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H F Bunn

Publications and source records attributed to H F Bunn.

At least 91 records · Page 5Linked to original sources
PubMed1984

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.

Adolescent↗
PubMed1984

Post-translational modifications of hemoglobin.

Post-translational modifications of hemoglobin can provide special insights into metabolic disorders. A variety of small molecules in health and disease can form covalent adducts with hemoglobin. The most abundant and best understood of these nonenzymatic modifications is the glycosylation of hemoglobin at the N-terminus of the beta chain (Hb AIc) as well as at the N-terminus of the alpha chain and at certain lysine residues. Glycosylated hemoglobins are elevated in diabetics and offer a useful way of monitoring diabetic control. Moreover, non-enzymatic glycosylation of other tissues may contribute significantly to the long term complications of diabetes. Additional minor hemoglobin components can be formed from other small reactive compounds. For example, cyanate, a breakdown product of urea, reacts with hemoglobin to form distinct minor components in red cells of uremic patients. Acetaldehyde can form hemoglobin adducts in red cells of alcoholics. Thus, hemoglobin can be viewed as a "reporter molecule", revealing metabolic perturbations in a variety of diseases.

Blood Protein Electrophoresis↗
PubMed1984

Electrolyte composition and equilibrium in hemoglobin CC red blood cells.

The red cells of two patients with homozygous hemoglobin C disease (CC) were found to have reduced cation and water content when compared to the red cells of two normal subjects that contained only hemoglobin A (AA). The reduction in cation content was of such a magnitude that the intracellular chloride and hydroxyl (and proton) concentrations were within normal limits despite a measured reduction in the concentration of impermeant negative anions in CC as compared with AA cells of 40 mEq/kg of dry cell solids. The osmotic coefficient of hemoglobin C in CC cells was found to be similar to that observed for hemoglobin A in AA cells. We found no evidence for increased amounts of bound or osmotically abnormal water in CC cells. The reduction in cell cation content in CC cells is mainly due to a reduction in cell K content. The reduced K content is probably related to an increased ouabain- and furosemide-insensitive pathway for K transport in CC cells.

Acid-Base Equilibrium↗
PubMed1983

Characterization of glycosylated hemoglobins. Relevance to monitoring of diabetic control and analysis of other proteins.

Boronate affinity chromatography and ion exchange chromatography were used to measure the levels of glycosylated hemoglobins in normal and diabetic hemolysates, as well as the distribution of glucose adducts on alpha-NH2-valine and epsilon-NH2-lysine residues. When analyzed by ion exchange chromatography on BioRex 70 resin, the Hb Alc peak comprised 4.4 +/- 0.6% of 15 normal hemolysates and 9.1 +/- 2.1% of 15 diabetic hemolysates. The "Hb Alc" was rechromatographed on GlycoGel B boronate affinity resin that binds vicinal hydroxyl groups of covalently linked sugars. Only 70 +/- 5% of the hemoglobin adhered to the resin. Analysis by the thiobarbituric acid colorimetric test confirmed that the affinity resin effectively separated glycosylated from nonglycosylated hemoglobin. When corrected for nonglycosylated contaminants, the mean level of Hb Alc in normal hemolysates was 2.9 +/- 0.4%, a value considerably lower than those previously reported. In addition to Hb Alc, 5.2 +/- 0.5% of the remaining hemoglobin (Hb Ao) was glycosylated. In diabetics, glycosylated Ao was increased in parallel with Hb Alc. After reduction with [3H]borohydride and acid hydrolysis, glycosylated amino acids were first purified on Affi-Gel boronate affinity resin and then analyzed by ion exchange chromatography. The glucose adducts on Hb Ao were distributed as follows: alpha-chain N-terminal valine, 14%; alpha-chain lysines, 40%; beta-chain lysines, 46%. This study has revealed several pitfalls in the analysis of nonenzymatically glycosylated proteins. Peaks isolated by ion exchange chromatography or electrophoresis are likely to be contaminated by nonglycosylated proteins. Furthermore, both the thiobarbituric acid test and [3H]borohydride reduction show variable reactivity depending upon the site of the ketoamine-linked glucose.

Blood Proteins↗
PubMed1983

The value of the peripheral blood smear in anemic inpatients. The laboratory's reading v a physician's reading.

Since physicians are routinely taught to review the peripheral blood smear results of all anemic patients, we analyzed the diagnostic value of the laboratory's blood smear reading and the incremental value of a physician's personal reading in anemic inpatients. Blood smear abnormalities, as reported by the laboratory and two hematologists, were poorly reproducible, with only five of the 11 types of abnormalities being more reproducible than could be expected by chance. The blood smear performed no better than RBC indices in detecting probable iron deficiency or low serum levels of folate or vitamin B12. In anemias not caused by deficiency states, the blood smear reading performed by the hospital laboratory provided unique information in 6% of the cases and helpful information in another 25%, but the additional reading performed by a hematologist never provided unique information and provided incremental helpful information in only 4% of the cases. The peripheral blood smears of all anemic inpatients should be read by the hospital laboratory, but in our hospital, a routine additional personal reading by a physician had limited incremental value and could be reserved for selected cases.

Anemia↗
PubMed1982

Molecular and cellular pathogenesis of hemoglobin SC disease.

Solution and cell studies were performed to ascertain why individuals with hemoglobin (Hb) SC have disease whereas those with Hb AS do not. The polymerization of deoxygenated mixtures containing sickle cell Hb (Hb S; alpha 2 beta 2(6)Glu leads to Val) and Hb C (alpha 2 beta 2(6)Glu leads to Lys) was investigated by measurements of delay times and solubilities. In mixtures containing more than 40% Hb S, polymerization takes place by the same mechanism as in solutions of Hb S alone, with no evidence for independent crystallization of Hb C. A detailed comparison of Hb S/Hb C and Hb S/Hb A mixtures with identical concentrations and proportions of Hb S show that there is no significant difference in the tendency of Hb C and Hb A to copolymerize with Hb S. In 50:50 Hb S/Hb C mixtures, polymerization is about 15 times more rapid than in 40:60 Hb S/Hb A mixtures at the same total Hb concentration. Measurements on density-fractionated erythrocytes show that SC cells contain a higher total Hb concentration and a more uniform distribution of reticulocytes compared to normal (AA) or sickle trait (AS) cells. The concentration distribution for C trait (AC) cells is much closer to that of SC cells than to AS or AA cells. It appears, therefore, that the presence of Hb C results in the SC cell beginning its life with an abnormally high Hb concentration. From these findings we conclude that both the larger proportion of Hb S and the higher intracellular Hb concentration contribute to the pathogenesis of Hb SC disease.

Anemia, Sickle Cell↗
PubMed1982

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.

Animals↗
PubMed1982

Sickle cell disease: advances in the 1970s and challenge in the 1980s.

During the past 10 years considerable progress has been made in understanding the molecular and cellular events responsible for sickling. This information should be valuable in designing effective and safe therapy for this disease. Current studies on the natural history of sickle cell disease should permit the development of improved methods of patient care. The techniques of modern molecular biology have been successfully applied to antenatal diagnosis of sickle cell disease. These techniques may eventually be applied to the control of fetal hemoglobin synthesis in sickle red cells and to the insertion of normal beta globin genes in to bone marrow.

Anemia, Sickle Cell↗
PubMed1982

Effects of red cell membrane on the polymerization of sickle hemoglobin.

We have investigated the effect of erythrocyte membrane preparations on the rate of polymerization and the solubility of deoxyhemoglobin S. The kinetics of polymerization were monitored by measuring changes in turbidity of hemoglobin S solutions as a function of time in a temperature-controlled cuvette. The solubility of deoxyhemoglobin S, an equilibrium measurement, was determined by measuring the hemoglobin concentration of the supernatant following centrifugation of the polymerized hemoglobin S solution. The solubility of deoxyhemoglobin S was unaffected by the presence of red cell membrane ghosts, inside-out red cell vesicles, or inside-out vesicles enriched in erythrocyte spectrin and actin (Bands 1, 2, and 5) in the hemoglobin solution. Furthermore, inside-out vesicles had no significant effect on the kinetics of polymerization. The addition of erythrocyte membrane ghosts to the hemoglobin solution, in ratios approximating that of the intact red cell, increased the rate of polymerization up to 8-fold. However, when corrections were made for the excluded volume contributed by the membrane ghosts, the enhancement of the rate of polymerization was reduced to less than 3-fold. These results fail to show significant facilitation of intracellular sickling by nucleation sites on the red cell membrane.

Anemia, Sickle Cell↗
PubMed1982

The treatment of sickle cell anemia with induced hyponatremia.

Since the rate of polymerization of sickle hemoglobin is exquisitely dependent on its concentration, a small reduction in intracellular hemoglobin concentration should cause a significant inhibition of sickling. In three patients with homozygous sickle cell anemia, sustained hyponatremia was induced by a program consisting of a high fluid intake, a low salt diet and a vasopressin analog, DDAVP. During periods of hyponatremia, mean corpuscular hemoglobin concentration (MCHC) fell 13% and in vitro sickling was reduced as assessed by morphology and oxygen affinity. The frequency and duration of sickle cell crises appeared to be decreased during periods when patients were hyponatremic. These preliminary results indicate that reduction in intracellular hemoglobin concentration is an effective approach to the treatment of sickle cell anemia.

Anemia, Sickle Cell↗
PubMed1981

Non-enzymatic glycosylation of protein: a form of molecular aging.

Aldoses and ketoses react non-enzymatically with amino groups on proteins. The rates for individual amino groups differ widely. Hb AIc, the product of reaction of glucose and the N terminal amino group on beta chains, is normally present inside erythrocytes at about 4% of total haemoglobin. It is 3--5 fold increase in diabetics. Other fast haemoglobins are glycosylation products as well. The condensation between glucose and amino group first and rapidly leads to the formation of an aldimine (Schiff base) which then is slowly rearranged to give a ketoamine. The importance of the above reaction is outlined for other amino groups on haemoglobin and for many other proteins. Its role in the diabetic complications and in molecular aging in general is discussed.

Aging↗
PubMed1981

Reaction of monosaccharides with proteins: possible evolutionary significance.

Measurements were made of the rate of condensation of various monosaccharides with amino groups of hemoglobin to form Schiff base linkages. The reactivity of each sugar was dependent on the extent to which it exists in the open (carbonyl) structure rather than in the ring (hemiacetal or hemiketal) structure. Among the 15 monosaccharides tested, aldoses showed higher reactivities than ketoses. Glucose was the least reactive of the aldohexoses. The emergence of glucose as the primary metabolic fuel may be due in part to the high stability of its ring structure which limits potentially deleterious nonenzymatic glycosylation of proteins.

Borohydrides↗
PubMed1981

Nonenzymatic glycosylation of bovine lens crystallins. Effect of aging.

We have investigated nonenzymatic glycosylation of crystallins from calf and mature bovine lenses (2-6 years old). The lens homogenates were treated with 200-fold molar excess of [3H]NaBH4 and the incorporation of radioactivity was determined. The extent of glycosylation was more precisely determined from the 6 N HCl hydrolysate of [3H]borohydride-treated proteins by analyzing the glucitol-lysine adduct on a high pressure cation exchange column. We found that the [3H]NaBH4 incorporation and the amount of glucitol-lysine detected increased with age, particularly in HM alpha crystallin, a high molecular weight aggregate which accumulates with aging. This age-related increase in nonenzymatic glycosylation was also demonstrated by a comparison of crystallins isolated from the cortex and nucleus of a single lens. Nonenzymatic glycosylation of lens crystallins exemplifies a new form of post-transitional modification of long-lived proteins in vivo.

Aging↗
PubMed1981

Kinetic analysis of the nonenzymatic glycosylation of hemoglobin.

The rate constants have been derived for (a) the condensation of glucose with hemoglobin to form the labile Schiff base intermediate, pre-AIc; (b) the dissociation of this complex to hemoglobin and glucose; (c) the rearrangement of this complex to form the stable ketoamine, Hb AIc. These measurements required the purification of commercially available D-[14C]glucose in order to remove a rapidly reacting contaminant. The initial condensation reaction rate (kappa'1) was measured by incubating column purified Hb A0 for up to 8 h under physiologic conditions with purified D-[14C]glucose in the presence of cyanoborohydride which traps the Schiff base and reduces it to a stable adduct. A parallel incubation utilizing Hb AIc revealed the contribution of the beta-NH2-terminal amino group (kappa 1) to the overall value for kappa'1. The reverse reaction rate (kappa -1) was determined from incubations carried ut in the absence of cyanoborohydride. The rate of the Amadori rearrangement (kappa 2) was determined from longer (6-21 days) incubations under identical conditions, followed by chromatographic isolation of Hb AIc. This value for kappa 2 agrees well with one we previously obtained from in vivo data. These experiments provide direct chemical evidence for an aldimine precursor in the nonenzymatic glycosylation of protein. Furthermore, the use of these rate constants provides a reasonable estimate of the distribution of the labile aldimine (pre-AIc) and the stable ketoamine (Hb AIc) in normal and diabetic red cells. This information is useful in the interpretation of measurements of glycosylated hemoglobin in diabetic patients.

Blood Glucose↗