Fingerstick glycosylated hemoglobin, plasma protein, and albumin.

We have developed techniques that permit the affinity-chromatographic determination of glycosylated hemoglobin, plasma protein, and albumin on fingerstick samples of whole blood. The fingerstick glycohemoglobin technique takes advantage of the high sensitivity of measurement of hemoglobin by absorbance at 414 nm. The glycosylated plasma protein is assayed by a highly sensitive method based on binding of Coomassie blue. An enzyme-linked immunosorbent assay is used to measure albumin in the bound and nonbound fractions of an aminophenylboronic acid chromatographic separation. The fingerstick method for assay of glycosylated plasma albumin gives results that are approximately 40% higher than comparable values obtained on the same patient with a 1-ml plasma sample determined with the bromcresol green technique. There is good correlation of fingerstick glycoalbumins with fingerstick glycohemoglobins and glycosylated plasma protein values. These procedures should be useful for children and for large-scale ambulatory screening for diabetes mellitus.

Comparison and contrast of affinity chromatographic determinations of plasma glycated albumin and total glycated plasma protein.

Techniques for affinity measurement of glycated albumin and for glycated total plasma protein have been developed. The two techniques were contrasted. Both techniques are linear over a 100-fold range of sample concentrations. There appears to be a non-specific early glucose binding phase to non-albumin plasma proteins. Although this phase is detected by radioactive incorporation and thiobarbituric acid, it does not interfere with the affinity determination, which does not appear to detect the early binding species. The correlation of glycated albumin levels with glycated hemoglobin levels is much stronger than that of glycated globulin levels with glycated hemoglobin levels. Due to the large contribution of glycated albumin levels to total glycated serum protein levels, the correlation of the latter with glycated hemoglobin levels is sufficiently strong to allow the use of either technique as an adequate index of glycation.

Clinical use and time relationship of changes in affinity measurement of glycosylated albumin and glycosylated hemoglobin.

Simple techniques for measurement of glycosylated hemoglobin and glycosylated albumin by affinity chromatography on m-aminophenylboronic acid agarose columns have recently been developed. This study explored the time course of changes in glycoalbumin versus those of glycohemoglobin in response to rapid changes in ambient glucose concentration. One would predict that glycoalbumin levels would change more rapidly than glycohemoglobin levels due to the shorter half-life of albumin than hemoglobin. This was found to be the case in a group of rabbits rendered diabetic with alloxan. Glycoalbumin levels plateaued 4 weeks after alloxan administration, while glycohemoglobin levels were still rising. In a group of diabetic patients in whom glucose levels were initially poorly controlled, strict diet or intensive insulin management were used to rapidly bring glucose levels under control. In this group of patients, the glycoalbumin values entered the normal range and plateaued, while glycosylated hemoglobin levels were still falling. Glycoalbumin determination by affinity chromatography is a valuable adjunct to glycosylated hemoglobin determination in evaluating near term control of blood sugar values.

Aminophenylboronic acid affinity chromatography and thiobarbituric acid colorimetry compared for measuring glycated albumin.

Two techniques originally developed for measurement of glycated ("glycosylated") hemoglobin but also applicable to determination of glycated albumin are the thiobarbituric acid colorimetric technique (I) and the aminophenylboronic acid affinity chromatographic procedure (II). The latter reliably distinguishes diabetics from nondiabetics, and concentrations of glycated hemoglobin and glycated albumin are linearly correlated. I is nonspecific; it neither correlates with diabetic status nor with values derived via the affinity technique. Most of the chromogenic material is present in the fraction of albumin that does not bind to aminophenylboronic acid. Glucose interferes significantly with I but only slightly with II. Prolonged incubation of plasma with glucose dramatically increases the II-determined glycated albumin. Reactivity with thiobarbituric acid increases much less, and mainly in the II-bound fraction. This fraction contains a high proportion of nonspecifically reactive material. The percentage of glycated albumin determined in crude plasma samples by II differs only slightly from the value determined by purifying the albumin from the plasma. This technique appears more promising than I for eventual clinical applications.

Use of aminophenylboronic acid affinity chromatography to measure glycosylated albumin levels.

A simple technique for the measurement of glycosylated albumin by affinity chromatography on m-aminophenylboronic acid agarose columns is presented. The technique relies on bromcresol green determination of albumin in the nonbound and bound fractions. There is a linear correlation between albumin concentration of the bound fraction and glycohemoglobin values in individuals. A control nondiabetic plasma pool with a glycohemoglobin value of 7.10% +/- 0.05% (mean +/- SEM) had a glycoalbumin value of 1.64% +/- 0.06%, while a diabetic control plasma pool with a glycohemoglobin value of 13.63% +/- 0.07% had a glycoalbumin value of 4.02% +/- 0.12%. Compared with results from the affinity technique, the preponderance of colorimetric reaction determined with the thiobarbituric acid procedure is nonspecific, in that it does not correlate with diabetic status or with values derived by the affinity procedure. The bulk of thiobarbituric acid-reactive material is present in the fraction of albumin that does not bind to aminophenylboronic acid. This nonbound fraction contains plasma glucose, which significantly interferes with thiobarbituric acid determinations but only very slightly interferes with the affinity procedure. Prolonged incubation of plasma with 500 mg/dl glucose dramatically increases affinity-determined glycosylated albumin. Thiobarbituric acid reactivity increases much less, the increase being mainly in the fraction bound to aminophenylboronic acid. The percentage glycosylated albumin determined by the affinity technique in crude plasma samples differs very slightly, if at all, from that determined by purification of the albumin from plasma. The affinity technique appears very promising for eventual clinical applications in the management of diabetes.

An interspecies comparison of normal levels of glycosylated hemoglobin and glycosylated albumin.

Aminophenylboronic acid affinity chromatography was used to measure glycosylated hemoglobin and glycosylated albumin levels in a variety of species. The highest glycosylated hemoglobin levels were found in man, the lowest in the chicken and the pig. The highest glycosylated albumin levels were found in avian species, the lowest in the mouse and the rat. A simple kinetic model was used to analyze the rates of formation of glycosylated hemoglobin and albumin in the various species. Rates of glycosylated albumin formation were very similar across the species while rates of glycosylated hemoglobin formation were quite different, presumably reflecting wide differences in erythrocyte permeability to glucose among the species.