Homocysteine HPLC Assay kit:
For Research Use Only
Size: 100 Tests
Incubation Time: 10 minutes
Sample Type: Serum, Plasma
Sample Size: 50 µL
Product Developed and Manufactured in Germany.
Product Support in the USA.
Reference Values (Normal)
< 15 µmol/l (Resch “Homocysteine” ISBN 3-920328-17-5)
It is suggested that each laboratory establish its own normal ranges
The Eagle Biosciences Homocysteine HPLC Assay Kit is intended for the quantitative determination of homocysteine in plasma and serum. The Homocysteine HPLC Assay Kit is for research use only and should not be used for diagnostic procedures.
For the determination of homocysteine, the sample is reduced and derivatized in one step. The albumin bound and the oxidized homocysteine is reduced and converted into a fluorescent probe. During a precipitation step high molecular substances are removed. The sample is cooled at 2-8°C, centrifuged and injected into the HPLC system. The isocratic separation via HPLC at 30°C uses a “reversed phase” column. One run lasts 5 minutes. The chromatograms are recorded by a fluorescence detector. The quantification is performed by the delivered plasma calibrator; the concentration is calculated via the “internal standard method” by integration of the peak areas resp. peak heights.
- Pipette into 1.5 ml reaction tubes: 50 µl sample, CAL or CTRL + 50 µl IS + 20 µl RED + 100 µl DERIVAT
- Vortex briefly. Incubate for 10 min at 60 °C
- Cool down the sample at 2-8 °C and add 100 µl PREC
- Vortex briefly, incubate for 5 min at 2-8°C and centrifuge at 10.000g for 5 minutes.
- Inject 20 µl of the supernatant into the HPLC-system. The supernatant is stable in the dark for 6 days at 2-8°C.
Homocysteine is an amino acid, which is produced out of methionine. It is not ingested by nutrition. Homocysteine is rapidly degraded to cysteine when vitamin B6, folic acid and vitamin B12 are sufficiently available. Increased levels of homocysteine will damage heart and blood vessels. Meanwhile it is an established risk marker for cardiovascular disease. US studies demonstrate that 40% of heart attacks are caused by increased homocysteine levels. Also the risk for stroke is increased by elevated homocysteine concentrations in plasma. A participation of homocysteine in Alzheimer and Parkinson diseases are under discussion.
The reason for high homocysteine levels are a lack of vitamin B6, folic acid and vitamin B12 and/or a defect in the enzyme cystathione synthase, which catalysis the reaction from homocysteine to cysteine. Increased homocysteine concentration have been found to be reduced by the supplementation with folic acid, vitamin B6 and vitamin B12.
The Eagle Biosciences Homocysteine HPLC Assay Kit makes it possible to determine homocysteine in an easy, fast and precise method. The Homocysteine HPLC Assay kit includes all reagents for preparation and separation of the samples with exception of the columns (IC2801rp) and the controls (IC2801ko). Both can be supplied by Eagle Biosciences. Beside the complete test kits it is possible to order all components separately. Please request our single component price list.
Limitations of the Method
Since the concentration of homocysteine in serum samples is depending on the clotting time, EDTA-plasma should be preferred as sample.