250 U/L 100–250 U/L

Child 60–170 U/L 60–170 U/L

LDH (P-L) (Mod. IFCC Method)

(Courtesy: Tulip Group of Companies)

For the determination of LDH activity in serum (For in

vitro diagnostic use only).

Summary

LDH is found in many body tissues particularly heart, liver,

skeletal muscle, kidney and RBCs. LDH is found in the form

of isoenzymes based on their electrophoretic mobility with

each isoenzyme being primarily from different organs.

Increased levels are found in myocardial infarction,

pulmonary diseases, hepatic diseases, hemolytic anemias,

renal diseases and muscular dystrophy.

Principle

LDH catalyzes the reduction of pyruvate with NADH to form

NAD. The rate of oxidation of NADH to NAD is measured

as a decrease in absorbance which is proportional to the

LDH activity in the sample.

 LDH

Pyruvate + NADH + H+ Lactate + NAD+

Normal Reference Values

Serum : 230–460 U/L at 37°C

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 25 mL 2 × 75 mL

L1 : Buffer Reagent 20 mL 2 × 60 mL

L2 : Starter Reagent 5 mL 2 × 15 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned.

Enzymology 539

Reagent Preparation

Reagents are ready to use.

Working reagent: For sample start assays, a single reagent

is required. Pour the contents of 1 bottle of L2 (Starter

Reagent) into 1 bottle of L1 (Buffer Reagent). This working

reagent is stable for at least 1 week when stored at 2–8°C.

Alternatively for flexibility as much of working reagent may

be made as and when desired by mixing together 4 parts

of L1 (Buffer Reagent) and 1 part of L2 (Starter Reagent).

Alternatively, 0.8 mL of L1 and 0.2 mL of L2 may also be

used instead of 1 mL of the working reagent directly during

the assay.

Sample Material

Serum. Free from hemolysis. Total LDH is reported to be

stable in serum for 1–3 days at 2–8°C. Freezing inactivates

the liver isoenzyme.

Procedure

Wavelength/filter : 340 nm

Temperature : 37°C/30°C/25°C

Light path : 1 cm

Substrate Start Assay

Pipette into a clean dry test tube labeled as test (T):

Addition

Sequence

(T)

25°C / 30°C

(T)

37°C

Buffer reagent 0.8 mL 0.8

Sample 0.05 mL 0.02

Incubate at the assay temperature for 1 minute and add

Starter reagent 0.2 mL 0.2 mL

Mix well and read the initial absorbance A0 and repeat

the absorbance reading after every 1, 2, and 3 minutes.

Calculate the mean absorbance change per minute

(∆A/min).

Sample Start Assay

Pipette into a clean dry test tube labeled as Test (T):

Addition

Sequence

(T)

25°C/30°C

(T)

37°C

Working reagent 1.0 mL 1.0 mL

Incubate at the assay temperature for 1 minute and add

Sample 0.05 mL 0.02 mL

Mix well and read the initial absorbance after 1 minute

and repeat the absorbance reading after every 1, 2, and

3 minutes. Calculate the mean absorbance change per

minute (∆A/min).

Calculations

Substrate/Sample Start

LDH activity in U/L = ∆A/min × 3333

25°C/30°C

37°C = ∆A/min × 8095

Temperature Conversion Factors

Assay Desired Reporting Temperature

Temperature 25°C 30°C 37°C

25°C 1.00 1.33 1.92

30°C 0.75 1.00 1.44

37°C 0.52 0.70 1.00

Linearity

The procedure is linear up to 2000 U/L at 37°C. If the

absorbance change (∆A/min) exceeds 0.250, use only the

value of the first 2 minutes to calculate the result, or dilute

the sample 1 + 9 with normal saline (NaCl 0.9%) and repeat

the assay (results × 10).

Note

Samples having a high activity, show a very low initial

absorbance as most of the NADH is consumed prior to the

start of measurement. If this is suspected then dilute the

sample and repeat the assay.

The working reagent or the combined reagent should

have an absorbance above 1.000 against distilled water at

340 nm. Discard the reagent if the absorbance is below 1.000.

RBCs have a very high LDH content and hence,

hemolyzed samples should not be used.

System Parameters

Reaction : UV Kinetic Interval : 60

Wavelength : 340 nm Sample volume : 0.02 mL

Zero setting : Distilled

water

Reagent volume : 1.00 mL

Incubation

temperature

: 37°C Standard :

Incubation time : — Factor : 8095

Delay time : 60 sec React. slope : Decreasing

Read time : 180 sec Linearity : 2000 U/L

No. of read : 4 Units : U/L

540 Concise Book of Medical Laboratory Technology: Methods and Interpretations Clinical Relevance

Myocardial Infarction

The elevation of LDH that follows an MI is characterized

by:

1. High levels within 12 to 24 hours of infarction (18

hours) and 2 to 10 times normal.

2. Elevation that may continue for 6 to 10 days (larger

than SGOT or CK). For this reason, LDH determination

may be useful in the late diagnosis of MI. Elevations

return to normal in 8 to 14 days.

Pulmonary Infarction

In pulmonary infarction, there is usually an increased LDH

within 24 hours of the onset of pain. The pattern of normal

SGOT and elevated LDH that levels 1 to 2 days after an episode

of chest pain provides evidence for pulmonary infarction.

Conditions in general and according to degree of

increase in levels.

1. Elevated levels of LDH are observed in a variety of

conditions:

a. Acute MI

b. Acute leukemia

c. Hemolytic anemias

d. Hepatic disease

e. Skeletal muscle necrosis

f. Sprue

g. Acute pulmonary infarction

h. Malignant neoplasms, extensive cancer

i. Acute renal infarction and chronic renal disease

j. Shock with necrosis of minor organs

k. Myxedema.

2. The greatest increase (2–40 times normal) is seen in:

a. Megaloblastic anemia

b. Extensive cancer (especially hepatic metastases)

c. Shock and anoxia.

3. Moderate increase (2–4 times normal) is seen in:

a. MI

b. Pulmonary infarction

c. Hemolytic anemia

d. Granulocytic or acute leukemia

e. Infectious mononucleosis

f. Progressive muscular dystrophy.

4. Slight increase occurs in:

a. Delirium tremens

b. Hepatitis

c. Obstructive jaundice/cholangitis

d. Cirrhosis

e. Nephrotic syndrome

f. Hypothyroidism.

Decreased LDH levels are associated with a good

response to cancer therapy

Elevated urine LDH levels occur in:

1. Cancer of kidney or bladder

2. Glomerulonephritis

3. Malignant hypertension

4. Lupus nephritis

5. Acute tubular necrosis

6. Renal transplantation and hemograft rejection

7. Pyelonephritis (sometimes).

Interfering Factors

1. Strenuous exercise and the muscular exertion involved

in childbirth will cause increased levels.

2. Skin diseases can cause falsely increased levels.

3. Hemolysis of RBCs due to freezing, heating, or shaking

the blood sample will cause falsely increased levels.

4. Drugs that may elevate levels comprise:

a. Codeine

b. Clofibrate

c. Meperidine

d. Mithramycin

e. Morphine

f. Procainamide.

5. Oxalate is known to cause decreased levels.

Electrophoresis of LDH Isoenzymes

Normal Values

Isoenzyme Organ related Percentage of

total LDH

Isoenzyme 1 Cardiac (25–40%)

Isoenzyme 2 Cardiac (35–46%)

Isoenzyme 3 Pulmonary (17–32%)

Isoenzyme 4 Hepatic (9–18%)

Isoenzyme 5 Hepatic (6–17%)

Variation of 2% to 4% are considered physiologically

normal (isoenzymes 1 to 5 are also present in human

skeletal muscle).

Test Significance

Electrophoresis or separation of LDH identifies the

5 isoenzymes or fractions of LDH, each with its own

characteristics physical and chemical properties.

Fractionating the LDH activity multiplies its diagnostic

relevance since LDH is found in many organs. The LDH

isoenzymes are released into the bloodstream when

tissue necrosis occurs. However, a complete knowledge of

the clinical history is necessary to properly interpret the

resulting patterns. The isoenzymes are evaluated in terms

Enzymology 541

of patterns established, not on the basis of the value of a

single isoenzyme. The 5 isoenzyme fractions of LDH show

different patterns in various disorders. Abnormalities in the

pattern suggest, which tissue has been damaged and help

to diagnose myocardial infarction, pulmonary infarction,

and liver disease. This test is sensitive enough to detect

hepatic fraction in infectious hepatitis before clinical

jaundice appears. It is in confirming the diagnosis of

suspected MI that the separation of LDH isoenzymes finds

its most frequent application, especially when a second

infarct occurs shortly after the first. In these cases, the

ECG is already abnormal, but the isoenzyme pattern will

show increased LDH1, indicating the release of more of the

cardiac enzyme.

Clinical Relevance

Abnormal patterns reflect damaged tissue

1. LDH1 and LDH2 are increased in MI and in some

hemolytic anemias.

2. LDH3 is increased in pulmonary infarction and

extensive pneumonia.

3. LDH5 is increased in liver disease.

4. An increase in LDH2, LDH3, LDH4 is common in

malignant disease.

5. The LDH pattern will be essentially the same in MI,

pernicious anemia, and renal infarction. This is

because RBC’s and the kidney have an isoenzyme

pattern similar to that of heart muscle.

6. In most cancers, one to three of the bands (LDH2,

LDH3, and LDH4) are frequently increased. A notable

exception is in seminomas and dysgerminomas when

LDH1, and LDH2 are increased. Frequently, an increase

in LDH3 may be the first indication of the presence of

cancer.

Creatine Kinase K (NAC Act) (Mod. IFCC Method)

(Courtesy: Tulip Group of Companies)

For the determination of CK activity in serum (For in vitro

diagnostic use only).

Summary

Creatine kinase (CK) is mainly found in all muscle and

brain tissue. It plays an important role in the energy storing

mechanism of the tissues.