Anticancer, Inc.

Li Tang

Yuying Tan

Mingxu Xu

Qinghong Han

Shigeo Yagi

A method to determine a total cysteine in biological fluids utilizes similarly treated portions of the fluid with a homocysteinase and a non-specific desulfurase.

Total cysteine assay

A method to detect and measure the concentration of hydrogen peroxide comprises adding reagents consisting essentially of peroxidase and a dialkyl phenylenediamine and detecting any color generated by the interaction of hydrogen peroxide with these reagents.

Method to determine hydrogen peroxide

An improved synthesis of N,N,-disubstituted-p-phenylene-diamine (DSPDA) preferably di-n-butyl-p-phenylenediamine is disclosed. The improvement rests on the discovery that DSPDA and its salts are extremely sensitive to oxygen so that once the product DSPDA or its salt is formed, oxygen must be excluded from all manipulations.

Synthesis of N,N-disubstituted-p-phenylenediamine

Biological fluid assay methods

Novel enzymatic methods to determine the concentration of homocysteine in biological fluids are described. In a typical embodiment of the invention, the biological fluid sample is from a patient, and the methods of the invention are useful to assess risk for cardiovascular disease. The novel methods of the invention involve use of particular homocysteinase enzymes that permit the determination of homocysteine concentrations in biological samples without interference from the concentrations of cysteine and/or of methionine that are routinely present in such samples. There is also provided a diagnostic kit for use in determining the amount of homocysteine in a biological sample comprising (a) a homocysteinase having the aforementioned characteristics, and (b) at least one reagent capable of being used to determine the amount of product formed in the homocysteinase reaction. In a further aspect, the homocysteinase is provided as a chimeric molecule that comprises amino acid subsequences derived from, or patterned on, more than one homocysteinase, and which is typically produced from a chimeric polynucleotide that encodes therefor. Additional enhancements in homocysteine assay methodology include use of the enzyme .gamma.-glutamylcysteine synthetase to further limit any interference from cysteine present in the biological samples.

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San Diego, CA, United States of America

Li Tang