Determination of Cholesterol Level in Serum by Enzymatic Colorimetric Method
Introduction
Clinical chemistry uses chemical processes to measure levels of chemical components in the blood.
It is very useful for the early diagnostic of disease and for monitoring organ function. The most common specimens used in clinical chemistry are blood and urine.
Table 1 shows the common blood tests and measurable items using UV/Vis spectrophotometers.In this application note, the cholesterol level in human serum was determined by the enzymatic method using the LAMBDA™ 465 UV/ Vis Spectrophotometer and UV Lab™ software
Principle
The cholesterol esters of the sample are hydrolyzed by cholesterol esterase. 4-Cholesten-3-one and H2O2 are then formed from the released free cholesterol by cholesterol oxidase.
A measurable red quinoneimine derivative, that has an absorbance at 500 nm, is formed from hydrogen peroxide (H2O2) and 4-amino-antipyrine in the presence of phenol and peroxidase.
Cholesterol levels in serum are calculated using Equation 1.
(Normal range : 130 - 250 mg/dL)
Reagents and Apparatus
1. Cholesterol buffer solution (Cholesterol kit, 100 mL)
- phenol 132 mg, NaH2PO4 0.78g, NaH2PO4 0.71 g
2. Enzyme reagent (Cholesterol kit, 100 mL dilution) -
cholesterol oxidase 12 unit, cholesterol esterase 3.5 unit,
peroxidase 6700 unit, 4-aminoantipyrine 17.0 mg/dL
3. Cholesterol standard solution (Cholesterol kit) - 300mg/dL
4. Human serum sample
5. D.I water
6. Water bath
7. LAMBDA 465 (UV/Vis Spectrophotometer)
8. UV Lab software
9. Cuvettes (10 mm pathlength)
Procedure
1. Prepare an enzyme solution by dissolving the enzyme
reagent to 100 mL in the cholesterol buffer solution.
2. Prepare the mixture as shown in Table 2.
3. Place in a water bath at 37 °C for five minutes.
4. Measure blank solution.
5. Measure standard solution.
6. Measure sample solution.
(Perform the measurement quickly, within one hour).
Instrument Parameters
The LAMBDA 465 instrument parameters are as follows. Figure 1
shows experimental method.
Experiment Setup
Data type: Absorbance
Sampling: Single cell
Mode: Scan no. : 30; Integration no.
Result
Figure 2 shows the spectra of cholesterol. The absorbance values
and cholesterol level of serum sample are shown in Table 3. The
determined cholesterol level in serum is 253.84 mg/dL calculated
using Equation 1.
Figure 2 shows the spectra of cholesterol. The absorbance values
and cholesterol level of serum sample are shown in Table 3. The
determined cholesterol level in serum is 253.84 mg/dL calculated
using Equation 1.
Conclusion
The determination of cholesterol level in human serum by enzymatic
colorimetric method was performed using the LAMBDA 465 UV/Vis
spectrophotometer and the UV Lab software. After the reaction,
the measurement time was minimized using the LAMBDA 465,
enabling us to collect data quickly over the full wavelength range
from 190 to 1100 nm. Data processing was performed effectively
by the powerful and easy to use software. The calculated cholesterol
level by Equation 1 was slightly higher than the normal expected
range (130 - 250 mg/dL).
The determination of cholesterol level in human serum by enzymatic
colorimetric method was performed using the LAMBDA 465 UV/Vis
spectrophotometer and the UV Lab software. After the reaction,
the measurement time was minimized using the LAMBDA 465,
enabling us to collect data quickly over the full wavelength range
from 190 to 1100 nm. Data processing was performed effectively
by the powerful and easy to use software. The calculated cholesterol
level by Equation 1 was slightly higher than the normal expected
range (130 - 250 mg/dL).
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