Method for determining a substance in a biological fluid and reagent combination for use in the method

A method for determining alpha-amylase present in a biological fluid also containing endogenous glucose, which comprises the steps of:

• (1) enzymatically hydrolyzing water-soluble starch with the alpha-amylase present in said fluid to produce oligosaccharides including maltose,
• (2)(a) concurrently consuming the glucose present in said fluid by reacting the glucose present in said fluid with adenosine triphosphate in the presence of hexokinase to convert the glucose to glucose-6-phosphate, and reacting the glucose-6-phosphate with beta-nicotinamide adenine dinucleotide in the presence of glucose-6-phosphate dehydrogenase to convert the glucose-6-phosphate to 6-phosphogluconate with the production of reduced beta-nicotinamide adenine dinucleotide, and
• (b) reacting the reduced beta-nicotinamide adenine dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the beta-nicotinamide adenine dinucleotide to its oxidized form and produce lactate,
• (3) thereafter inhibiting the lactate dehydrogenase enzymatic activity in said fluid, and
• (4) thereafter continuing said hydrolysis with the ampha-amylase present in said fluid, enzymatically hydrolyzing the maltose produced in the presence of alpha-glucosidase to produce glucose, reacting the glucose thus produced with adenosine triphosphate in the presence of hexokinase to produce glucose-6-phosphate, and reacting the glucose-6-phosphate thus produced with beta-nicotinamide adenine dinucleotide in the presence of glucose-6-phosphate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the alpha-amylase activity.

A method for determining alpha-amylase present in a biological fluid also contacting endogenous glucose, which comprises the steps of:

• (1)(a) consuming the glucose present in said fluid by reacting the glucose present in said fluid with adenosine triphosphate in the presence of hexokinase to convert the glucose to glucose-6-phosphate, and reacting the glucose-6-phosphate with beta-nicotinamide adenine dinucleotide in the presence of glucose-6-phosphate dehydrogenase to convert the glucose-6-phosphate to 6-phosphogluconate with the production of reduced beta-nicotinamide adenine dinucleotide, and
• (b) reacting the reduced beta-nicotinamide adenine dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the beta-nicotinamide adenine dinucleotide to its oxidized form and produce lactate,
• (2) thereafter inhibiting the lactate dehydrogenase enzymatic activity in said fluid, and
• (3) thereafter enzymatically hydrolyzing soluble starch with the alpha-amylase present in said fluid to produce oligosaccharides including maltose, enzymatically hydrolyzing the maltose produced in the presence of alpha-glucosidase to produce glucose, reacting the glucose thus produced with adenosine triphosphate in the presence of hexokinase to produce glucose-6-phosphate, and reacting the glucose-6-phosphate thus produced with beta-nicotinamide adenine dinucleotide in the presence of glucose-6-phosphate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the alpha-amylase activity.

A method for determining triglycerides present in a biological fluid also contacting endogenous glycerol, which comprises the steps of:

• (1)(a) consuming the glycerol present in said fluid by reacting the glycerol with beta-nicotinamide adenine dinucleotide in the presence of glycerol dehydrogenase to convert the glycerol to dihydroxyacetone with the production of reduced beta-nicotinamide adenine dinucleotide, and
• (b) reacting the reduced beta-nicotinamide adenine dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the beta-nicotinamide adenine dinucleotide to its oxidized form and produce lactate,
• (2) thereafter inhibiting the lactate dehydrogenase enzymatic activity in said fluid, and
• (3) thereafter enzymatically hydrolyzing the triglycerides in said fluid with lipase to produce glycerol, and reacting the glycerol thus produced with beta-nicotinamide adenine dinucleotide in the presence of glycerol dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the triglyceride content of said fluid.

A method for determining triglycerides present in a biological fluid also containing endogenous glycerol, which comprises the steps of:

• (1)(a) consuming the glycerol present in said fluid by reacting the glycerol with adenosine triphosphate in the presence of glycerokinase to convert the glycerol to glycerol phosphate, reacting the glycerol phosphate with beta-nicotinamide adenine dinucleotide in the presence of glycerophosphate dehydrogenase to convert the glycerol phosphate to dihydroxyacetone phosphate with the production of reduced beta-nicotinamide adenine dinucleotide, and
• (b) reacting the reduced beta-nicotinamide adenine dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the beta-nicotinamide adenine dinucleotide to its oxidized form and produce lactate,
• (2) thereafter inhibiting the lactate dehydrogenase enzymatic activity in said fluid, and
• (3) thereafter enzymatically hydrolyzing the triglycerides in said fluid with lipase to produce glycerol, reacting the glycerol thus produced with adenosine triphosphate in the presence of glycerokinase to produce glycerol phosphate, and reacting the glycerol phosphate thus produced with beta-nicotinamide adenine dinucleotide in the presence of glycerophosphate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the triglyceride content of said fluid.

In a method for determining the alpha-amylase present in a biological fluid by reacting glucose-6-phosphate produced in the course of the determination in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glucose-6-phosphate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the activity of the alpha-amylase in the fluid, said fluid also contacting an endogenous material which or a derivative of which produced during the determination likewise reacts in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glucose-6-phosphate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide, thereby interfering with the determination, the improvement for obviating the interference caused by the endogenous material which comprises:

• reacting said endogenous material or derivative thereof present in said fluid in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glucose-6-phosphate dehydrogenase to consume the endogenous material or derivative by conversion thereof to an oxidation product of the reaction with the production of reduced beta-nicotinamide adenine dinucleotide,
• reacting the resulting reduced beta-nicotinamide adenine dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the reduced beta-nicotinamide adenine dinucleotide to its oxidized form with the reduction of pyruvate to lactate,
• thereafter inhibiting the lactate dehydrogenase enzymatic activity in said fluid, and
• thereafter producing in said fluid a quantity of glucose-6-phosphate for said reaction thereof and production of reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the alpha-amylase activity.

In a method for determining a transaminase present in a biological fluid by reacting L-glutamate produced in the course of the determination in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glutamate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the activity of the transaminase in the fluid, said fluid also containing an endogenous material which or a derivative of which produced during the determination likewise reacts in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glutamate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide, thereby interfering with the determination, the improvement for obviating the interference caused by the endogenous material which comprises:

• reacting said endogenous material or derivative thereof present in said fluid in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glutamate dehydrogenase to consume the endogenous material or derivative by conversion thereof to an oxidation product of the reaction with the production of reduced beta-nicotinamide adenine dinucleotide,
• reacting the resulting reduced beta-nicotinamide dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the reduced beta-nicotinamide adenine dinucleotide to its oxidized form with the reduction of pyruvate to lactate,
• thereafter inhibiting the lactate dehydrogenase enzymatic activity in said fluid, and
• thereafter producing in said fluid a quantity of L-glutamate for said reaction thereof and production of reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the transaminase activity.

In a method for determining triglycerides present in a biological fluid by reacting glycerol produced in the course of the determination in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glycerol dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the content of the triglycerides in the fluid, said fluid also containing an endogenous material which or a derivative of which produced during the determination likewise reacts in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glycerol dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide, thereby interfering with the determination, the improvement for obviating the interference caused by the endogeneous material which comprises:

• reacting said endogeneous material or derivative thereof present in said fluid in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glycerol dehydrogenase to consume the endogenous material or derivative by conversion thereof to an oxidation product of the reaction with the production of reduced beta-nicotinamide adenine dinucloeotide,
• reacting the resulting reduced beta-nicotinamide adenine dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the reduced beta-nicotinamide adenine dinucleotide to its oxidized form with the reduction of pyruvate to lactate,
• thereafter inhibiting the lactate dehydrogenase enzymatic activity in said fluid, and
• thereafter producing in said fluid a quantity of glycerol for said reaction thereof and production of reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the triglyceride content.

In a method for determining triglycerides present in a biological fluid by reacting glycerol phosphate produced in the course of the determination in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glycerophosphate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the content of the triglycerides in the fluid, said fluid also containing an endogenous material which or a derivative of which produced during the determination likewise reacts in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glycerophosphate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide, thereby interfering with the determination, the improvement for obviating the interference caused by the endogenous material which comprises:

• reacting said endogenous material or derivative thereof present in said fluid in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glycerophosphate dehydrogenase to consume the endogenous material or derivative by conversion thereof to an oxidation product with the production of reduced beta-nicotinamide adenine dinucleotide,
• reacting the resulting reduced beta-nicotinamide adenine dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the reduced beta-nicotinamide adenine dinucleotide to its oxidized form with the reduction of pyruvate to lactate,
• thereafter inhibiting the lactate dehydrogenase enzymatic activity in said fluid, and
• thereafter producing in said fluid a quantity of glycerol phosphate for said reaction thereof and production of reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the triglyceride content.

A two-reagent assay system for determining alpha-amylase present in a biological fluid also containing endogenous glucose, which system includes a first reagent comprising an aqueous solution of the following materials in proportions per liter of solution between about 80% and 120% of the proportions indicated:

• ______________________________________ Materials Amount ______________________________________ Water-soluble starch 1.25 grams Adenosine triphosphate 1.25 millimoles Beta-nicotinamide adenine dinucleotide 2 millimoles Alkali metal pyruvate 0.75 millimoles Hexokinase 1500 I.U. Glucose-6-phosphate dehydrogenase 2500 I.U. Lactate dehydrogenase 250 I.U. Alkali metal phosphate buffer, pH 6.3-7.1 25 millimoles Alkali metal chloride 50 millimoles Magnesium salt 3 millimoles ______________________________________
• said first reagent to be admixed with said fluid initially for hydrolyzing the starch and converting glucose present to 6-phosphogluconate in a product containing the beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising an aqueous solution of the following materials in proportions per liter of solution between about 80% and 120% of the proportions indicated:
• ______________________________________ Material Amount ______________________________________ Alpha-glucosidase 7000 I.U. Alkali metal oxamate 180 millimoles Alkali metal phosphate buffer, pH 6.3-7.1 25 millimoles ______________________________________
• said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase and then producing reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the alpha-amylase activity.