Optimized methods to measure acetoacetate, 3-hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment.

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 mul 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.

A multiwell lectin-binding assay using lotus tetragonolobus for measuring different glycosylated forms of haptoglobin.

Using the fucose-specific lectin, Lotus tetragonolobus, we recently isolated abnormally-glycosylated forms of haptoglobin (FHp) that are useful for monitoring cancer patients and for detecting active disease in rheumatoid arthritis. FHp is detected by electrophoresis and silver staining. In order to use FHp clinically, a better assay is required. A lectin-binding assay (LBA) is described, in which FHp is captured by lotus bound to multi-well plates, and the amount captured is measured by an enzyme-labelled antibody system. The LBA results correlate with those obtained with electrophoresis. The method also gives good precision and low background values. In the presence of 1 mol/l fucose the bound-FHp was reduced by between 60-100%. This confirms that the method is detecting abnormally-fucosylated forms of haptoglobin. This approach opens up exciting possibilities for investigating large numbers of pathological sera and it suggests that other combinations of lectin and antibody may be worth investigating in the future.

Blood glucose, lactate, pyruvate, glycerol, 3-hydroxybutyrate and acetoacetate measurements in man using a centrifugal analyser with a fluorimetric attachment.

Methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, 3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood, using the Cobas Bio centrifugal analyser fitted with a fluorimetric attachment. Intra-assay and inter-assay coefficients of variation ranged from 1.9 to 7.9% and from 1.0 to 7.2% respectively. Correlation coefficients ranged from 0.96 to 0.99 against established continuous-flow and manual spectrophotometric methods. All seven metabolites can be measured using a single perchloric acid extract of 20 microliter of blood. The versatility of the assays is such that as little as 100 pmol pyruvate, 3-hydroxybutyrate or as much as 15 nmol glucose can be measured in the same 20 microliter extract.

Perchloric acid interference in enzymatic-fluorimetric-continuous-flow assay methods for measuring glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate in blood.

Perchloric acid is commonly used to denature and precipitate proteins in samples before various metabolites are measured in tissue, blood, and other body fluids. However, perchloric acid can interfere in the analytical process, possibly by inhibiting the enzymes used. We have determined the effects of perchloric acid on measurements of glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate in blood by enzymatic-fluorimetric-continuous-flow assays. There was a net increase or decrease in the apparent concentration of some of these metabolites when the perchloric acid concentration in the samples differed from that of the reference standards-some of these differences were due to the concentration of perchlorate ion and some to the pH of the acid extracts. The results show the need either to add a fixed amount of blood to perchloric acid or to neutralize and remove the perchlorate.