CLINICAL CHEMSTRY 2 H2O2 + 4-aminophenazone

LABORATORY REVIEWER + phenol quinoneimine +

4H2O
EXERCISE 8: TOTAL CHOLESTEROL
DISCUSSION:
ASSAY REQUIREMENT
Cholesterol
major lipid in body Wavelength: 500 nm
precursor of steroid
Temperature: 20-25 C
hormones
usually associated with Measurement: Reagent Blank (0.01)
coronary heart disease
Unsaturated steroid alcohol Incubate 10 mins at 20-25 C
2/3 in esterified form CALCULATION:
transported by lipoprotein
such as HDL, LDL, VLDL
REAGENT:
Phosphate Buffer
4-Aminophenazole
EXERCISE 9: HDL-C
Phenol
Peroxidase DISCUSSION:
Cholesterol Oxidase
Cholesterol esterase High Density Lipoprotein
(HDL)
STANDARD: 200 mg/dL or 5.17 good cholesterol
mmol/L removes excess cholesterol
from cells
REFERENCE VALUE: <200 mg/dL or
smallest and most dense
<5.2
lipoprotein
PRINCIPLE comprises of both lipid and
protein
Determined by Enzymatic Apolipoprotein A-1 (apo A-1)
Hydrolysis and Oxidation used a as index of the
Uses cholesterol esterase, amount of HDL
cholesterol oxidase, peroxidase larger the lipoprotein, the more
Quinoneimine formed from H2O2 lipid is present relative to
+ 4-aminophenazone in protein lighter the density
presence of (Phenol and
peroxidase) REAGENT:
ENZYME

Cholesterolester + H2O Goods Buffer (6.6)

Cholesterol + Fatty Acid Sodium Chloride
Cholesterol + O2 Cholesterol Esterase
cholestene-3-one + H2O2 Cholesterol Oxidase
POD Catalase
 Ascorbate oxidase Triglycerides
Preservative contains 3 fatty acid
attached to glycerol by ester
SUBSTRATE
bonds
Goods Buffer (7.0) Fatty Acid can be saturated
Peroxidase or unsaturated
4-aminoantipyrin Saturated (Solid in RT,
Sodium Azide animals)
Detergents Unsaturated (Liquid in
RT, plants)
STANDARD: Check the bottle
REAGENT:
REFERENCE VALUE: >35.14 mg/dl or
>0.91 mmol/L PIPES buffer (7.5)
4-chlorophenol
PRINCIPLE 4-aminoantipyrine
Magnesium Ions
Determined by Direct
ATP
homogenous assay
Lipase
elimination of chylomicrons,
Peroxidase
VLDL, LDL using Enzymatic
Glycerol Kinase
reaction with cholesterol
Glycerol-3-Phosphate oxidase
esterase, cholesterol oxidase,
and catalase STANDARD: 200 mg/dL or 2.28
surfactant is added together mmol/L
with chromogen and enzyme
peroxidase REFERENCE VALUE: <194 mg/dL or
< 2.2 mmol/L

ASSAY REQUIREMENT PRINCIPLE

Wavelength: 593 nm major enzyme used in methods

of measurement is Lipase
Temperature: 37 C
Triglyceride glycerol + fatty
Measurement: Reagent Blank (0.01)
substrate (0.25); Enzyme (0.75) acids
Glycerol + ATP glycerol-3-
Incubate 5 mins at 37 C phosphate +ADP
Glycerol-3-phosphate + O2
CALCULATION: dihydroxyacetone phosphate +
H2O2
H2O2 + 4-aminoantipyrine +4
chlorophenol quinoneimine +
HCL + H20
ASSAY REQUIREMENT
EXERCISE 10: Triglyceride
Determination Wavelength: 500 nm
DISCUSSION: Temperature: 20-25C
Measurement: Reagent Blank (0.01) Wavelength: 520-580 nm
Incubate 10 mins at 20-25C Temperature: 20-25C
CALCULATION: Measurement: Reagent Blank (0.01)
Incubate 10 mins at 20-25C standing
time 30 mins
CALCULATION:

EXERCISE 11: Total Protein

Determination EXERCISE 12: Albumin
Determination
DISCUSSION:
DISCUSSION:
Protein
synthesized in liver Albumin
significant in the assessment major protein found in
of synthetic function of liver plasma
Protein Deficiency: also significant in assessing
Malabsorption and liver function
Malnutrition Other function: oncotic
Most requested: Total Protein function and pH
and Albumin also binds in various
substance such as thyroid
REAGENT:
hormones, bilirubin, iron,
Sodium Hydroxide fatty acids
Potassium Sodium Tartrate
REAGENT:
Copper Sulfate
Potassium Iodide Citrate Buffer (4.2)
Bromcresol green
STANDARD: 80 g/L or 8 g/dL
STANDARD: 40 g/L or 4 g/dL
REFERENCE VALUE: 66-87 g/L or 6.6-
8.7 g/dL REFERENCE VALUE: 38-51 g/L or 3.8-
5.1 g/dL
PRINCIPLE
PRINCIPLE
proteins comprises of several
peptides bonds capacity of albumin to bind with
Cupric ions- chelates peptide various
bonds to for Violet colored Albumin binds with certain dyes
compound in alkaline solution froms color complex measured
Biurets Reaction- Absorbance spectrophotometrically
of this is complex is proportional Bromcresol Green mixed with
to the protein concentration in albumin in the sample pH is
the sample maintained by citrate buffer
Potassium Iodide (KI)- The absorbance of this complex
formed is proportional to the
ASSAY REQUIREMENT
 albumin concentration in Incubate 10 mins at 20-25C standing
sample time 30 mins
ASSAY REQUIREMENT CALCULATION:
Wavelength: 520-580 nm
Temperature: 20-25C
Measurement: Reagent Blank (0.01)