Triglycerides assay and reagents therefor

• US 4,245,041 A
• Filed: 12/07/1977
• Issued: 01/13/1981
• Est. Priority Date: 12/07/1977
• Status: Expired due to Term

• Current Assignee: Indiana Development Finance Authority
• Original Assignee: American Monitor Corporation
• Inventors: Denney, Jerry W.
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US05/858,187
• Time in Patent Office: 1,133 Days
• Field of Search: 195/103.5 R, 195/99, 435/15, 435/19, 435/26
• US Class Current: 435/15
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/61 involving triglycerides

Abstract

An improved triglyceride assay and reagent system which provides the introduction of iron into a reaction mixture in which glycerol from hydrolyzed triglycerides is coupled to a NAD/NADH indicator system, and measuring the resultant change of the oxidation state of the iron by use of an iron chelator, thereby improving the sensitivity and performance of the assay.

Claims

1. In a method for the determination of triglycerides in biological fluids by enzymatically hydrolyzing the triglycerides with lipase, converting the product thus formed to glycerol-1-phosphate with adenosine triphosphate (ATP) and the enzyme glycerol kinase (GK), and converting the glycerol-1-phosphate to dihydroxyacetone phosphate by the use of the enzyme glycerol phosphate dehydrogenase (GPDH) with the concomitant reduction of nicotinamide adenine dinucleotide (NAD) to the reduced form NADH,

the improvement, in said triglyceride assay, of reacting the NADH thus formed with iron in the oxidized (ferric) state to form reduced (ferrous) iron, said ferric ion being included in the same reaction mixture as the lipase while the lipase is present therein and is exerting its enzymatic effect, said reaction being mediated by an electron transfer agent, and reacting the reduced iron with an iron chelator to form a chromophore of high intensity, and thereafter quantitating the amount of triglyceride present in the biological fluid by measuring the amount of chromphore formed.

2. The method of claim 1 wherein the electron transfer agent is phenazine methosulfate.

3. The method of claim 1 wherein the iron chelator is 9-(2-pyridyl)-acenaphtho[1,2-e]-as-triazine sulfonate.

4. The method of claim 1 wherein the iron chelator is bathophenanthroline sulfonate.

5. The method of claim 1 wherein the iron chelator is 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'"'"'-disulfonic acid, disodium salt trihydrate.

6. In a method for the determination of triglycerides in biological fluids in which the triglycerides are enzymatically hydrolyzed by use of the enzyme lipase, and by involving the products of the lipase-catalyzed reaction in a further reaction or set of reactions which ultimately yields a change in the oxidation state of an intermediate compound, the improvement of including iron, in the form of ferric ion, in same reaction mixture as the enzyme lipase, and subsequently using the change in the oxidation state of the iron as a means of quantitating the triglyceride content.