Enzyme conversion immunoassay for determining total homocysteine in plasma or serum.

A rapid and precise immunoassay for quantification of total homocysteine in blood samples is presented. The method avoids the use of radioisotopes and chromatographic separations and relies on enzymatic conversion of homocysteine to S-adenosyl-L-homocysteine, followed by quantification of S-adenosyl-L-homocysteine by an enzyme-linked immunoassay in microtiter format. The within- and between-assay imprecision is < 6% and 8%, respectively, and results by the method show good correlation with those by HPLC. Including controls and calibrators in duplicates, 82 samples can be analyzed within 2.5 h. The procedure can be fully automated.

Chromatographic and electrophoretic methods for modified hemoglobins.

The discovery of the clinically important glycohemoglobin adducts and their relation to diabetes mellitus have greatly stimulated the study of other minor post-translational modifications of hemoglobin. Chromatographic and electrophoretic procedures have played an important role in these studies. Today several hemoglobin adducts are known and the formation of adducts with glucose, phosphorylated carbohydrates, urea/cyanate, aspirin, vitamins, acetaldehyde, penicillin and acetyl CoA have been described. Furthermore, new adducts, such as those observed using hemoglobin as a biochemical marker monitoring environmental, occupational and lifestyle exposures to reactive toxic chemicals are constantly being reported. This review deals with chromatographic and electrophoretic separation methods available for the study of non-enzymatic post-translational modifications of hemoglobin. Suitability, perspectives and biomedical applications are discussed.

Soluble highly coloured phenylboronic acids and their use in glycohemoglobin quantification.

Water soluble dye-phenylboronic acid conjugates (dye-PBAs) possessing strong absorption of visible light are introduced as new reagents for the determination of glycohemoglobin. Their functionality and prospective use are demonstrated in a semi-homogenous glycohemoglobin assay. The assay is based on cis-diol esterification of dye-PBA to glycohemoglobin followed by selective precipitation of hemoglobin from solution, co-precipitating bound dye-PBA. Quantification of the molar "dye-PBA/Hb"-ratio in redissolved precipitates using either absorption or fluorescence spectroscopy, reflects the glycation level of the blood samples used. Future development of the assay principle is illustrated in a filter based assay, collecting the precipitated hemoglobin on a filter followed by reflectometric readings directly on the precipitate. The significance of this work lies first, in the demonstration of a new principle for the determination of glycohemoglobin, and second, as an illustration of the prospective use of water soluble, signal-forming non-immobilised boronic acids in the determination of cis-diol containing analytes.

Selective precipitation of human hemoglobin by organic solvents and metal cations.

The interaction of human hemoglobin with several organic solvents and metal cation has been studied in order to obtain selective precipitation of hemoglobin from solution. Alcohols, and preferably the mixture ethanol: butanol added to a final concentration of 8% (v/v) 1-butanol was found to be superior in this respect, giving close to selective precipitation of hemoglobin from whole blood lysates. An equally specific precipitation was achieved by using zinc-chloride in 10-15 molar excess to hemoglobin. Contrary to organic solvents, complex formation with Zn2+ resulted in a reversible precipitation enabling renaturation using strong chelating agents. Specificity of the hemoglobin-precipitating agents was verified by chromatographic and electrophoretic studies. Applications of the presented methods in analytical chemistry and in the isolation and purification of blood proteins are discussed.

Glycohemoglobin filter assay for doctors' offices based on boronic acid affinity principle.

We present a new filter assay for the determination of glycohemoglobin as a unique application of the boronic acid affinity principle. With the use of a water-soluble blue-colored boronic acid derivative and a specific precipitation method for hemoglobin, total hemoglobin including bound boronic acid is precipitated and collected on a filter strip before quantification. Hemoglobin and boronic acid are quantified by a dual-wave-length reflectometric measurement, and the result is reported directly as percent glycohemoglobin. The test is simple, quick, and designed as a doctors' office test for the monitoring and management of diabetes. The imprecision of the assay is < 4% over the range 3-18% Hb A1c, and the method is linear up to at least 20% Hb A1c. Comparisons with four well-established glycohemoglobin methods yielded correlation coefficients ranging from 0.94 to 0.99, with slopes from 0.94 to 1.01.

Failure of glucose-binding lectins Con A and Lentil Lectin to identify glycation of haemoglobin.

We have studied the interaction of Concanavalin A and Lentil Lectin with glycohaemoglobin by a nephelometric lectin-glycogen/dextran precipitation system and monitored the inhibitory effect of glycohaemoglobin on the precipitation. Although inhibitory effects were clearly demonstrated using simple sugars and transferrin, no effect was observed by glycohaemoglobin in relevant concentrations. This is compared to affinity chromatography, binding studies using gel filtration and electrophoresis, and affinity studies using Concanavalin A immobilised on magnetisable polymer particles. Lack of interaction between glycohaemoglobin and lectins is discussed in view of steric constraints and reduced availability of the glycated residues and the stereochemical form of the glycated 1-amino-1-deoxy-fructosyl residues in glycohaemoglobin.

Radiolabelling of human haemoglobin using the 125I-Bolton-Hunter reagent is superior to oxidative iodination for conservation of the native structure of the labelled protein.

Both disruption of the native protein structure and oxidation of iron in the haem/iron(II)-proto-porphyrin-IX residues were observed using the Iodo-gen (1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycouril) method in 125I-labelling of haemoglobin. The reactions taking place affect the native structure of haemoglobin and result in a more acidic molecule. The detrimental effects were unaffected by the presence of iodine. Electrophoretic studies demonstrate that 125I-labelling of haemoglobin using the Bolton-Hunter reagent is the method of choice in order to preserve the native protein.

Protein-boronic acid conjugates and their binding to low-molecular-mass cis-diols and glycated hemoglobin.

Different methods for covalent linkage of phenylboronic acid (PBA) to structural proteins and enzymes are presented. Protein-PBA conjugates, free in solution or immobilised on magnetizable polymer particles, were tested for their binding of D-sorbitol, D-mannose and glycohemoglobin (GHb). Similarly, alkaline phosphatase-PBA conjugates were used in an attempted enzyme-linked sorbent assay for the detection of GHb. Affinity chromatography on immobilised D-mannose and gel chromatographic studies of protein-PBA complexes with [14C]sorbitol, clearly illustrated a low affinity of the interaction studied. Glycated hemoglobin could not be detected using the enzyme-linked sorbent assay approach. However, GHb was found to be specifically retained on columns filled with protein-PBA-coated particles as affinity matrix, enabling the glycation level of blood samples to be determined.