5-hydroxymethylfurfural and thiobarbituric-acid

Topics: Collagen; Colorimetry; Cornea; Furaldehyde; Glycosylation; Humans; Hydrogen-Ion Concentration; Hydrolysis; Microchemistry; Oxidation-Reduction; Sclera; Thiobarbiturates

Skin and aorta collagen specimens of Wistar rats, white mice, beagle dogs, cats, horses, and human necropsies of different ages were examined with respect to the content of glycated products. The data presented show that (a) glycation and accumulation of the chromophore(s) are comparable in collagen samples from different species of comparable age; (b) glycation and pigmented accumulation increase markedly during the first 5-10 years of age; (c) the extent of glycation is different in different tissues (in particular, glycation of aortal collagen is about twice that of skin collagen); and (d) collagen pigmentation as followed by fluorescence is comparable in aortal and skin collagen (except below 10 years); pigmentation measured by absorbance at 350 nm is, on the contrary, lower in aortal than in skin collagen. Based on the assumption of constant blood glucose level during the life span, it appears feasible to conclude that the degree of nonenzymatic collagen glycation reflects the time period for which the protein was exposed to the action of sugars. This period, because of increased cross-linking, is likely to be extended in older animals. Other factors, such as differences in collagen turnover between different tissues and the intensity of the removal process of the glycated products, should be taken into consideration as well.

Topics: Aging; Animals; Aorta; Cats; Chromatography, Affinity; Collagen; Dogs; Furaldehyde; Glycosylation; Horses; Humans; Lysine; Mice; Skin; Species Specificity; Spectrometry, Fluorescence; Sugar Acids; Thiobarbiturates; Ultraviolet Rays

A comparison of methods, two based upon 5-hydroxymethylfurfural determination (colorimetric using a semi-automated procedure and HPLC) and ion exchange chromatography (HbA1 using BioRad method) was performed. The influence of variables on 5-hydroxymethylfurfural determination is reported. The dialysis step appears to be essential for an accurate determination of 5-hydroxymethylfurfural released from glycated haemoglobin. The adjustment of haemoglobin concentration to an accurately known level is required for the colorimetric method, in order to achieve the same background colour absorbance. For the HPLC method this step is not necessary as 5-hydroxymethylfurfural liberation was proved to be stoichiometric. The Sep Pak C-18 cartridge purification appears to be an improvement for the accurate determination of 5-hydroxymethylfurfural using HPLC. When the three methods were compared the overlap between normals and diabetics was found to be less, using the 5-hydroxymethylfurfural HPLC method, suggesting that this procedure, although more time consuming, seems to be more useful in the management of diabetes mellitus.

Topics: Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Colorimetry; Furaldehyde; Glycated Hemoglobin; Humans; Thiobarbiturates

A colorimetric method for the measurement of glycosylated hemoglobin is described. The method is based on the detection of hydroxymethylfurfural liberated from the ketoamine-linked hexose of glycosylated hemoglobin. Rapid hydrolysis is achieved by heating at 120-124 degrees C under pressure, and simplification of the procedure enables the test to be performed in one disposable test tube. Standardization is by means of easily prepared lyophilized human hemoglobin preparations. The test has proven to be reliable and economical in routine use.

Topics: Chromatography; Colorimetry; Furaldehyde; Glycated Hemoglobin; Hemoglobinometry; Humans; Hydrolysis; Thiobarbiturates

We describe here some useful modifications of the thiobarbituric acid (TBA) assay for measurement of nonenzymatic glucosylation of serum protein. The modified assay minimizes interference by glucose without a lengthy dialysis step, and does not require an independent blank determination. These modifications should make the TBA assay more convenient for evaluating glycemic control in diabetes. Serum protein is first precipitated with cold ethanol to remove endogenous glucose. The protein is then hydrolyzed in an oxalic acid solution to release glucose as hydroxymethylfurfural (HMF). The HMF is reacted with TBA to form a chromophore which is extracted into isobutanol for spectrophotometric analysis (lambda max = 435 nm). The absorbance at 435 nm is corrected by subtracting a blank reading at 500 nm, and the nmol HMF released is determined using a standard curve prepared with pure HMF. Normal values of this assay for both adults and children are 0.38 +/- 0.10 nmol HMF/mg serum protein (means +/- 2 SD). When the assay was applied to serum samples from a group of 39 Type I diabetic children more than 90% of the children exceeded the normal range of the assay.

Topics: Adolescent; Adult; Blood Proteins; Chemical Precipitation; Child; Diabetes Mellitus, Type 1; Furaldehyde; Humans; Hydrolysis; Methods; Reference Values; Spectrophotometry; Thiobarbiturates

This study shows that the thiobarbituric acid (TBA) reaction as applied to the measurement of non-enzymatically glycosylated serum proteins(s) [1], yields erroneous results unless strictly standardized conditions are followed. Critical points are in particular (a) the concentrations of NaBH4 required for the preparation of the blanks by reduction of the ketoamine linkages; (b) the amounts of protein which should be identical in all serum samples to be compared; (c) dialysis for removal of glucose and NaBH4 prior to hydrolysis; (d) increase in the yield of 5-hydroxymethylfurfural (HMF) by appropriate conditions of hydrolysis. From our data obtained with an improved TBA-assay it appears that about 90% of total glycosylated serum protein is accounted for by glycosylated albumin. Thus changes in the latter used for diagnostic purposes in diabetes, may well be reflected by total serum glycosylprotein measurement.

Topics: Blood Glucose; Blood Proteins; Borohydrides; Dialysis; False Positive Reactions; Furaldehyde; Glycated Serum Proteins; Glycoproteins; Humans; Hydrolysis; Spectrum Analysis; Thiobarbiturates