1. 1. The paper describes a new principle for the assay of metabolites which depends upon enzymatic catalysis and isotope dilution; the new principle is derived theoretically and is shown to hold for the enzymes glycerol kinase and hexokinase. The principle is used to measure serum glycerol and the values are compared to those obtained with the spectrophotometric assay. It is suggested that the principle may be of value for precise measurement of metabolic compounds that are present difficult or impossible to assay. 2. 2. The addition of unlabelled substrate to an enzyme that is converting isotopically labelled substrate into product has an effect of the incorporation of label into the product, and the magnitude of this effect is dependent upon two factors: firstly the incorporation is decreased according to the isotope dilution principle; and secondly the incorporation may be increased according to the relationship between the substrate concentration and the Km of the enzyme for the substrate. If only a small fraction (10-20%) of the substrate is converted into product by the action of the enzyme, these effects can be described mathematically according to the isotope dilution principle and the Michaelis-Menten equation, respectively. In the theoretical section of this paper these two effects are combined and a simple mathematical relationship is derived which related the changes in incorporation of label into the product to the construction of unlabelled substrate and the Km of the enzyme: this is termed the enzymatic-isotope dilution principle. The predictions of this principle are tested using the enzymes glycerol kinase (EC 2.7.1.30) and hexokinase (EC 2.7.1.1); very close agreement is found between the theoretical predictions and the experimental observations. 3. 3. The principle could be used as a basis for estimation of the concentrations of compounds of biological interest, the main requirement for such an assay are that the compound can be isotopically labelled, that an enzyme can react specifically with this compound, and that this compound and the product of the enzymatic reaction can be physically separted, so that incorporation of label into the product can be measured. 4. 4. The principle has been used for the assay of serum glycerol using the enzyme glycerol kinase and following the incorporation of radioactivity into α-glycerophosphate; both human and rat serum were used without prior protein precipitation. The concentrations obtained by this method are compared to those obtained by the usual enzymatic spectrophotometric assay for glycerol; there is reasonable agreement between the two methods. 5. 5. The enzymatic-isotope dilution principle can be used to extend the number of compounds which can be assayed enzymatically but which cannot be coupled to changes in the oxidation or reduction of pyridine neucleotides. The methods offfers both enzymatic specificity and precision for compounds which are at present difficult to assay. © 1968.
