VETERINARY LABORATORY DIAGNOSIS I & II

(VLD 421 & VLD- 511)

Practical
Lesson Plan

S.No Topic
01 Pathological Components of Urine
02 Estimation of Blood Glucose
03 Estimations of Total Proteins and Albumin
04 Estimation of Total and HDL Cholesterol
05 Estimation of Urea and BUN
06 Estimation of Serum Creatinine
07 Estimation of Serum Bilirubin
08 Estimation of Serum Calcium
09 Estimation of Serum Phosphorus
10 Estimation of SGPT
11 Estimation Of Serum Triglycerides
12 Estimation of Serum Uric acid
13 Estimation of Serum Sodium
14 Estimation of Serum Potassium
15 Estimation of Serum Chlorides
16 Estimation of SGOT
17 Estimation of Serum Alkaline Phosphates
 Practical 1

PATHOLOGICAL COMPONENTS OF URINE

In any pathological condition, analysis of urine is a useful diagnostic aid.

Commonly analyzed pathological constituents (substances) of urine are:
Blood, protein reducing sugars, ketone bodies, bile salts and bile pigments.
Most of these substances are present in trace amounts in normal urine also.

1.Benzindine Test: (Psuedo peroxidase reaction) for detection of blood in urine.

Principle:

Hemoglobin present in the blood catalyses the release of oxygen from H2 O2 leading to the
oxidation of toluidine (benzidine reagent) and formation of blue color.

Procedure:
To 2 ml urine add 1-2 drops of benzidine reagent and 2 ml of 3% H2 O2.
Hematuria (RBC in urine) occur in conditions like pyelonephritis, cystitis, urolithiasis,
neoplasms of kidney, presence of parasites such as Dictophyma renale, Dirofilaria immitis larvas
in dogs and also in heavy metal poisonings.

Hemoglobin (Hb in urine) is a common symptom in leptospirosis, piroplasmosis. Sometimes,

they are caused by chemical hemolytic agents like Cu,Hg,etc. and bacteria like clostridium
hemolyticum.
Examine the sediment of the centrifuged urine under microscope:
If the RBCs are intact it is hemoaturia; otherwise it is hemoglobinuria.

2. Tests for Proteins:

(a) Hellers Nitric acid test: Take 2 ml of urine in a tube and add 2 ml of Conc.HNO3along the
sides of the tube. An appearance of a white ring at the junction of the two liquids indicates the
presence of proteins.
(b) Biuret Test: Take 2 ml of urine in a test tube, and 1 ml of 5% NaOH and 1-2 drops of 1%
copper sulphate. Production of a violet color indicates the presence bonds in proteins. Amino
acids will give a negative result.
In general albuminuria (presence of albumin in urine) occurs in the following conditions:
(i)Excessive muscular (exertion of albumin through kidney) occurs in nephrosis, poisoning by
lead, arsenic, mercury, sulfonamides, ethers and phenols.
Accidental proteinuria occurs under conditions like cystitis, urolithiasis and trauma.

3.Benedicts test:
This test is for the detection of reducing sugars (Glucose).
To 2.5 ml of Benedicts qualities reagent add 4 drops of urine, boil for 2 minutes and then cool.
If glucose is present, gradually, a precipitate with green, yellow, orange or brick red colors may
be formed depending on the concentration of glucose present.
Glucosuria occurs in conditions such as diabetes mellitus, acute or chronic pancreatitis,
hyperthyroidism, over activity of adrenal cortex, increased intracranial pressure, enterotoxaemia
in sheep caused by toxin of clostridium.

4.Rotheras test:

For detection of ketone bodies (acetonelacetoacetic acid only)

Saturate 5 ml of urine with (NH)So and add few drops of 5% sodium nitroprusside.
Add 2 ml of conc. NHOH or liquid NH along the inner walls of the tube.
Appearance permanganate color indicates the presence of ketone bodies.
Ketone bodies include acetone, acetoacetic acid and hydroxy butyric acid.
They are formed due to the derangement in lipid and carbohydrate metabolism.
They accumulate as acetyl CoA and are not utilized in, lipogenesis or in citric acid cycle.
Their accumulate in the blood and urine produces a condition known as ketosis.
In small animals(dog and cat) diabetes mellitus and starvation may result in ketonuria.

5.Hays test:

For detection of bile salts

Over 5 ml of urine, sprinkle sulphur powder and view the tube without shaking it.
If bile salts are present, sulphur sinks tot the bottom of the tube.
This is due to the reduction of surface tension of urine by the salts.
Bile salts are found in urine of all animals when liver disorder occur.

6. Fouchets test:

For detection of bile pigments

To 5 ml of urine add an equal volume of 10%barium chloride solution.
Filter, spread the filter paper and dry the precipitate and add 2 drops of Faucets reagent.
A green or bluish green coloration indicates the presence of bile pigment.
Bile pigments are found in urine of all animas when liver disorder occurs.

Practical 2

ESTIMATION OF BLOOD GLUCOSE

(GOD/POD Method)

Principle:
Glucose is oxidized by the enzyme glucose oxides (GOD) to give D-gluconic acid and hydrogen
Peroxide (HO). Hydrogen peroxide in presence of enzyme peroxidase (POD) oxidizes
phenol, which combines with 4-aminoantipyrine to produce a red colored quinoneimine dye. The
intensity of the colour developed is proportional to the glucose concentration in the sample.
D-Glucose + H O D-Gluconic acid + HO
H O+4-am + Phenolinoantipyrine + phenol Red quinoneimine dye + H O

Sample :
Serum
Reagents Provided:
(i) Enzyme Regent
(ii) Buffer solution
(iii) Glucose standard (100 mg%)
Regent Preparation:
Dilute one vial of enzyme (i) in one bottle solution (2)Mix gently to dissolve. The
prepared working enzyme reagent is stable for at least a month at 2- 8C.

Procedure:
Pipette into clean dry test tubes labeled blank (B), Standard(S) and test(T).

S.No Reagent B S T
1. Working Enzyme 3.0ml 3.0 ml 30. ml
Reagent
2. Distilled Water 30l - -
3. Glucose standard - 30l -
4. Serum / Plasma - - 30l

Mix well and incubate at 37C for 15 minutes. Then measure the absorbance of Test (T) and
Standard (S) against Blank(B) on a Photocolorimeter with a green filter or on a
Spectrophotometer at 505.nm.

Calculation ;
OD of (T)
Glucose = ---------------- X 100

OD of (S)

Result:
The amount of glucose present in the given serum.

Clinical Significance:
Glucose determination is mainly useful in the diagnosis of Diabetes mellitus in which blood
glucose
Levels are elevated. Other diseases like hyperthyroidism, hyperpituitarism, severe nephritis,
pancreatitis, vomiting during pregnancy asphyxia, anesthesia. Pneumonia, dehydration and
certain hepatic disorders also lead to hyperglycemia.
 Hypoglycemia occurs frequently as a result of over sage if insulin or antidiabetic treatment
.hypoglycemia may also be noticed in conditioned like hyperinsulinemia, hypothyroidism,
hypopitutarism and hypoadrenocorticism. Hypoglycemia may also occur in certain physiological
conditions like pregnancy, starvation, severe prolonged exercises and lactation.

Practical 3

ESTIMATION OF TOTAL PROTEINS AND ALBUMIN

(Biuret and BCG Dye Method)

Principles:
Total Proteins: Proteins in serum react with copper of Biuret Reagent in alkaline medium to
form a blue purple complex with absorption maximum at 550 ml.

Albumin: Albumin in serum binds with the dye Bromicresol Green (BCG) at Ph 3.8 to forma
green
Colored complex, the absorbance of which is measured at 600 ml.

Sample:
Serum

Reagents:
1.Biuret Reagent 2.Bromicresol Green Dye-Buffered Reagent 3.Protein Standard
(BCG Reagent)

Procedure:
Total proteins: Mark three test tubes as Blank, Standard and test proceed as follows.

S.No Reagents Blank(B) Standard(S) Test(T)

1. Proteins Standard - 60 l -
2. Serum - - 60 l
3. Biuret Reagent 3.0 ml 3.0 ml 3.0 ml

Mix well and allow the tubes to stand at R.T for 5 min.
Measure the O.D of all tubes at 550 nm or using an yellow green filter.

Albumin: Mark three test tubes as Blank, Standard and Test and proceed as follows.

S.No Reagents Blank (B) Standard Test (T)

(S)
1. BCG Reagent 3.0 ml 3.0 ml 3.0 ml
 2. Proteins Standard - 20 l -
3. Serum - - 20 l

Mix well and allow the tubes to stand at R.T for 1 min.
Measure the O.D of all the tubes at 600 nm or using a red filter.

Calculations:

Total Proteins (g/dL) (X) = OD of (T)

---------------- X Conc.of the Total Proteins Standard.

OD of (S)
Albumin (g / dL) (Y) = OD of (T)
-------------- X Conc of Albumin Standard

OD of (S)

Globulins (g/dl) = (X) (Y)

A/G Ratio (Y)

= ---------
(X) (Y)

Results:
The given serum contains:
Total proteins:
Albumins:
Globulins:
A/G ratio:

Clinical Significance:
A/G ration is quite important in the diagnosis of many diseases.
Elevated levels of total proteins and albumin may be found in dehydration.
Elevated levels may be observed in multiple myeloma.
Hypoproteinemia and hypoalbuminemia are seen in renal disease and nephritic syndrome.
Hypoalbuminemia with normal total proteins may be observed in hepatic damage.
 Practical 4

ESTIMATION OF TOTAL CHOLESTEROL AND HDL CHOLESTEROL

(Wybenga & Pillegis Method)

Principles:
Cholesterol reacts with hot solution of ferric perchlorate, ethyl acetate and sulphuric acid
(cholesterol
and gives lavender colored complex, which is measured at 560 nm.

Sample:
Serum / plasma

Reagents:
1.Cholesterol Reagent 2.Working cholesterol standard, 200 mg% 3.precipitating reagent

Procedure:
(A) For total Cholesterol: Mark three test tubes as Bank. Standard and test and proceed as follows.

S.No Reagents Blank(B) Standard(S) Test(T)

1. Cholesterol 3.0 ml 3.0 ml 3.0 ml
Reagent
2. Standard - 15 l -
3. Serum - - 15 l

Mix well and keep the tubes immediately in the boiling water bath exactly for the 90 seconds
(1 minutes). Cool them immediately to R.T under running tap water. Measure the O.D. of the
tubes at 560 or using a yellow green filter.

(B) For HDL Cholesterol:

(I) HDL Cholesterol separation:

Pipette into centrifuge tube Quantity

Sample 200 l
Precipitating reagent 2000 l

Mix well keep at R.T for 10 minutes.

Then centrifuge at 200 rpm for 15 minutes to obtain a clear supernatant.
Proceed to step (II)
(II)HDL- Cholesterol estimation: Mark three test tubes, Blanks, Standard and test and proceed as
follows.

Reagents Blank(B) Standard(S) Test(T)

Cholesterol 3.0 ml 3.0 ml 3.0 ml
Reagent
Standard - 15 l -
Serum - - 120 l

Mix well and keep the tubes immediately in the boiling water bath exactly for 90 seconds (1
minutes).
Cool them immediately to R.T. under running tap water. Measure the O.D of all the tubes at 560 nm
using a yellow green filter.

Calculations:
1.Total Cholesterol (mg/dl) = O.D of T
-------------- X 200
O.D. of S
2.HDL Cholesterol (mg/dl) = O.D of T
-------------- X 50
O.D. of S

Results:
The give serum contains:
Total cholesterol =
HDL cholesterol =

Clinical Significance:
High values of total cholesterol may be found in diabetes mellitus, hypothyroidism, obstructive
jaundice,
Nephritic syndrome, biliary cirrhosis, atheroslerosis, etc.
Low values of total cholesterol may be found in hyperthyroidisms, malnutrition, gauchers disease
and
acute hepatitis.

Decreased level of HDL cholesterol lead to increased chance of coronary heart disease (CHD) while
increased levels reduce these chances. Lower values of HDL cholesterol and increased ration of total
cholesterol to HDL cholesterol are taken as risk factor for CHD.
 Practical 5

ESTIMATION OF UREA AND BUN

(DAM method)
Principle:
Urea reacts with hot acidic diacetylmonoxime in presence of thiosemicarbazide and produces a rose-
purple colored complex, which is measurede colorimetrically.

Specimen:a
Serum / plasma

Reagents:
1. Working urea standard 2. Urea Reagent 3. Diacetylmonoxime (DAM)

Preparation of working solution:

Solution I:
Dilute one ml of Urea Reagent to 5ml with distilled water.

Procedure:
Mark three test tubes as Blank, Standard and Test and proceed as follows:

Blank Standard Test

S.No. Reagents
(B) (S) (T)
1. Solution I 2.5 ml 2.5 ml 2.5 ml
2. Working Urea Standard (30 mg%) - 10 l -
3. Serum / Plasma - - 10 l
Mix Well
4. Diacetylmonoxime (DAM) 250 l 250 l 250 l

Mix well and keep the tubes in boiling water, exactly for 10 minutes. Cool them immediately under
running water for 5 minutes and mix by inversion. Measure the optical density within 10 minutes at
525nm or using a green filter.

Calculations:

1. Blood urea concentration (mg/dl) = OD of the Test

------------------- x 30
OD of the Standard

2. Blood Urea Nitrogen (BUN) (mg/dl) = Concentration of Blood Urea (mg/dl) x 0.467

Result:

The amount of urea in the given serum = mg / dl

The amount of BUN in the given serum = mg / dl

Clinical Significance

Elevated values are found in renal disorders like nephrosis, nephritis, anuria etc.
Decreased levels are observed in low protein intake, liver disorders etc.
 Practical 6

ESTIMATION OF SERUM CREATINIE

Alkaline Picrate Methods

Principles:
Picric acid reacts with creatinine in alkaline medium to form a red colored complex, whose
absorbance is proportional to creatinine concentration. Picric acid reagent has dual role as a
deproteinizing reagent and as a reactant. Sample:
Serum
1.Picric acid reagent
2.Sodium hydroxide 0.75 n
3.Creatinine Standard (150 mg%)

Preparation of working Standard:

Dilute 0.1 ml of urea Reagent to 10 ml with distilled water.

Procedure:
Deproteinization of Serum:
Pipette into a clean dry test tubes labeled balnk (B), Standard (S) and test(T) as follows:

S. Reagents (B) (S) (T)

No (ml) (ml) (ml)
1. Standard creatinine - 0.5 -
2. Supernatant from above step - - 2.0
3. Distilled water 0.5 - -
4. Picric acid reagent 1.5 1.5 -
5. Sodium hydroxide 0.75 N 0.5 0.5 0.5

Mix well and allow the reaction mixture to stand at room temperature for exactly 20 minutes.
Measure the absorbance of the standard (S) and test (T), blank (B) against Distilled water at 520 nm
or with a filter in a photometric colorimeter.
Calculation:
OD of (T) OD of (B)
Serum creatinine (mg / dl) = -------------------------------- X 3
OD of (S) OD of (B)
Results:
The creatinine content of the given serum is:

Clinical significance:
An increased serum level is usually associated with various renal diseases In the earlier stages.
Creatinine clearance test is a sensitive index of impaired renal function. The prerenal factors that
increase blood urea have less influences on creatinine concentration. Hence for diagnosis of renal
diseases, serum creatinine is preferred over urea estimation. In addition to renal disease, elevated
level of serum creatinine and creatinuria may be observed in extensive muscle destruction.
 Practical 7

ESTIMATIONOF SERUM BILIRUBIN

(Malloy and Evelyn Method)

Principles:
Bilirubin reacts with diazotised sulfanilic acid in acidic medium to form azobilirubin, a pink
complex whose absorbance is proportional to the bilirubin concentration. Direct bilirubin, being
water soluble is allowed to react with doazotised sulfanilic acid in the absence of methanol
while for total bilirubin (direct and indirect), the diazotisation is carried in the presence of
methanol.

H
Bilirubin + Diazotised Sulfanilic acid Azobilirubin Red-Purple complex.

Sample
Serum
Reagents:
1.Diazo A reagent 2.Diazo B reagent 3.Diazo Blank 4. Methanol 5.Artificial standard
(10 mg/dl)

Preparation of working Diazo reagent : just before use, mix 1.0 ml of Diazo A with 0.03 ml of
DoazoB.

Procedure:
Pipette into clean dry test tubes labeled as T T (for total bilirubin) and D D 9for direct
bilirubin) as follows:
S.No Reagent T T D D
(ml) (ml) (ml) (ml)
1. Serum 0.1 0.1 0.1 0.1
2. Methonal 1.25 1.25 - -
3. Diazeo blank - 0.25 - 0.25
4. Diazo reagent 0.25 - 0.25 -
5. Distilled Water - - 1.25 1.25

Mix well and read the absorbance of Dand D exactly after one minute against distilled water at
540 nm or with a green filter in a photoelectric colorimeter.

Mix well and keep the tubes T and T in a dark at room temperature for 30 minutes.
The read the absorbance of the artificial standard and T and T against distilled water at 540 nm
or with a green filter in a photoelectric colorimeter within 30 minutes.
Calculation:

(OD (T) OD(T) OD(D) OD

(D)
A.Total Bilirubin (mg/dl): = ---------------------X10 B.Direct Bilirubin (mg/dl):
------------------- X 10
OD (S) OD (S)
C.Indirect Bilirubin (mg/dl): = (A) - (B)

Result: In the given serum a) Total bilirubin is:

b) Direct bilirubin is:
C) Indirect bilirubin is:

Clinical Significance:
Hyperbilirubin is a characteristic of jaundice. In hemolytic and neonatal jaundice, unconjucated
bilirubin level increase without corresponding increase in conjugated bilirubin.. In viral hepatitis,
toxic hepatitis and cirrhosis, there is overall damage to liver cell and hence the ability of the liver
to form conjugated bilirubin decrease resulting in an increase in unconjugated bilirubin in serum.
In obstructive jaundice, there is an increase in both conjugated and unconjugated bilirubin levels.

Practical 8

ESTIMATION OF SERUM CALCIUM

(OCPC Method)

Principle:
Calcium in alkaline medium reacts with o-cresolphthalein complex one to form a bluish purple
colored complex whose absorbance is proportional to the calcium concentration. Interference
due to Magnesium and form eliminated by using - hydroxy quinoline.

Sample:
Serum

Reagent:

1.OCPC reagent 2. AMP buffer 3. Calcium standard (10 mg/dl)

Procedure:
Working reagent preparation:
Just prior to use, mix COPC reagent and AMP buffer in equal volumes. This is Working
Calcium Reagent.

Pipette into clean, dry acid washed test tubes labeled as blank (B), Standard (S), and test (T) as
follows.
 S.No Reagent B S T
(ml) (ml) (ml)
1. Standard calcium - 0.03 -
2. Serum - - 0.03
3. Working calcium 1.0 1.0 1.0
reagent
4. Distilled water 2.0 2.0 2.0

Mix well incubates at 37 C or 15 25 C for 5 minutes. Then measure the absorbance of the
standard (S) and test (T) against the blank (B) at 570 or a yellow green filter in a photoelectric
colorimeter.

Calculations:

O.D (T)
Calcium (mg/dl) = ------------ X 10
O.D (S)

Result:
The calcium content of given serum = mg/dl

Clinical significance:
The metabolism of calcium is closely related to that of phosphorus. Calcium in plasma occur in
two forms- non- diffusible proteins bound and free or ionized calcium.
Increased serum calcium levels are observed in primary hyperparathyroidism, hypervitaminosis
D and multiple myeloma. In addition some neoplastic disease of bones may also be associated
with increased serum calcium.
Decreased serum levels are observed in hypoparathyroidism, tetany, steatorrhea and nephritis.

Practical 9

ESTIMATION OF SERUM PHOSPHORUS

(UV Molybdate Method)

Principles:
Phosphorus reacts with molybdate to form phoshomolybdate. The increase in absorbance due to
formation of this complex is measured at 340 nm and is proportional to the concentration of
phosphorus in the sample.

Sample:
Serum

Reagents:
1. Molybdate reagent 2. Sample Blank reagent 3. Phosphorus standard (5 mg/dl)

Procedure:
Pipette into clean, dry acid-washed test tubes labeled as bland (B), Standard (S), and test (T) as
follows.

S.No Reagent B S (ml) T

(ml) (ml)
1. Standard calcium - 0.01 -
2. Serum - - 0.01
3. Molybdate Reagent 1.0 1.0 1.0
4. Deionized water 2.0 2.0 2.0

Mix well and incubate at 37 C for 5 minutes. Read absorbance against Reagent Blank.

Calculation:

Phosphorus (mg/dl) = O.D (T)

------------ X 5
O.D (S)
Results:
The phosphorus content of given serum = mg/dl

Clinical significance:
An increased serum phosphorus level is found in hypoparathyroidism, disease of bones,
acromegaly, myelogenous leukemia, neoplasm, magnesium deficiency massive blood
transfusions, chronic renal failure and child hood.
Low serum phosphorus level is seen in alcoholism, diabetes mellitus, hyper alimentation,
acidosis (ketoacidosis) primary hyperparathyroidism, Vitamin D decency and hypokalemia.

Practical 10

ESTIMATION OF SGPT
(Reitman and Frankels method)

Principle:
SGPT (ALT) catalyses the following reaction:
A - Ketoglutarate + L-Alanine L-Glutamate + Pyruvate

pyruvate so formed is coupled with 2,4 dinitrophenyl hydrazine (2,4 DNPH) to give the
corresponding hydrazone, which gives brown color in alkaline medium and this, can be
measured colorimetrically.
 Specimen:
Serum / plsam

Reagents:

1.Buffered alkaline a Ketoglutarate substrate pH 7.4 2. DNPH Reagent

3.Sodium hydroxide (4N) stock 4. Working pyruvate standard, 2
mM

Preparation of working solution:

Dilute 1 ml Sodium hydroxide (4N0 stock to 10ml with purified water. Other reagents are
ready to use.

Procedure:
Mark 6 test tubes 1,2,3,4,5 for standards and T for test and proceed as follows:

Tube No. 1 2 3 4 5 Test

Enzyme activity 0 28 57 97 150 Find out from
(Units/ml) standard graph
Buffered substrate 0.50 0.45 0.40 0.35 0.30 0.50
(ml)
Pyruvate standard - 0.05 0.10 0.15 0.20 Serum 0.10 ml
2 mM (ml)
Distilled water 0.10 0.10 0.10 0.10 0.10 0.10
(ml)
Mix well and incubate at 37 for 30 min
DNPH reagent 0.50 0.50 0.50 0.50 0.50 0.50
(ml)
Mix well and allow to stand at R.T for 20 minutes
Working NaOH 5.0 5.0 5.0 5.0 5.0 5.0
(ml)
Mix well; After 10 minutes measure the O.D at 505 nm
OD values at 505
nm

Standard graph:
Plot standard graph by taking the enzyme units on X-axis and OD values in the Y-axis.

Calculation:
For test:
From the standard curve obtained in the graph, extrapolate the test OD value to the
corresponding.
enzyme activity in the X-axis.

Clinical Significance:
 ALT is increased in hepatocellular injury in dog and cat. It is not useful in evaluating chronic
liver disease. It may be elevated in corticosteriod treatment. This enzyme is not useful in
evaluating hepatic disease in horse, cow, sheep goat, and pig.

Practical 11

ESTAMATION OD SERUM TRIGLYCERIDES

(GPO/POD ENZYMATIC METHOD)

Principles:
Triglycerides are hydrolyzed by lipase to glycerol and free fatty acids. Glycerol is
phosphorylated by ATP in the presence of glycerokinase to glycerol-3 P, which is oxidized by
Gly 3-P oxidase (GPO) producing HO. HO so formed reacts with 4 amino-antipyrine and
3,5-dechloro-2-hydroxy benzene sulfonic acid in the presence of peroxidase (POD) to produces a
red quinoneimine dye. The intensely of color developed is proportional to the triglyceride
concentration.

Reagents:
1. Enzyme Reagent 2.Buffer solution 3.Triglyceride standard (200 mg/dl)

Procedure:
Pipette into clean, dry acid-washed test tubes labled as blank (B), standard (S), and test (T) as
follows:

S.No Reagent B (ml)S (ml) T(ml) OD at

510
1 Working enzyme reagent 0.5 0.5 0.5
2 Triglyceride standard - 0.01 -
3 Serum - - 0.01
4 Mix well and incubate at 37 C for 10 minutes
5 Distilled water 2.0 2.0 2.0

Measure the absorbance of standard (S) and test (T) against the Blank (B) at 510 or with a green
filter in a photoelectric colorimeter within 30 minutes.

Calculation:
Serum triglycerides (mg/dl) = O.D (T)
------------ X 200
O.D (S)
Results:
The content of trigylcerides in the given serum= mg/dl

Clinical significance:
Levels of both cholesterol and triglycerides in blood have been identified as risk factors related
to atherosclerotic disease. The levels of cholesterol and triglycerides can very independently and
hence evaluation of hyperlipidemia included determination of both levels.
 Elevated levels of triglycerides are found in atherosclerotic disease, Diabetes mellitus,
biliary obstruction and other metabolic disorders associated with endocrine disturbances.

Practical 12
ESTIMATION OF SERUM URIC ACID
(Phosphotungstic Acid Method)

Principles:
Tungstic acid deproteinzes serum with least loss of uric acid. In alkaline medium, uric acid
reduces phosphotungstate reagent to form a blue color (Tungsten Blue complex), whose
absorbance is proportional to uric acid concentration.

Reagents:
1. Sulphuric acid 2/3 N 2.Sodium tungstate 3. Sodium carbonate
4. Phosphotungstate 5.Uric acid standard stock (100 mg/dl).

Preparation of working standard:

Dilute 0.1 ml of stock standard with 0.9 ml distilled water. Prepare fresh on day of assay.
Procedure:

I. Deproteinization of serum pipette into a clean dry test tube labeled (T) as follows:

Reagent (T)
Serum 0.5ml
Distilled water 4.0ml
Sulphuric acid 2/3 N 0.25ml
Sodium tungstate 0.25

Mix well and let stand for 10 minutes at room temperature. Centrifuge at 3000 rpm for 5
minutes.

II. Color development:

Pipette into clean, dry test tube labelled blank (B), standard (S), and test (T) as follows:

S.No Reagent B (ml) S (ml) T (ml) OD values

1. Supernatant from - - 1.5
above step
2. Working standard - 1.5 -
3. Sodium carbonate 0.5 0.5 0.5
 reagent
4. Phosphotungstate 0.5 0.5 0.5
reagent

Mix well and let stand in the dark at room temperature for 15 minutes. Then measure the
absorbance of blank (B), standard (S) and test (T) against the distilled water at 710 nm or with a
red filter in a photoelectric colorimeter.
Calculation:
O.D (T) O.D (B)
Uric acid (mg/dl) = ----------------------- X 10
O.D (S) O.D (B)
Result:
The uric acid content of the given serum = mg /dl
Clinical Significance:
In man and all primates, uric acid is the end product of purine metabolism. Its determination
helps in differentiating gout form non-gout arthritis. Increased levels are observed in gout,
leukemia, broncho and lobar pneumonia and polycythemia.

Practical 13

ESTIMATION OF SERUM SODIUM

(Colorimetric Method)

Principles:
Sodium Is quantitatively precipitated as uranyl magnesium sodium acetate and the excess of
uranyl salt reacts with potassium ferrocyanide to produce a brownish color. The intensity of the
color is inversely proportional to the sodium concentration in the sample.

Uranyl ions + Mg ions + Na UranylMgNa Precipitate

Free Uranyl ions +K Fe (CN) Brown colored complex

Reagents:
1.Precipitating reagent 2. Acid Reagent 3.Color reagent 4.Sodium standard
(150 mmol/l)
Procedure:
I. Precipitation of sodium and protein:
Pipette into two clean dry test tubes labelled as standard (S), and test (T) as follows:
Reagent S T
Standard 0.01 -
Serum - 0.01ml
Precipitating reagent 0.50ml 0.50ml

Mix well and allow to stand at room temperature for 5 minutes. Then centrifuge at 3000 rpm for
5 minutes to obtain a clean supernatant.
II. Color development:
Pipette into clean, dry tubes labelled blank (B), standard (S), and test as follows:

S.No Reagents B (ml) S (ml) T (ml) OD

values
1. Supernatant from - 0.01 0.01
above step
2. Precipitating reagent 0.01 - -
3. Acid Reagent 0.50 0.50 0.50
4. Color reagent 0.05 0.05 0.05
5. Distilled water 2.00 2.00 2.00

Mix well and allow the reaction mixture to stand at room temperature for 5 minutes. Then
measure the absorbance of blank (B), standard (S) and test (T) against distilled water at 530nm
or with a green filter in a photoelectric colorimeter within 10 minutes.
Calculations:
Sodium (mmol/dl) = O.D of B (-) OD of T
-------------------------- X 150 mmol/l
O.D of B (-) OD of S
Result:
The sodium content of given serum = mmol / l or meq /l

Clinical Significance:
Elevated sodium level is mainly found in conditions like hyperadrenalism (Cushings syndrome)
and dehydration, while low sodium level is found in the following conditions: hyperadrenalism
(Addisons disease), severe polyuria, metabolic acidosis, diarrhea and renal diseases in which
sodium reabsorbtion is defective.

Practical 14

ESTIMATION OF SERUM POTASSIUM

(Turbidimetric Method)

Principles:
Potassium ions react with sodium tetra phenyl boron to produce a colloidal suspension, which is
stabilized in a special matrix. The extent of turbidity is measured I items of absorbance and is
proportional to potassium concentration.

Tetra phenyl Boron + K White turbidity

Reagents:

1.Boron reagent 2. Potassium standard (5 mmol/l)

Procedure:
Pipette into two clean, dry test tubes labelled standard (S) and test (T) as follows:

S.No Reagents S (ml) T (ml) OD

Values
1. Standard Potassium 0.02 -
2. Serum - 0.02
3. Boron (K) reagent 1.00 1.00
4 Distilled water 1.50 1.50

Mix well wait for 5 minutes at room temperature and read the absorbance of standard (S) and test
(T) against distilled water at 620nm or with a red filter in a photoelectric colorimeter within 10
minutes.

Calculations:

Potassium (mmol/dl) = O.D (T)

------------ X 5
O.D (S)
Result:the potassium content of given serum = mmol/l or meq.l

Clinical Significance:
Sodium is the major intracellular cation. Both play an important role in the maintenance of
normal distribution between cells and plasma and correct environment for muscles contraction..
In the kidney, sodium is filter red by the reabsorbed is mainly governed by aldosterone, sodium
assay is used in the diagnosis and prognosis of hyper-and hyperadrenalism.
An increased in serum potassium is more potassium may occur in renal failure, anuria
and severe oilguria. An increase in serum potassium is less common than the decreased level.
decrease in serum potassium is more commonly seen in severe vomiting, diarrhea and
hyperadrenalsim. A decreased is also found during attacks of paralysis. Apathy and muscles
weakness are the predominanat feature of low potassium levels.

Practical 15

ESTIMATION OF SERUM CHLORIDE

(Thiocyante method)

Principles:
Chloride ions react with a solution of metric thiocyanate and ferric nitrate to form undissoicated
metric chloride and a red brown ferric thiocyanante complex, which is quantitatively
proportional to the chloride ion concentration.

2Cl + Hg (SCN) Hg Cl + 2 (SCN)

3 (SCN) + Fe Fe (SCN)
Reagents
1. Color Reagent 2. Chloride standard (100 mmol/l)

Procedure:
Pipette into two clean, dry test tubes labelled blank (B), Standard (S) and test as follows:

S.No Reagents S (ml) T OD

B (ml (ml) values)
1. Standard Chloride - 0.01 -
2. Serum - - 0.01
3. Chloride reagent 1.0 1.0 1.0
4. Distilled water 2.0 2.0

Mix well wait for 2 minutes at room temperature and read the absorbance of standard (S) and test
(T) against blank at 505nm or with a green filter in photoelectric colorimeter within 310 minutes.

Calculations:

Chloride (mmol/dl) = O.D (T)

----------- X 100
O.D (S)

Result :
The chloride content of given serum = mmol/or meq l

Clinical Significance:
Chloride is major extra cellular anion. Which is significantly involved in the maintenance of
water distribution, osmotic pressure and cation-anion balance in the extra cellular fluid
compartment.
High serum values may be observed in dehydration and in conditions causing decreased
renal blood flow such as congestive heart failure.
Low values may be observed in salt-losing nephritis, diabetic acidosis and renal failure.
Lower CSF chloride values may be seen in meningitis, especially untreated tubercular
meningitis.
Practical 16

ESTIMATION OF SGOT
(Reitman and frankels method)

Principle:
 SGOT (AST) catalyses the following reaction:
a-Ketoglutarate + L Aspartate L- Glutamate + Oxaloacetate

Oxaloacetate so formed is coupled with 2,4 Dinitrophenyl hydrazine (2,4-DNPH) to

give the corresponding hydrazone, which gives brown color in alkaline medium and this can be
measured calorimetrically.

Specimen:
Serum / plasma

Reagents:
1.Buffered aspartate a Ketoglutarate substrate p H 7.4 2.DNPH colour reagent
3.Sodium hydroxide (4N) stock 4. Working pyruvate standard, 2mM

Preparation of working NaOH solution:

Dilute 1 ml of Sodium hydroxide (4N) stock to 10 ml with purified water. Other reagents are
ready to use.
Tube No 1 2 3 4 5 Test
Enzyme activity (Units/ml) 0 24 61 114 190 Find out from standard graph
Buffered substrate (ml) 0.50 0.45 0.40 0.35 0.30 0.50
Pyruvate standard 2mM (ml) - 0.05 0.1 0.15 0.20 Serum 0.10
Distilled water (ml) 0.10 0.10 0.10 0.10 0.10 0.10
0
Mix well and Incubate for 1 hr at 37 C
DNPH reagent (ml) 0.50 0.50 0.50 0.50 0.50 0.50
Mix well and allow to stand for 20 minutes at R.T
Working NaOH (ml) 5.0 5.0 5.0 5.0 5.0 5.0
Mix well; After 10 minutes measure the O.D at 505 nm
OD values

Procedure:
Mark 6 test tubes as 1,2,3,4,5 for standards and T for test and proceed as follows:

Standard graph:
Plot a standard graph by taking the OD values in the Y axis and the enzyme units in X-axis.

Calculation:
From the standard curve obtained in the graph extrapolate the test OD value to the corresponding
enzyme activity in the X axis.

Clinical Significance:
Elevated levels of SGOT are found in myocardial infarction, in liver disease, skeletal muscle
trauma and some times in renal diseases.
 Practical 17

ESTIMATION OF SERUM ALKALINE PHOSPHATASE

(KIND & KINGS METHOD)

Pprinciple:
Alkaline phosphatase (ALP) hydrolyzes phenyl phosphate into phenol and inorganic
phosphate at pH 10.0. The phenol so found reacts with 4-amino antipyrine in alkaline medium in
the presence of oxidizing agent, potassium ferricyanide to form a red complex whose absorbance
is proportional to the enzyme activity.

Reagents:

1. Buffered substrate reconstitute with 4.5 ml of distilled water.

2. Colour reagent
3. Phenol standard (10 mg/dl)

Procedure:
Pipette into clean, dry test tubes labelled as blank (B), standard (S), control(C) and test (T) as
follows:

S.No Reagent B (ml) S (ml) C (ml) T (ml)

1. Working buffered substrate 0.5 0.5 0.5 0.5
2. Distilled water 1.5 1.5 1.5 1.5
0
3. Mix well and incubate 37 C for 3 minutes
4. Serum - - - 0.05
5. Phenol standard - 0.05 - -
0
6. Mix well and incubate 37 C for 15 minutes
7. Colour reagent 0.5 0.5 0.5 0.5
8. Serum - - 0.05 -
9. OD values at 510

Mix well and measure the absorbance of standard (S), control(C) and test (T) and blank (B)
against distilled water at 510nm or with a green filter in a photoelectric colorimeter.

Calculation:
Serum ALP (KA units) = O.D (T) OD (C)
----------------------- X 10
O.D (S) OD (B)

Result:
The ALP activity of the given serum= KA units (KA= King-Armstrong units)
Clinical Significance:
Serum Alp estimation is useful in diagnosis of hepatobiliary diseases also disease associated with
increased osteoblastic activity. It is elevated in osteomalacia and rickets. Very high levels are
found in patients with bone cancer.

Elevated levels are also found in intra hepatic obstruction due to stone or spasm and also
in extra hepatic obstruction (cholestasis) where the levels are still higher.