Seiichi Era

Conformational changes in seventeen cystine disulfide bridges of bovine serum albumin proved by Raman spectroscopy

Conformational changes in cystine disulfide bridges of bovine serum albumin during acid‐induced isomerization (N→F and F→E transitions) have been studied with Raman spectroscopy. In an X‐ray crystallographic study of human serum albumin, Carter and Ho reported that all disulfide bridges of the albumin molecule are in the gauche‐gauche‐gauche conformation [1] . On the other hand, the solution structure of bovine serum albumin examined by Raman spectroscopy differs from its crystal structure in the conformation of some of the disulfide bridges. Two Raman bands were detected at 520 and 505 cm−1 in the disulfide stretching mode region, suggesting that the 17 disulfide bridges in the N‐form of bovine serum albumin solution take both the gauche‐gauche‐gauche and gauche‐gauche‐trans conformations. The ratio of the peak intensities at 520 and 505 cm−1 (I505/I520) is increased from 1.6 to 2.1 and from 2.1 to 6.3 on going from the N‐ to the F‐form and from the F‐ to the E‐form, respectively, indicating that the gauche‐gauche‐trans conformation of the disulfide bridges is converted to a gauche‐gauche‐gauche one which is the most energetically stable form during the acid‐induced isomerization. However, small amounts of gauche‐gauche‐trans conformation still remain even in the E‐form.

Increased oxidized form of human serum albumin in patients with diabetes mellitus

High-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on Asahipak GS-520H columns at neutral pH (6.87) showed a clear resolution of human mercaptalbumin (HMA) and nonmercaptalbumin (HNA), which are reduced and oxidized form of HSA, respectively. We studied the conversion of HMA to HNA (mercapt-nonmercapt conversion) as an index of oxidative change of the tissues and organs in 28 normal subjects and in a total of 47 patients with non-insulin dependent diabetes mellitus (NIDDM). Mean (+/- SD) values of the HMA fraction of HSA, f(HMA), [HMA/(HMA + HNA)], was significantly lower in NIDDM patients than in normal subjects (0.63 +/- 0.067 vs 0.75 +/- 0.028, P < 0.001). It was lower in poorly controlled NIDDM patients (0.63 +/- 0.058, n = 20) than in well controlled NIDDM patients (0.67 +/- 0.032, n = 9) (P < 0.05). Plasma glucose values sampled on occasions including overnight fasting and postprandial ones (r = -0.441, n = 47, P < 0.01), but not plasma glucose values sampled on overnight fasting (r = -0.345, n = 29) or postprandial (r = -0.467, n = 18) conditions and HbA1c (r = -0.211, n = 34), negatively correlated with the f(HMA) values, indicating that mercapt-nonmercapt conversion may not be due to cumulative hyperglycemia over a month, but due to short-term alteration in blood glucose level. The presence or absence of diabetic complications including nephropathy, retinopathy and neuropathy did not affect the f(HMA) values. In conclusion, decreased f(HMA) values in the diabetic patients suggested the presence of a rapidly altered oxidative change of albumin due to hyperglycemia.

High-performance liquid chromatographic studies on non-mercapt α mercapt conversion of human serum albumin. II

Abstract High-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on a GS-520 column with 0.03 M sodium phosphate buffer—0.15 M sodium sulphate (pH 6.87) showed three peaks, the principal component corresponding to human mercaptalbumin (HMA) and the secondary and tertiary components to nonmercaptalbumin (HNA). Using HPLC analysis, the nonmercapt → mercapt conversion of HSA during haemodialysis and the mercapt → nonmercapt conversion after haemodialysis in chronic renal failure were re-confirmed, indicating that HMA is a covalent carrier protein for sulphur-containing amino acids. Fractions of HMA in various liver diseases were significantly lower than those of healthy male adults.

Observation for redox state of human serum and aqueous humor albumin from patients with senile cataract

Abstract Human serum albumin is known to be a mixture of mercaptalbumin (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form), i.e. a protein redox couple in serum. In order to examine the redox state of human albumin in aqueous humor together with serum, we have studied 51 senile cataract patients, including 16 men and 35 women with ages ranging between 54 and 96 years (mean±S.D. 73.9±8.51). We used a high-performance liquid chromatography combined with fluorescence detection with improved sensitivity. The patients had no detectable evidence of other eye diseases. Of considerable interest is the finding that there was an extremely small amount of HMA fraction (f(HMA)) (f(HMA), 3.50%) as well as a markedly large amount of HNA fraction (f(HNA)) (f(HNA-1), 84.5%; f(HNA-2), 12.0%) in the aqueous humor from patients with senile cataract, compared to the corresponding serum values (f(HMA), 66.4%; f(HNA-1), 31.0%; f(HNA-2), 2.62%). It is anticipated that significantly decreased f(HMA) and increased f(HNA) values in aqueous humor from patients with senile cataract may clearly reflect defective antioxidative defense systems which may lead to the formation of a cataract. To the best of our knowledge, this is the first study observing the redox state of human albumin in aqueous humor.

Alteration of redox state of human serum albumin before and after hemodialysis.

Background:Persistent hypoalbuminemia is a predictor of death in long-term maintenance hemodialysis patients, although cardiovascular diseases remain the leading cause of death. A decreased serum antioxidant activity in maintenance hemodialysis patients may contribute to increased oxidative damage, and may be associated with accelerated atherosclerotic changes. Methods:The aim of this study was to examine the redox state of human serum albumin in maintenance hemodialysis patients by high-performance liquid chromatography (HPLC) using a fluorescence detector. Results: HPLC of human serum albumin on a Shodex-Asahipak ES-502N column at pH 4.85 showed a clear resolution of human mercaptalbumin (HMA) and nonmercaptalbumin (HNA), which are the reduced and oxidized forms of human serum albumin, respectively. The mean ± SD percentage of the HMA fraction of human serum albumin was significantly lower in maintenance hemodialysis patients than in age-matched normal subjects. The percentage of HMA increased 3–5 h after starting the hemodialysis and then decreased to subnormal levels. Conclusion: This suggests that serum albumin may be a major extracellular antioxidant in maintenance hemodialysis patients, and that hemodialysis may rescue serum albumin reduction by inducing intermolecular sulfhydryl-disulfide exchange reaction.

Oral branched‐chain amino acid supplementation improves the oxidized/reduced albumin ratio in patients with liver cirrhosis

Aim:  Branched‐chain amino acid (BCAA) supplementation improves hypoalbuminemia in decompensated cirrhotics. Recently, it was clarified that the ratio of oxidized albumin within total albumin rises with progression of liver cirrhosis. We conducted a feasibility study to investigate whether BCAA supplementation might improve this ratio.

THE CHARACTERIZATION OF HUMAN BRAIN TUMOR USING MAGNETIZATION TRANSFER TECHNIQUE IN MAGNETIC RESONANCE IMAGING

The clinical applicability of magnetization transfer (MT) technique in magnetic resonance imaging (MRI) for the estimation of the histological and constitutional feature of brain tumors was investigated. MT effect was evaluated by measuring the MT ratio (MTR). The parameters in 1.5-tesla MRI system were as follows: TR, 50 msec; TE, 5 msec; flip angle, 30 degree; offset frequency of off-resonance MT pulse, 1000 Hz. The sequence was performed in 20 normal volunteers and 45 patients with brain tumors which were characterized histologically and surgically. The MTR for brain tumors was significantly lower than that for normal brain tissue (p < 0.05). The MTR for meningioma was higher than that for the other brain tumors (p < 0.05). In the meningiomas, MTR for fibrous type was higher than that for meningothelial type, but there was no statistical significance. Regarding the physical consistency for the brain tumors, as classified by surgery, there was a statistically significant difference in MTR between the soft tumor group (0.22 +/- 0.03, n = 6) and the hard tumor group (0.36 +/- 0.04, n = 10) (p < 0.01). This study suggested that the MT technique for patients with brain tumor may be useful to understand the characteristics of the tumors presurgically, based on the degree of intermolecular interaction of macromolecule such as protein.

Alteration of redox state of human serum albumin in patients under anesthesia and invasive surgery

Human serum albumin is a mixture of mercapt- (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form). We studied the mercapt<-->nonmercapt conversion of human serum albumin, which reflects the redox state of the extracellular fluids, in cardiac and other common surgical_ patients using high-performance liquid chromatography. Mean values of [(HMA)/(HMA+HNA)]+/-standard deviation, fHMA+/-sigma], for patients who received common surgery (group 1) and cardiac surgery (group 2) at the start of anesthesia were 0.636+/-0.050 (n = 83) and 0.615+/-0.062 (n = 14), respectively. fHMA values were markedly lower than those for healthy male adults of 0.750+/-0.028 (n = 28). fHMA values increased at 24 h after the start of anesthesia and decreased on the 4th postoperative day in most of the patients. These postoperative changes were prominent in cardiac surgical patients. Although fHMA values after the 7th postoperative day recovered to those at the start of anesthesia in almost all of common surgical patients, those in cardiac surgical patients never recovered even on the 21st postoperative day.

The redox state of albumin and serious cardiovascular incidence in hemodialysis patients.

Human serum albumin is composed of human mercaptoalbumin (HMA) with cysteine residues having reducing powers and oxidized human non‐mercaptoalbumin. The aim of this study is to clarify whether such redox state of albumin (HSA‐redox) influences the incidence of cardiovascular disease (CVD) in chronic kidney disease patients on regular hemodialysis (HD). We measured HSA‐redox using high‐performance liquid chromatography in 86 anuric HD patients. The association between HSA‐redox and incidental CVD events was evaluated. Twenty patients experienced symptomatic CVD events (16 patients died) at the 2‐year follow‐up. The fraction of HMA (f(HMA)) showed a significantly lower value in patients with CVD than that without CVD, in both pre‐HD (36.5 ± 5.8% and 44.6 ± 9.8%, respectively) and post‐HD (57.2 ± 6.2% and 67.2 ± 7.4%, respectively). The adjusted odds ratio (OR) for the incidental CVD event in patients with pre‐HD f(HMA) < 40% was 5.0 (95% CI; 1.2 to 21.3), and that in patients with post‐HD f(HMA) < 60% was 20.6 (3.2 to 134.7). Likewise, the adjusted OR for the CVD death in patients with pre‐HD f(HMA) < 40% was 2.5 (0.6 to 12.5), and that in patients with post‐HD f(HMA) < 60% was 25.6 (2.5 to 262.8). In conclusion, HSA‐redox is closely related to serious CVD incidence and mortality among HD patients.

Temperature-dependent near-infrared spectra of bovine serum albumin in aqueous solutions: spectral analysis by principal component analysis and evolving factor analysis.

Fourier transform near-infrared (FT-NIR) spectra have been measured for bovine serum albumin (BSA) in an aqueous solution (pH 6.8) with a concentration of 5.0 wt % over a temperature range of 45–85 °C. Not only conventional spectral analysis methods, such as second-derivative spectra and difference spectra, but also chemometrics, such as principal component analysis (PCA) and evolving factor analysis (EFA), have been employed to analyze the temperature-dependent NIR spectra in the 7500–5500 and 4900–4200 cm−1 regions of the BSA aqueous solution. Intensity changes of bands in the 7200–6600 cm−1 and 4650–4500 cm−1 regions in the difference spectra indicate variations of the hydration and secondary structure of BSA in the aqueous solution, respectively. The plot of a band intensity at 7080 cm−1 in the different spectra shows a clear turning point at 63 °C, revealing that a significant change in the hydration occurs at about 63 °C. The forward and backward eigenvalues (EVs) from EFA suggest that marked changes in the hydration and secondary structure of BSA take place in the temperature ranges of 61–65 °C and 59–63 °C, respectively. In addition, the temperature of 71 °C marked in the EFA plots may correspond to the onset temperature of increase in the intermolecular β-sheet structure.