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The biosynthesis of human hemoglobin A1c. Slow glycosylation of hemoglobin in vivo.

Hemoglobin A1c, the most abundant minor hemoglobin component in human erythrocytes, is formed by the condensation of glucose with the N-terminal amino groups of the beta-chains of Hb A. The biosynthesis of this glycosylated hemoglobin was studied in vitro by incubating suspensions of reticulocytes and bone marrow cells with [3H]leucine or 59Fe-bound transferrin. In all experiments, the specific activity of Hb A1c was significantly lower than that of Hb A, suggesting that the formation of Hb A1c is a posttranslational modification. The formation of Hb A1c in vivo was determined in two individuals who were given an infusion of 59Fe-labeled transferrin. As expected, the specific activity of Hb A rose promptly to a maximum during the 1st week and remained nearly constant thereafter. In contrast, the specific activity of Hb A1c and also of Hbs A1a and A1b rose slowly, reaching that of Hb A by about day 60. These results indicate that Hb A1c is slowly formed during the 120-day life-span of the erythrocyte, probably by a nonenzymatic process. Patients with shortened erythrocyte life-span due to hemolysis had markedly decreased levels of Hb A1c.

The interaction of 2,3-diphosphoglycerate with various human hemoglobins

Abstract Oxygen equilibria were measured on a number of human hemoglobins, which had been “stripped” of organic phosphates and isolated by column chromatography. In the presence of 2 × 10-4 M 2,3-diphosphoglycerate (2,3-DPG), the P50 of hemoglobins A, A2, S, and C increased about twofold, signifying a substantial and equal decrease in oxygen affinity. Furthermore, hemoglobins Chesapeake and MMilwaukee-1 which have intrinsically high and low oxygen affinities, respectively, also showed a twofold increase in P50 in the presence of 2 × 10-4 M 2,3-DPG. In comparison to these, hemoglobins AIC and F were less reactive with 2,3-DPG while hemoglobin FI showed virtually no reactivity. The N-terminal amino of each β-chain of hemoglobin AIC is linked to a hexose. In hemoglobin FI the N-terminal amino of each γ-chain is acetylated. These results suggest that the N-terminal amino groups of the non-α-chains are involved in the binding of 2,3-DPG to hemoglobin.

Regulation of Transcription by Hypoxia Requires a Multiprotein Complex That Includes Hypoxia-Inducible Factor 1, an Adjacent Transcription Factor, and p300/CREB Binding Protein

ABSTRACT Molecular adaptation to hypoxia depends on the binding of hypoxia-inducible factor 1 (HIF-1) to cognate response elements in oxygen-regulated genes. In addition, adjacent sequences are required for hypoxia-inducible transcription. To investigate the mechanism of interaction between these cis-acting sequences, the multiprotein complex binding to the lactate dehydrogenase A (LDH-A) promoter was characterized. The involvement of HIF-1, CREB-1/ATF-1, and p300/CREB binding protein (CBP) was demonstrated by techniques documenting in vitro binding, in combination with transient transfections that test the in vivo functional importance of each protein. In both the LDH-A promoter and the erythropoietin 3′ enhancer, formation of multiprotein complexes was analyzed by using biotinylated probes encompassing functionally critical cis-acting sequences. Strong binding of p300/CBP required interactions with multiple DNA binding proteins. Thus, the necessity of transcription factor binding sites adjacent to a HIF-1 site for hypoxically inducible transcription may be due to the requirement of p300 to interact with multiple transcription factors for high-affinity binding and activation of transcription. Since it has been found to interact with a wide range of transcription factors, p300 is likely to play a similar role in other genes, mediating interactions between DNA binding proteins, thereby activating stimulus-specific and tissue-specific gene transcription.

A rapid method for the determination of glycosylated hemoglobins using high pressure liquid chromatography

Hemoglobin (Hb) Alc is a minor component of Hb found in normal individuals but elevated two or threefold in patients with diabetes mellitus. Limited studies have suggested that the level of Hb Alc is proportional to the integrated concentration of glucose over time. Thus it could serve as an index of hyperglycemia. Its measurement may enable a more objective approach to assessing whether or not the control of hyperglycemia can be correlated with the severity of complications of diabetes. Large scale clinicab studies of Hb Alc have not been undertaken for lack of a rapid assay system. This article describes a method of high pressure liquid chromatography (HPLC) which enables the isolation of Hb Alc in 27 min using only 12 microgram of Hb (100 microliter of blood) and a second method for the isolation of total fast Hb components (also elevated in diabetes) in 11 min. Using the first method, a total of 36 assays were performed on the blood of a single normal volunteer over a one month period. the mean level of Hb Alc was 4.95 +/- 0.12% (SD) +/- 0.02% (SEM), while the coefficient of variation (C.V.) was 2.4%. The mean Hb Alc & b level was 1.65 +/- 0.06% +/- 0.01% (C.V. = 3.6%). Values for Hb Alc in 10 normal individuals were 5.06 (mean) +/- 0.32% (SD) +/- 0.01% (SEM). Hb Alc values in 15 patients with diabetes mellitus ranged from 6.8 to 20.0%. The second method was designed to assay Hb Ala, Hb Alb, and Hb Alc as a single peak and yielded results identical to the sum of these components as determined by the first method ( r = 0.98; p less than 0.001).

Nonenzymatic glycosylation of erythrocyte membrane proteins. Relevance to diabetes.

Nonenzymatic glycosylation of proteins of the erythrocyte membrane was determined by incubating erythrocyte ghosts with [3H]borohydride. The incorporation of tritium into protein provides a reliable assay of ketoamine linkages. The membrane proteins from 18 patients with diabetes incorporated twice as much radioactivity as membrane proteins from normal erythrocytes. After acid hydrolysis, amino acid analysis showed that the majority of radioactivity was localized to glucosyllysine. Autoradiograms showed that all of the major proteins of the erythrocyte membrane, separated by electrophoresis on sodium dodecyl sulfate gels, contained ketoamine linkages. No protein bands in either normal or diabetic erythrocytes showed significant preferential labeling. Erythrocyte membranes from three patients with hemolytic anemia showed reduced incorporation of tritium from [3H]-borohydride, indicating decreased nonenzymatic glycosylation. Two patients with diabetes and hemolytic anemia had incorporation of radioactivity similar to that of normal individuals. In these groups of patients the incorporation of tritium into erythrocyte membrane proteins correlated with levels of hemoglobin AIc. Thus the modification of membrane proteins like that of hemoglobin depends on blood glucose levels as well as erythrocyte age. These studies show that the enhanced nonenzymatic glycosylation of proteins in diabetics extends beyond hemoglobin to the proteins of the erythrocyte membrane and probably affects other proteins that have slow turnover and are exposed to high concentrations of glucose.

Glycosylated Hemoglobin in Human and Animal Red Cells: Role of Glucose Permeability

We have compared red cells from man and selected animals in order to determine the effect of glucose permeability on nonenzymatic glycosylation of hemoglobin. Glucose permeability was highest in the primates (human, baboon, rhesus monkey), lower in dogs and rabbits, and nearly zero in pigs. Glycosylation of hemoglobin was measured by three independent methods: cation-exchange chromatography on Bio-Rex 70 (Bio-Rad, Inc., Richmond, California), agar gel electrophoresis, and affinity chromatography. The colorimetric thiobarbituric acid test did not provide valid data on animal hemolysates. However, this test was useful for identifying glycosylated hemoglobin (HbA1c) components isolated on Bio-Rex chromatography. In all animals tested, levels of HbA1c (from Bio-Rex chromatography) and total glycosylated hemoglobin (from affinity chromatography) correlated well with glucose exposure, the product of intracellular glucose concentration, and red cell life span. These results indicate that nonenzymatic glycosylation of hemoglobin in mammals is determined by three major variables: mean plasma glucose concentration, red cell life span, and red cell glucose permeability.

A Study of Induced Hyponatremia in the Prevention and Treatment of Sickle-Cell Crisis

Because the formation of sickle cells is dependent on the intracellular concentration of deoxyhemoglobin S, we investigated the possibility of altering or preventing sickle-cell crises by reducing serum sodium so as to cause red cells to swell. In three patients with sickle-cell anemia who had been disabled by recurrent painful crises, sustained dilutional hyponatremia was induced by 1-desamino-8-D-arginine vasopressin (DDAVP) in combination with a high fluid intake. Mean corpuscular hemoglobin concentration fell, and the degree of sickling at low partial oxygen pressure was reduced, as determined by morphologic criteria and by increased oxygen affinity of blood. Chronic hyponatremia (serum sodium, 120 to 125 mmol per liter) reduced the frequency of painful crises, whereas acutely induced hyponatremia abbreviated the duration of crises. These results, although preliminary, are encouraging enough to warrant further study of the safety and effectiveness of induced hyponatremia in the prevention and treatment of sickle-cell crises.

Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus.

We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.

National Diabetes Data Group: Report of the Expert Committee on Glucosylated Hemoglobin

National Diabetes Data Group: Report of the Expert Committee on Glucosylated Hemoglobin The slow chemical reaction between glucose and proteins leads to continuous production of nonenzymatically glucosylated proteins in blood and tissues. The steady-state level of glycosylation of specific proteins is proportional to both the average glucose concentration and the lifespan of the protein in the circulation or tissues. Because the level of glucosylated hemoglobin has been shown to provide an index of blood glucose concentration during the previous 1-2-mo period, it is being used increasingly in the clinical management of diabetes. There has been considerable confusion in this field regarding terminology, methodology, and interpretation of assays of glucosylated hemoglobin. In this report we review the relative merits and limitations of various procedures that have been developed for the measurement of glucosylated hemoglobins. We have attempted to identify preferred methods and reliable standards for the accurate and reproducible measurement of glucosylated hemoglobins. In addition, we present an assessment of the current value of glucosylated hemoglobin measurements in the detection and management of diabetes and guidelines for the use and interpretation of these measurements.

The acetylation of hemoglobin by aspirin. In vitro and in vivo.

The chemical modification of hemoglobin by aspirin (ASA) has been studied, both in intact human red cells and in purified hemoglobin solutions. After incubation of red cells with 20 mM [acetyl-1minus14C]ASA, incorporation of radioactivity into hemoglobin was observed in agreement with the results of Klotz and Tam (1973. Proc. Natl. Acad. Sci. U. S. A. 70: 1313-1315). In contrast, no labeling of hemoglobin was seen when [carbosyl-14-C]ASA was used. These results indicate that ASA acetylates hemoglobin. The acetylated hemoglobin was readily separated from unmodified hemoglobin by both gel electrofocusing and by column chromatography. Quantitation of the extent of acetylation by densitometric scanning of gels agreed very well with estimates obtained from radioactivity measurements. Hemolysates prepared from red cells incubated with ASA showed normal oxygen affinity and heme-heme interaction. Purified acetylated hemoglobin had a slightly increased oxygen affinity and decreased heme-heme interaction. There was no difference in the rate of acetylation of oxy- and deoxyhemoglobin. ASA acetylated column-purified hemoglobin A more readily than hemoglobin in crude hemolysate, but less rapidly than purified human serum albumin. The rate of acetylation of hemoglobulin increased with pH up to approximately pH 8,5. Structural studies were done on hemoglobin incubated with 2.0 mM and 20 mM [acetyl-1-14-C]ASA. Alpha- and beta-chains were acetylated almost equally. Tryptic digests of purified acetylated subunits were fingerprinted on cellulose thin layer plates and autoradiographed. Both alpha- and beta-chains showed a number of radioactive spots that were either ninhydrin negative or weakly ninhydrin positive. These results indicate that hemoglobin is acetylated at a number of sites, probably at the epislon-amino group of lysine residues. To determine whether ASA acetylates hemoglobin in vivo, hemolysates of 14 patients on long-term high-dose ASA therapy were analyzed by gel electrofocusing and compared to specimens of individuals not receiving ASA. The ASA-treated group had a twofold increase in a minor hemoglobin component having an isoelectric point lower than that of hemoglobin A, and indistinguishable from the minoe component which appears when hemoglobin is incubated with ASA in vitro.