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Hcy OEM with cystathionine cyling

Category:Recombinant Proteins



Hcy detection Kit OEM ( Original EquipmentManufacturer ) with Cystathionine Enzyme Cycling Method

TheLiquid Stable 2 Part Homocysteine Reagent is intended for in vitro quantitativedetermination of total homocysteine in human serum and plasma. The device canassist in the diagnosis and treatment ofpatients suspected of having hyperhomocysteinemia and homocystinuria.

Homocysteine (HCY) is a thiol- containing amino acidproduced by the Intracellulardemethylation of methionine. Homocysteine is exported into plasma where itcirculates, mostly in its oxidized form, bound to plasma proteins as aprotein-HCY mixed disulfide with albumin (protein-SS-HCY). Smaller amounts ofreduced homocysteine and the disulfide homocystine (HCY-SS-HCY) are present.Total homocysteine (tHCY) represents the sum of all the HCY species found inserum or plasma (free plus protein bound). Homocysteine is metabolized toeither cysteine or methionine. In the vitamin B6 trans-sulphuration pathway,homocysteine is irreversibly catabolized to cysteine. A major part ofhomocysteine is remethylated to methionine, mainly by the folate andcobalamin-dependent enzyme methionine synthase. Homocysteine accumulates and isexcreted into blood when these reactions areimpaired. Severely elevated concentrations of total homocysteine are found insubjects with homocystinuria, a rare genetic disorder of the enzymes involvedin the metabolism of homocysteine. Patients with homocystinuria exhibit mentalretardation, early arteriosclerosis and arterial and venous thromboembolism.Other less severe genetic defects which lead to moderately elevated levels oftotal homocysteine are also found.

Epidemiologicalstudies have investigated the relationship between elevated homocysteinelevels and cardiovascular disease (CVD). A meta-analysis of 27 of thesestudies, including more than 4000 patients, estimated that a 5 umol/L increasein total homocysteine was associated with an odds ratio for coronary arterydisease (CAD) of 1.6 (95% confidence interval [CI], 1.4 to 1.7 for men and 1.8(95% CI 1.3 to 1.9) for women;the oddsratio for cerebrovascular disease was 1.5 (95% CI 1.3 to 1.9). The riskassociated with a 5 umol/L increase in total homocysteine was the same as that associatedwith 0.5 mmol/L (20 mg/dL) increase in cholesterol. Peripheral arterialdisease also showed a strong association.

Hyperhomocysteinemia,elevated levels of homocysteine, can be associated with an increased risk ofCVD. There have also been many published reports of prospective studies on therelationship between hyperhomocysteinemia and risk of CVD in men and women whowere initially healthy. End points were based on a cardiovascular event such asacute myocardial infarction, stroke, CAD, or mortality. The results of elevenof these nested case-control studies reviewed by Cattaneo were equivocalwhere five of the studies support the association with risk and six do not.More recently homocysteine levels were determined in a prospective study ofpost-menopausal women who participated in the Women’s Health Study. Specimensfrom 122 women, who subsequently developed cardiovascular events, were testedfor homocysteineand compared to a control group of 244 women who were matched for age andsmoking status. The women in the control group remained free of disease duringthe three year follow-up period. The results demonstrated that post-menopausalwomen who developed cardiovascular events had significantly higher baseline homocysteinelevels. Those with levels in the highest quartile had a two-fold increase inrisk of any cardiovascular event. Elevated baseline homocysteine levels wereshown to be an independent risk factor. Also, homocysteine levels weredetermined in 1933 elderly men and women for the Framingham Heart Study cohortand demonstrated that elevated levels of homocysteine are independently associatedwith increased rates of all-cause and CVD mortality.

Patientswith chronic renal disease experience an excess morbidity and mortalitydue to arteriosclerotic CVD. Elevated concentration of homocysteine is afrequently observed finding in the blood of these patients. Although suchpatients lack some of the vitamins involved in the metabolism of homocysteine,the elevated HCY levels are mainly due to impaired HCY removal from the bloodby the Kidneys. Recentevidence has also implicated elevated blood levels of homocysteine in miscarriagesand birth defects.Drugs such as methotrexate, carbamazepine, phenytoin, nitrousoxide, and 6-azauridine triacetate interfere with HCY metabolism and may giveelevated levels of HCY.

Test Summary andPrinciple

Boundor dimerized homocysteine (oxidized form) is reduced to free homocysteine,which then reacts with serine catalyzed by cystathionine beta-ynthase(CBS) to form cystathionine. Cystathionine in turn is broken down by cystathioninebeta-lyase (CBL) to form homocysteine, pyruvate and ammonia. Pyruvateis then converted by lactate dehydrogenase (LDH) to lactate with nicotinamideadenine dinucleotide (NADH) as coenzyme. The rate of NADH conversionto NAD+ is directly proportional to the concentration of homocysteine (?A340 nm).

Reduction:Dimerized homocysteine, mixed disulfide, and protein-bound forms of HCYin the sample are reduced to form free HCY by the use of tris [2-carboxyethyl]phosphine (TCEP).


EnzymaticConversion:FreeHCY is converted to cystathionine by the use of cystathioninebetasynthase and excess serine. The cystathionine is then broken downto homocysteine, pyruvate and ammonia. Pyruvate is converted to lactate vialactate dehydrogenase with NADH as coenzyme. The rate of NADH conversionto NAD+ (? A340 nm) is directly proportional to the concentration of homocysteine.



R1reagent: NADH (0.47 mM), LDH (38 KU/L), Serine (0.76 mM), Trizma Base 1- 10%,Trizma Hydrochloride 1-10%, Sodium Azide < 1%. Reductant (TCEP:2.9 mM) R2reagent: Cycling Enzymes CBS (0.748 KU/L)and CBL (16.4 KU/L) SodiumAzide < 1%.

Calibrator1: Aqueous homocysteine blank ( 0 umol/L).

Calibrator2: Aqueous homocysteine solution ( 28 umol/L).

Reagent Preparation

R1and R2 are packaged ready to use. The reagents are stable until the expiration datespecified on the label.

Indications ofDeterioration

Thereagents should be clear of particulate material. They should be discarded ifthey become turbid.

CalibratorPreparation and Use Thecalibrators are prepared gravimetrically and are traceable to Standard ReferenceMaterial NIST SRM 1955, confirmed by a designated measurement procedure(HPLC). The calibrators are supplied inthe kit and are provided ready to use. Values are printed on the labels.


1. Adhere strictly to the instructions in thisinsert, particularly for handling andstorage conditions.

2. Reagent 1 and Reagent 2 contain sodium azidewhich can react withleador copper plumbing to form highly explosive metal azides. O disposal,flush with large quantities of water to prevent azide buildup.

3. Material safety data sheets for all hazardouscomponents contained inthiskit are available upon request.


1. Store kit components at 2-8?C and use untilthe expiry date on the labels. Do not use expired reagents.

2. Reagents may be used on multiple occasionsuntil the expiry date on the labels. Reagents must be returned to 2-8°C storagebetween use.

3. Do not mix different reagent kit lot numbers.


5. Do not expose Reagent 1 and Reagent 2 tolight during on-board use.

6. Avoid contamination of reagents. Use a newdisposable pipette tip foreachreagent or sample manipulation.

SpecimenCollection and Handling

1. Serum (collected in serum or serum separatortubes) and plasma (collected in potassium EDTA or lithium heparin tubes) may beused for the measurement of homocysteine. However, it is not recommended to useindividual patient results from serum,heparinized plasma and EDTA plasma interchangeably. Additionally matrixdifferences between serum and serum separator tubes and plasma tubes have beenreported. To minimize increases in homocysteine concentration from synthesis by redblood cells, process specimens as follows:

- Place all specimens (serum and plasma) on iceafter collection and prior to processing. Serum may clot more slowly and the volume may be reduced.

- All specimens may be kept on ice for up to 6hours prior to separation by centrifugation.

- Separate red blood cells from serum or plasmaby centrifugation and transfer to a sample cup or other clean container.

Note:Specimens not placed on ice immediately may exhibit a 10-20% increase inhomocysteine concentration.

2. If the assay will be performed within 2 weeksafter collection, the specimen should be stored at 2-8?C. If the testing willbe delayed more than 2 weeks,thespecimenshould be stored frozen at -20?C or colder. Specimens have been shown to bestable at -20?C for 8 months.

3. It is the responsibility of the operator toverify the correct specimen type(s) is (are) used in the liquid stable 2-PartHomocysteine Reagent.

4. Inspect all samples (specimens, calibratorsand controls) for bubbles. Remove bubbles prior to analysis.

5. Specimens containing particulate matter (fibrin,red blood cells, or other matter) and visibly lipemic specimens should not beused with the assay. Results from these specimens may be inaccurate.

6. Mix specimens thoroughly after thawing by lowspeed vortexing or by gentle inversion to ensure consistency in results. Avoidrepeated freezing and thawing. Specimens showing particulate matter,erythrocytes, or turbidity should be centrifuged before testing.

7. On-board instrument storage. EDTA plasmasamples can be stored for 3hoursonboard the AU400. The other recommended sample tubes for useonthe assay have not been tested.


Homocysteine Reagent R1 andR2, Calibrators, Controls .