Rudolf Flückiger

Hemoglobin Carbamylation in Uremia

CHROMATOGRAPHY of hemolysates of red cells resolves four minor hemoglobin components from the main hemoglobin A (HbA) fraction. These minor components — HbA1a1, HbA1a2, HbAlb, and HbA1c — collectiv...

Glycosylated Hemoglobins: Increased Glycosylation of Hemoglobin A in Diabetic Patients

The components of the hemoglobin-A1 fraction —hemoglobins A1a-c—arise from nonenzymatic glycosylation of hemoglobin A at the β-chain N-terminal amino groups and can be resolved from hemoglobin A by cation exchange chromatography. Glycosylation can also occur at the α-chain N-terminals as well as the E-amino groups of lysine residues of both α- and β-chains; this results in glycosylated species appearing in the hemoglobin-A fraction. In this study, we determined the extent of hemoglobin-A glycosylation using a colorimetric chemical method specific for the detection of ketoamine-linked hexoses in proteins. We demonstrate increased glycosylation of the main hemoglobin-A fraction in diabetic patients, which correlates significantly (r = 0.72, P < 0.001) with the hemoglobin-A, percentage determined by column chromatography in the corresponding hemolysates. This finding provides the basis for the application of this chemical procedure to the measurement of total glycosylation of hemoglobin.

Progress in targeting HIV-1 entry.

Current HIV entry inhibitors target the binding of the viral envelope glycoprotein gp120 to cellular CD4 and co-receptors, or block a late stage of the fusogenic activation of adjacent gp41. New targets are suggested by the role of cell surface protein disulfide isomerase (PDI), which attaches to the primary receptor CD4 close to the gp120-binding site. This could enable PDI to reduce gp120 disulfide bonds, which triggers the major conformational changes in gp120 and gp41 required for virus entry. Inhibiting cell surface PDI prevents HIV-1 entry. The new potential targets outlined are PDI activity as well as the sites of PDI-CD4 and PDI-gp120 interaction.

Quantitation of glycosylated hemoglobin by boronate affinity chromatography.

Total hemoglobin glycosylation and the contribution of glycosylation at the N-terminus of the beta-chains and at “non”-beta-N-terminal positions were quantitated by use of boronate affinity and ion exchange chromatography. Glycohemoglobin (y) was found to correlate linearly (y = 1.92x + 0.53; r = 0.96) with HbA1c (x) and to contain approximately 50% beta-N-terminally glycosylated hemoglobin. This result is in agreement with the binding on boronate agarose of the various hemoglobin components resolved by cation exchange chromatography. An amount of glycohemoglobin similar to that of HbA1c was isolatable from HbA. A slope of less than 2 results because HbA1c is retained only to 93% and the intercept of the regression line reflects the partial adherence (65%) of HbA1a+b to the resin. These results confirm the occurrence of significant “non”-beta-N-terminal glycosylation and show that under optimal chromatographic conditions total glycohemoglobin can be determined with boronate affinity chromatography.

Accelerated Healing of the Rat Achilles Tendon in Response to Autologous Conditioned Serum

Background Despite advances in the treatment of ruptured Achilles tendon, imperfections of endogenous repair often leave patients symptomatic. Local administration of autologous conditioned serum (ACS) in patients with inflammatory, degenerative conditions has shown beneficial effects. Purpose Because ACS also contains growth factors that should accelerate tendon healing, we studied the effect of ACS on the healing of transected rat Achilles tendon. Study Design Controlled laboratory study. Methods In preliminary in vitro experiments, rat tendons were incubated with ACS and the effect on the expression of Col1A1 and Col3A1 was assessed by real-time quantitative polymerase chain reaction. To test its effect in vivo, the Achilles tendons of 80 Sprague Dawley rats were transected and sutured back together. Ten rats from each group (ACS group, n = 40; control group, n = 40) were euthanized at 1, 2, 4, and 8 weeks postoperatively for biomechanical (n = 7) and histologic (n = 3) testing. Lysyl oxidase activity was assayed by a flurometric assay. The organization of repair tissue was assessed histologically with hematoxylin and eosin- and with Sirius red-stained sections, and with immunohistochemistry. Results Tendons exposed to ACS in vitro showed a greatly enhanced expression of the Col1A1 gene. The ACS-treated tendons were thicker, had more type I collagen, and an accelerated recovery of tendon stiffness and histologic maturity of the repair tissue. However, there were no differences in the maximum load to failure between groups up to week 8, perhaps because lysyl oxidase activities were unchanged. Conclusion and Clinical Relevance Overall, our study demonstrates that treatment with ACS has the potential to improve Achilles tendon healing and should be considered as a treatment modality in man. However, as strength was not shown to be increased within the parameters of this study, the clinical importance of the observed changes in humans still needs to be defined.

Chemical quantitation of hemoglobin glycosylation: Fluorometric detection of formaldehyde released upon periodate oxidation of glycoglobin☆

Abstract A sensitive fluorometric method for the quantitation of hemoglobin glycosylation, based upon periodate oxidation of the carbohydrate moieties present on both the α- and ϵ-amino groups of globin is described. The formaldehyde product is measured as the fluorescent 3,5-diacetyl-1,4-dihydrolutidine formed from the condensation of formaldehyde with acetylacetone and ammonia. This method is rigorously designed to assay glycosylated hemoglobin levels and to give a direct measure of the number of glycogroups per milligram of hemoglobin. It requires only 1 mg of protein and may also be used to determine the extent of the nonenzmatic glycosylation of other proteins.

[7] Measurement of nonenzymatic protein glycosylation

Publisher Summary This chapter discusses the methodology for the detection of nonenzymatic protein glycosylation. It describes proteins other than hemoglobin, which are enzymatically glycosylated. In the nonenzymatic glycosylation reaction, glucose and other aldoses and ketoses react with unprotonated amino groups to initially form a Schiffs base (aldimine) which can undergo the Amadori rearrangement to the corresponding 1-amino-1-deoxy-2-keto compound (ketoamine). The charge-dependent separations employing ion exchange chromatography, isoelectric focusing, and agar gel electrophoresis rely on the decrease in positive charge caused by glycosylation of the N-terminal amino groups of the β chains of hemoglobin. Glycosylation at all other sites does not alter the charge sufficiently to allow resolution of adducts from unglycosylated hemoglobin. On ion exchange chromatography, the hemoglobins glycosylated at the N-terminus of α chains and at some ɛ-amino groups elute in the leading edge of the main HbA peak. Total hemoglobin glycosylation may be determined chromatographically with boronate affinity chromatography or by one of several chemical techniques. Under certain conditions, the nonenzymatic glycosylation may even be determined in proteins which are also enzymatically glycosylated with these techniques.

PQQ, the elusive coenzyme

The recently discovered redox coenzyme, PQQ (methoxatin), is widely distributed. Quantitation of protein-bound PQQ has been difficult, but unique redox cycling reactions, which reflect its striking biological properties, reveal trace amounts. PQQ is a potential target for drugs.

Quantitation of Glycosylated Hemoglobin: Elimination of Labile Glycohemoglobin During Sample Hemolysis at pH 5

A simple method for the elimination of labile glycohemoglobin in the chromatographic quantitation of glycosylated hemoglobin is described. Use is made of the instability of Schiff base adducts in acidic solution. Erythrocytes are lysed with a pH 5 buffer. At this pH dissociation reaches completion during sample preparation.

Glycosylation of variant hemoglobins in normal and diabetic subjects

The extent of in vivo glycosylation of variant hemoglobins was examined in individuals with S-, C-, and D-trait. Chromatographie estimates of glycosylation for nondiabetic individuals with S-trait were significantly lower than those for nondiabetic black subjects with normal hemoglobin (P < 0.001). However, chemical determinations of glycosylation (thiobarbituric acid or TBA technique) were similar for these groups (P > 0.10). The Chromatographie elution pattern of hemoglobin S (HbS) was determined, and on this basis an adjustment procedure was performed for Chromatographie data. A regression line was calculated for the relationship between Chromatographie and colorimetrie estimates of glycosylated hemoglobin in S-trait individuals with and without diabetes. The slope of this line was significantly different (P < 0.001) from that for the relationship in individuals with normal hemoglobin. However, after adjustment of Chromatographie values from S-trait individuals, the slopes were similar (P > 0.10). Findings from individuals heterozygous for HbC and D were similar to those for individuals with S-trait. These data indicate that the extent of glycosylation of HbS, C, and D is similar to that of HbA in both the normoglycemic and hyperglycemic range. The TBA technique is the most direct method for determining the extent of glycosylation in individuals with HbS, C, or D. However, adjustment of column Chromatographie values is feasible.