Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

A simplified and efficient procedure for the purification of apolipoprotein AI from human serum high-density lipoprotein-3 by preparative isoelectric focussing on polyacrylamide gel beads

An improved method is described for the purification of milligram amounts of apolipoprotein AI from serum apo-HDL₃ by isoelectric focussing on polyacrylamide gel beads. The procedure involves a single focussing over a narrow (1.3 unit) pH gradient, and permits isolation of apo-AI of exceptional purity and in high yield (75% recovery of HDL₃ protein, ca. 50% corresponding to pure apo-AI). The electrophoretic mobility, pI values, molecular weight, antigenicity and amino acid composition of such apo-AI were indistinguishable from those reported in the literature. A rabbit antiserum to apo-AI isolated by focusing exhibited similar immunological reactivity to one prepared from an antigen isolated by gel filtration chromatography; moreover, apo-AI purified by the respective procedures reacted identically with both antisera. We conclude that isoelectric focussing on a support of polyacrylamide gel beads (as Bio-Gel P60) presents certain advantages for the isolation of highly purified apo-AI over both conventional chromatographic procedures and isoelectric focussing on a Sephadex support.     

Apolipoprotein-E degradation in human very low density lipoproteins by plasma protease(s): chemical and biological consequences

Serine proteases coisolate with human very low density lipoproteins (VLDL) which degrade apolipoprotein E and cause hypertriglyceridemic VLDL to lose the ability to interact with the LDL receptor of human skin fibroblasts. We identified proteolytic fragments of apolipoprotein-E in isolated VLDL which can be produced by the action of thrombin on purified apoE. There are two major thrombin cleavage products: M_r ～ 22,000 (E-22) and M_r ～ 12,000 (E-12), the N- and C-terminal fragments, respectively, of apoE. We conclude that the structural integrity and the ability of VLDL to interact with cell receptors are a function of not only VLDL constituents but also of the extent to which VLDL apoprotein E has been degraded.     

Effect and mechanism of the Ang-(1-7) on human mesangial cells injury induced by low density lipoprotein

Hyperlipidemia is an independent risk factor for renal disease, and lipid deposition is associated with glomerulosclerosis. The angiotensin converting enzyme 2-angiotensin-(1-7)-Mas axis (ACE2-Ang-(1-7)-Mas axis) has been reported to participate in lipid metabolic regulation but its mechanism remains unclear. We hypothesized Ang-(1-7) would reduce lipid uptake in human mesangial cells (HMCs) by regulating the low density lipoprotein receptor–sterol regulatory element binding proteins 2–SREBP cleavage activating protein (LDLr–SREBP2–SCAP) negative feedback system, and improve glomerulosclerosis by regulating the transforming growth factor-β1 (TGF-β1). In this study we found that ACE2 was undetected in HMCs. The administration of LDL caused normal LDLr–SREBPs–SCAP negative feedback effect. Exogenous Ang-(1-7) enhanced this negative feedback effect via down-regulating LDLr, SREBP2, and SCAP expression, and effectively inhibited LDL-induced lipid deposition and cholesterol increases. This enhanced inhibitory effect was reversed by the Mas receptor antagonist A-779. Meanwhile, Ang-(1-7) significantly decreased the high LDL-induced production of TGF-β1, an effect blocked by A-779. Interestingly, HMCs treated with Ang-(1-7) alone activated the TGF-β1 expression. Our results suggested that Ang-(1-7) inhibits LDL accumulation and decreases cholesterol levels via modulating the LDLr–SREBPs–SCAP negative feedback system through the Mas receptor. Moreover, Ang-(1-7) exhibits a dual regulatory effect on TGF-β1 in HMCs.