II.5.1.4- Quantitative determination of VLDL and LDL
Cholesterol
Calculation
According to the Friedwald formula (Friedwald et
al., 1972):
VLDL Cholesterol = 
LDL Cholesterol (mg/dL) = Total Cholesterol - HDL Cholesterol -
 
II.5.2. Determination of Markers of hepatic and renal
toxicity
II.5.2.1. Determination of total protein (Biuret method)
Principle: Copper ions react in alkali
solution, with protein peptide bonds to give a purple coloured Biuret complex.
The amount of complex formed is directly proportional to the amount of protein
in the specimen (Henry et al., 1974).
Reagents (see annexe 5)
Procedure: Total protein (TP) content was
measured by preparing reagent blank (20 ul distilled water and 1000 ul Biuret
reagent), standard (1000 uL Biuret reagent and 20 uL calibrator) and the sample
(1000uL Biuret reagent and 20 uL sample). After mixing and incubating for 30
min at 20-25°C the absorbance (A) of the samples and standard were read
against the blank at 550nm.
Calculation: Total protein concentration
(g/l) = 190 × Sample absorbance
II.5.2.2. Determination of creatinine level (chronolab
kits)
Principle: The assay is based on the reaction
of creatinine with sodium picrate as described by Jaffe. Creatinine reacts with
alkali picrate forming a red complex. The time interval chosen for measurement
avoids interference from other serum constituents (Murray et al.,
1984a).
+
+ H2O
NaOH
Creatinine
Picric acid
Red complex
.
The intensity of the colour formed is proportional to the
creatinine level in the sample.
Reagents (see annexe 6)
Procedure: The blank (1 ml WR), standard (1
ml WR and 100 uL standard) and sample (1 ml WR and 100 ul sample) were pipette
into cuvettes. After mixing, the stop watch was started and the absorbance (A
1) read after 30 seconds and 90 seconds (A 2) at
500nm.
Calculation: ?A =A2 -
A1
Creatinine in the sample (mg/dl) =  
II.5.2.3. Determination of transaminase activities (aspartate
aminotransferase ASAT and alanine aminotransferase ALAT). (chronolab kits)
Principle: Aspartate aminitransferase (ASAT)
or Glutamate oxaloacetate (GOT) catalyses the reversible transfer of an amino
group from aspartate to á-ketoglutarate forming glutamate and
oxaloacetate. The oxaloacetate produced is reduced to malate by malate
dehydrogenase (MDH) and NADH (Murray et al.,
1984b).
CH2 - COOH
H2N - CH - COOH
Aspartate
Glutamate
CH2 - COOH
CH2
H2N - CH - COOH
á -ketoglutarate
CH2 - COOH
CH2
C = O
COOH
CH2 - COOH
C = O
COOH
Oxaloacetate
+
+
ASAT
Oxaloacetate + NADH + H? MDH
Malate + NAD?
Alanine aminotransferase (ALAT) or Glutamate pyruvate
transaminase (GPT) catalyses the reversible transfer of an amino group from
alanine to á-ketoglutarate forming glutamate and pyruvate. The pyruvate
produced is reduced to lactate by lactate dehydrogenase (LDH) and NADH.
Alanine
CH3
H2N - CH - COOH
Pyruvate
CH3
O = C - COOH
á -ketoglutamate
CH2 - COOH
CH2
C = O
COOH
+
+
Glutamate
CH2 - COOH
CH2
H2N - CH - COOH
Pyruvate + NADH + H? LDH
Lactate + NAD?
The rate of decrease in concentration of NADH, measured
photometrically is proportional to the catalytic concentration of ASAT and ALAT
respectively.
Reagents (see annex 7)
Procedure: The same procedure was use both
for ASAT and ALAT where after pipetting 1mL of the WR and 100 uL of sample into
a cuvette it was mixed and incubates for 1 minute. The spectrometer was
adjusted to zero with distilled water. The initial absorbance (A) of the sample
was read, stopwatch started and the absorbance at 1 minute intervals read
thereafter for 3 minutes at 340nm. The difference between absorbance and the
average absorbance differences per minutes (?A/min) were calculated.
Calculation: ?A/min x 1746 =U/L of ASAT or
ALAT respectively
Units: one international unit (IU) is the amount of enzyme
that transforms 1umol of substrate per minute, in standard conditions. The
activity is expressed in units per litre of sample (U/L).
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