A synergistic composition of microbial lipases from Rhizopus arrhizirs and Candida cylindracea is used for the hydrolysis of glycerol esters such as triglycerides in the determination of glycerol esters present in an aqueous medium such as body fluid.

In processes for measuring glycerides in a fluid, which processes require initial conversion thereof to glycerol, this conversion is achieved according to an improved and simplified saponifying reaction in which a reaction temperature between 35.degree. and 40.degree. C. is used.

A method of determining triglycerides in biological fluids which comprises reacting a biological fluid sample with a lipolytic enzyme to liberate free glycerol, reacting said free glycerol with a coupled enzyme series in an oxygenated aqueous solution containing ATP, GK, .alpha.-GPDH, NAD and an electron transfer agent, and measuring the uptake of oxygen by the oxidation of the resulting NADH with an oxygen-sensing electrode.

Triglycerides are determined by enzymatic saponification with lipase in a buffer in the presence of a carboxylesterase and an alkali metal or alkaline earth metal (10-15 carbon atom) alkyl sulfate, in the additional presence of a compound of the general formula RO(CH.sub.2 CH.sub.2).sub.n H, in which R is alkyl or alkenyl containing 14 to 20 carbon atoms and n is a number of from 7 to 20; this determination prevents turbidity and does not disadvantageously influence enzyme activity.

An analytical process for enzymatic determination of a substrate in biological fluids. The fluid sample is reacted first in the absence of the specific kinase but in the presence of an NADH -- regenerating enzymatic system with the reagents needed for the reaction sequence; then the enzymes are separated from the reaction solution, and thereafter the necessary enzymes are added to the filtrate and NADH consumption is measured. The method is very accurate, economical in requiring less enzymes and only one measurement is necessary.