Enzymatic cycling assays for homocysteine and cystathionine

An enzymatic cycling assay for assessing the amount of homocysteine and/or cystathionine in a solution comprising:

• (a) contacting the solution containing homocysteine and/or cystathionine to form a reaction mixture, with cystathionine β-synthase, or a derivative thereof, L-serine, and cystathionine β-lyase, or a derivative thereof, for a time period sufficient to catalyze the cyclical conversion of homocysteine to cystathionine and the reconversion of cystathionine to homocysteine with the production of pyruvate and ammonia;
• (b) determining the amount of pyruvate and/or ammonia present in the reaction mixture;
• and
• (c) assessing the amount of homocysteine and/or cystathionine present in the solution based on the amount of pyruvate and/or ammonia produced, wherein said assessing step is completed within about 15 minutes after said contacting step.

A method for assessing the amount of homocysteine in a sample, comprising the steps of:

• (a) contacting the sample with a reducing agent for a time period sufficient to release reduced homocysteine from a homocysteine binding protein;
• (b) contacting said reduced homocysteine with a homocysteine metabolite binding transcription factor under conditions conducive for complex formation between said reduced homocysteine and said homocysteine metabolite binding transcription factor;
• (c) admixing said sample with a consensus polynucleotide sequence specifically recognized by said homocysteine/transcription factor complex in order to bind said homocysteine/transcription factor complex with said concensus polynucleotide sequence;
• (d) determining the amount of homocysteine/transcription factor complex bound by the concensus polynucleotide sequence;
• and
• (e) correlating the amount of homocysteine present in said sample with said determined amount.

An assay for assessing the amount of homocysteine in a sample comprising the steps of:

• creating a reaction mixture including said sample, serine, lactate dehydrogenase, NADH and a reductant capable of liberating protein-bound homocysteine in the sample, or their respective derivatives;
• incubating said reaction mixture for a time sufficient to reduce a preponderance of said protein-bound homocysteine;
• adding an enzyme mixture including cystathionine β-synthase and cystathionine β-lyase, or their respective derivatives, and cyclically converting homocysteine to cystathionine and reconverting cystathionine to homocysteine with attendant production of pyruvate;
• and
• measuring the production of NAD+ over time as a measure of homocysteine in the sample.

A method of sequentially assaying a plurality of liquid homocysteine-containing samples comprising the steps of:

• (a) creating a reaction mixture by placing in a container one of said samples together with serine, cystathionine β-synthase, cystationine β-lyase, lactate dehydrogenase, NADH and a reductant capable of liberating protein-bound homocysteine in the sample, or their respective derivatives;
• (b) cyclically converting homocysteine to cystathionine and reconverting cystathionine to homocysteine in said reaction mixture, with attendant production of pyruvate;
• (c) assessing the amount of pyruvate present in the reaction mixture by monitoring the production of NAD+ over time therein;
• and
• (d) repeating steps (a)-(c) for each of said samples, the time interval between the respective reaction mixture-creation steps for each of said samples being up to about 15 minutes.

A method of using cystathionine as a homocysteine assay calibrator comprising the steps of:

• (a) taking a known concentration of cystathionine;
• (b) adding said known concentration to a biological sample;
• (c) using said sample containing cystathionine in an enzymatic reaction which converts homocysteine to cystathionine and reconverts cystathionine to homocysteine with the attendant production of pyruvate and ammonia;
• (d) measuring the amounts of pyruvate or ammonia produced;
• and
• (e) using said measured amounts of pyruvate or ammonia as referenced standards for said homocysteine assay.