Abstract
Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were 7.1 and 12% respectively. All seven metabolites can be measured in a perchloric acid extract of 20 μl of blood. The methods have been optimized with regard to variation in the perchloric acid content of the samples. These variations arise from the method of sample preparation used to minimize changes occurring in metabolite concentration after venepuncture.
Highlights
Economical and specific measurement of the blood concentrations of intermediary metabolites such as acetoacetate, 3-hydroxybutyrate, glycerol, alanine and pyruvate, as well as the more frequent measurement of lactate and glucose, is of interest to the clinical chemist
This paper reports on an attempt to overcome the perchlorate interference and optimize the centrifugal fast analyser methods
The concentrations used were within the range likely to be found in extracts using the standard method of sample collection where a variable quantity of blood is added to 5 ml of 0"77 mol/1 perchloric acid
Summary
Economical and specific measurement of the blood concentrations of intermediary metabolites such as acetoacetate, 3-hydroxybutyrate, glycerol, alanine and pyruvate, as well as the more frequent measurement of lactate and glucose, is of interest to the clinical chemist. To examine the possibility that the apparent difference in metabolite concentration in the presence of variable concentrations of perchlorate is due to interference in fluorescence, rather than by a decreased enzyme activity, measurements were made photometrically at 340 nm using a LKB 8600 Reaction Rate Analyser at 37C (Pharmacia LKB Biotechnology Division, Central Milton Keynes, Buckinghamshire, UK).
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