BIOCHEMISTRY PRACTICAL
SERUM TOTAL PROTEIN
1. Fibrinogen is present in plasma but not in serum, so serum total protein estimation does not involve
fibrinogen.
2. Biuret test is interfered into by haemoglobin in the urine.
3. For finding the A:G ratio,
(a) Serum is taken in test tube and reacted with 28% sodium sulphite to precipitate out the
globulins.
(b) The filtrate collected contains mainly albumin. It is estimated by the Biuret method.
(c) The serum total protein before precipitation was found out.
(d) Globulin= Total protein – Albumin
Normal A:G ratio= 1.5- 2.5 [A:G ratio in normal CSF sample= 3:1]
A:G ratio reversal occurs in chronic liver disease (less albumin production) & multiple myeloma
(increased production of globulins).
4. Albumin can be directly measured by using bromocresol green in acidic medium. The green
coloured albumin-BCG complex’s OD is measured at 620 nm.
5. If protein values > 8 g/dl, dilute with 0.9% saline in 1:1 ratio, rerun and then multiply the
obtained value by 2.
6. Bisalbuminemia is a normal phenomenon where 2 bands of albumin are observed in the
electrophoretogram – one for the normal albumin & the other for albumin with a single amino acid
alteration.
7. Nephrotic syndrome:
(a) Edema
(b) Proteinuria
(c) Lipiduria
(d) Hyperlipidemia
8. Multistix method is most sensitive for albumin, less sensitive for globulin & insensitive for Bence-
Jones protein.
9. Better method for total protein estimation is pyrogallol red method; based on the shift in the
absorbance when the pyrogallol red- molybdate complex binds with the basic group of amino acids.
10. Microalbuminuria is used for identifying diabetic nephropathy.
SERUM ALP
1. Measured in KA units. King-Armstrong unit corresponds to the liberation of 1 mg of
phenol per 100 ml of serum.
2. Increased in:
(a) Physiological- children, puberty, pregnancy
(b) Pathological- bone diseases(like rickets, osteomalacia, Paget’s disease), liver &
biliary tract diseases.
3. Decreased in:
Kwashiorkor, anaemia, scurvy, cretinism, achondroplasia.
SERUM AST & ALT
1. AST- from heart, liver, skeletal muscle, kidney
ALT- from liver mainly (hence more specific for liver diseases)
2. Method used is modified Reitman & Frankel method.
3. It is a kinetic method, because significant amounts of reaction products accumulate
during incubation and increased reaction in the reverse direction.
BLOOD UREA
1. Urea estimation can be done by the Nessler’s method (using urease & Nessler’s
reagent; potassium mercuric iodide; brown colour is noted) or Berthelot reaction
(using urease, phenol, hypochlorite & sodium nitroprusside; blue indophenol is
formed). But the preferred one is the urease-GLDH method.
2. Can be estimated non-enzymatically by DAM (diacetyl monoxime).
3. Serum samples which are highly icteric or hemolysed cannot be used for urea
estimation.
4. If excretion rate>= 2ml/min, maximum clearance= UV/P [normal= 75 ml/min]
If excretion rate< 2ml/min, standard clearance= U√V/P [normal= 54 ml/min]
5. Urea concentration increases with age, due to decrease in GFR, causing more urea
to accumulate in blood.
SERUM CREATININE
1. Commonly used adsorbent is Lloyd’s reagent, that adsorbs creatinine in acidic
conditions.
2. Estimated creatinine clearance is given by the Cockcroft-Gault equation.
3. Interfering substances in the Jaffe’s method are: glucose, bilirubin, acetoacetate,
cefoxitin.
To get a near accurate value for creatinine, the OD is calculated for all the
chromagens in the serum after the colour formation. Then acetic acid is added that
destroys the creatinine picrate complex formed, resulting in a loss of colour. The non-
creatinine serum components retain their colour, and their combined OD after
acidification is measured.
The difference between the 2 ODs gives the OD due to creatinine only.
SERUM URIC ACID
1. Uric acid levels are higher in males than in females, because the higher plasma
estradiol levels in females leads to increased excretion & lesser retention of uric acid
in blood, compared to males.
Due to the same reason, uric acid levels tend to rise in women after menopause.
2. High protein diet is harmful for a patient of gout, because increased supply of
amino acids through diet will lead to the increased production and thus, increased
breakdown of purines, producing more and more uric acid – precipitating the attack
of gout.
BLOOD SUGAR
1. Difference between Dawn phenomenon & Somogyi phenomenon:
In both dawn & Somogyi phenomenon, there is rise in blood sugar (hyperglycemia)
in response to low sugar initially. But they differ in that, dawn phenomenon is
naturally seen in normal individuals. Here, the insulin production at night leads to
hypoglycaemia at night. But after around 2 am, the insulin production wanes and
there is production of anti-insulinistic hormones like GH, which lead to hyperglycemia.
But Somogyi effect is seen in the diabetics in response to low blood sugar following
an overdose of insulin at night.
2.
GLUCOSE TOLERANCE TEST
1.
2.
3. HbA1c & fructosamine both give an idea about the blood glucose levels. But
fructosamine gives an earlier indication than HbA1c.
Also, abnormal Hb do not interfere with fructosamine determination. The
fructosamine method can be used where HbA1c cannot be reliably measured, like
haemolytic anaemia, thalassemia & genetic Hb variants.
BLOOD LIPIDS
1. Friedwald formula:
Total cholesterol= HDL-C + LDL-C + (TG/5) [provided TG<390 mg/dl]
2. How does fatty liver lead to cirrhosis?
Fatty liver occurs due to accumulation of excess TG in liver cells. As more & more
fat molecules enter the cells, they merge together, occupying the cytoplasm & shifting
the nucleus to one side. This finally leads to the disintegration of the cell. This is
followed by the laying down of fibrous tissue as a healing process, that however
leads to cirrhosis.
3. In obese individuals, the fat molecules overload the cell, pushing the nucleus to one
side, causing cell lysis. This TG becomes extracellular, that cannot be metabolically
reutilised- forming the dead bulk in obese persons.
4. Hypocholesterolemia might lead to nervous system manifestations. This is because
cholesterol in the membrane controls its permeability, and thus of the Na+-K+-
ATPase pump. So, less content of cholesterol is harmful.
TEST FOR CARBOHYDATES
1. In Benedict’s solution, sodium citrate is used to chelate the cupric hydroxide to keep
it in solution. It forms a complex, which dissociates sufficiently to provide Cu2+ ions
for oxidation. The same purpose is served by Rochelle salt (sodium potassium
tartrate) in Fehling’s solution.
2. Barfoed test is used to distinguish between monosaccharides & disaccharides. As
the reaction with copper acetate & glacial acetic acid followed by heating is time-
specific, so it is positive for monosaccharides & negative for disaccharides.
3. Seliwanoff’s test is given by ketose sugars like fructose.