Schiff-base conjugate of n, n-dibutyl-p-phenylenediamine with pyridoxal 5'-phosphate for improved homocysteine assays using pyridoxal 5'-phosphate-dependent enzymes

A method of producing N,N-dibutyl-p-phenyldiamine-pyridoxal 5′-phosphate comprising the steps of:

• providing a solution of N,N-dibutyl-p-phenyldiamine (DBPDA) in an anhydrous alcohol;
• mixing the solution with a pyridoxal 5′-phosphate (PLP) solution, producing a mixture;
• forming a precipitate by adjusting a pH of the mixture to approximately 4.0;
• and
• centrifuging the precipitate.

A method of producing N,N-dibutyl-p-phenyldiamine-pyridoxal 5′-phosphate comprising the steps of:

• providing a solution of N,N-dibutyl-p-phenyldiamine (DBPDA) in an anhydrous alcohol;
• adding the solution to a pyridoxal 5′-phosphate (PLP) solution, producing a mixture;
• and
• crystallizing the mixture.

A kit comprising:

• a first reagent, the first reagent including a mixture of a reducing agent, homocysteinase (HCYase) and N,N-dibutyl-p-phenyldiamine-pyridoxal 5′-phosphate;
• a second reagent, the second reagent including an Fe(III) containing compound;
• a first buffer solution;
• and
• a second buffer solution.

A method for detecting the risk of a total homocysteine (tHCY) dependent adverse condition comprising the steps of:

• extracting plasma or serum containing homocysteine from a patient;
• reducing the homocysteine with a reducing agent;
• enzymatically reacting the homocysteine with homocysteinase (HCYase), producing hydrogen sulfide (H2S);
• providing N,N-dibutyl-p-phenylenediamine-pyridoxal 5′-phosphate (DBPDA-PLP Schiff-base)
• combining DBPDA released from the DBPDA-PLP with the H2S in the presence of an Fe(III) containing compound producing at least one chromophore;
• and
• measuring the at least one chromophore wherein the step of measuring the at least one chromophore determines a tHCY concentration between 5-15 μmol/L or greater than 15 μmol/L.