Takaya Mio

Glycated hemoglobin and lipid peroxidation in erythrocytes of diabetic patients.

In diabetes, glycation and subsequent browning (or glycoxidation) reactions are enhanced by elevated glucose concentrations. It is unclear whether the diabetic state per se also induces an increase in the generation of oxygen-derived free radicals (OFRs). However, there is some evidence that glycation itself may induce the formation of OFRs. OFRs cause oxidative damage to endogenous molecules, including cholesterol. 7-Oxocholesterol is known to be one of the major products of cholesterol oxidation. The level of cholesterol peroxidation products was assessed in erythrocyte membrane lipid by monitoring the peak height ratio of 7-oxocholesterol, one of the products of cholesterol peroxidation, to cholesterol with gas chromatography/mass spectrometry (GC/MS). The peak height ratio of 7-oxocholesterol to cholesterol was used as a biomarker of lipid peroxidation. The hemoglobin A1c (HbA1c) value, an index of glycemic stress, was measured by high-performance liquid chromatography. We examined the relationship between the levels of cholesterol peroxidation products and HbA1c in erythrocytes of diabetic and healthy subjects. There was a significantly increased ratio of 7-oxocholesterol to cholesterol in diabetic erythrocytes compared with control erythrocytes. The ratio of 7-oxocholesterol to cholesterol was significantly correlated with the level of HbA1c. This suggests that glycation of hemoglobin via chronic hyperglycemia is linked to cholesterol peroxidation in erythrocytes of both diabetic and healthy subjects.

Levels of lipid peroxidation product and glycated hemoglobin A1c in the erythrocytes of diabetic patients

In diabetes, the glycation and subsequent browning (or glycoxidation) reactions are enhanced by elevated glucose concentrations. It is unclear whether or not the diabetic state per se also induces an increase in the generation of oxygen-derived free radicals (OFRs). There is some evidence, however, that glycation itself may induce the formation of OFRs. OFRs could cause oxidative damage to endogenous molecules. We examined the relationship between the levels of lipid peroxidation and the levels of glycated hemoglobin A1c (GHbA1c) in erythrocytes of diabetic and healthy subjects. Lipid peroxidation was assessed in erythrocyte membrane lipids by monitoring peak height ratios of conjugated linoleic acid (CLA), one of the products of lipid peroxidation, to linoleic acid (LA) using gas chromatography-mass spectrometry (GC/MS). CLA is a collective term used to designate a mixture of positional and geometric isomers of LA in which the double bonds are conjugated. The peak height ratio of CLA to LA was used as a biomarker of lipid peroxidation. GHbA1c, an index of glycemic stress, was measured by high-performance liquid chromatography. There were significantly increased ratios of CLA to LA in diabetic erythrocytes compared with control erythrocytes. These ratios of CLA to LA were also significantly correlated with GHbA1c values. This suggests that glycation via chronic hyperglycemia links lipid peroxidation in the erythrocytes of both diabetic and healthy subjects.

Influence of admission functional status on functional change after stroke rehabilitation.

Inouye M, Hashimoto H, Mio T, Sumino K: Influence of admission functional status on functional change after stroke rehabilitation. Am J Phys Med Rehabil 2001; 80:121–125. ObjectiveTo determine whether the admission functional score influences the functional change after stroke rehabilitation. DesignTwo hundred forty-three patients who had received the Functional Independence Measure (FIMTM) assessment at admission and at discharge were enrolled in the study. The patients were stratified into three groups according to their FIM total scores at admission, i.e., ≦36, 37 to 72, and ≧73. ResultsThe Scheffé’s multiple comparison test showed that patients with FIM total scores of ≧73 at admission were significantly younger (58 ± 11 [SD] yr) than those who had scores of 37 to 72 (64 ± 11 yr) or ≦36 (66 ± 12 yr). Patients with FIM total scores of 37 to 72 at admission showed significantly higher FIM gain (37 ± 15) compared with those patients who had scores of ≧73 (20 ± 10) or ≦36 (29 ± 23). ConclusionThe functional levels of affected patients at admission stratified by the FIM scale roughly predict the degree of functional gain after rehabilitation in survivors with a first episode of ischemic stroke. Moderately affected patients will benefit from intensive rehabilitation. These findings may be useful for rehabilitation triage.

Link between glycation and lipoxidation in red blood cells in diabetes.

Oxidative stress is postulated to be increased in patients with diabetes mellitus. Glycation enhanced by elevated glucose concentrations may induce the formation of oxygen-derived free radicals (OFRs). OFRs would cause oxidative damage to endogenous molecules, including cholesterol. Accumulating evidence suggests that oxidative cell injury caused by OFRs contributes to the development of both macroangiopathy and microangiopathy in diabetes. Our previous studies have shown that 7-keto cholestadien is one of the major products of cholesterol peroxidation in diabetic erythrocyte membrane and its levels correlate with hemoglobin Alc (HbAlc) values. We have newly identified 3-cholesten-6-one, one of the minor products of cholesterol peroxidation, in it. The aim of our study is to investigate whether 3-cholesten-6-one levels also correlate with HbAlc values. Levels of 3-cholesten-6-one were assessed in erythrocyte membrane lipid by monitoring peak areas of 3-cholesten-6-one to cholesterol with gas chromatography-mass spectrometry. The peak area ratio of 3-cholesten-6-one to cholesterol was used as a marker of cholesterol peroxidation. The HbAlc value, an index of both glycemic stress and glycation, was measured by high-performance liquid chromatography. In this study, we evaluated 33 diabetic and 29 healthy subjects, matched for age (59.3+/-14.5 vs. 57.3+/-13.7 years, mean+/-S.D.) and sex (15 males and 14 females vs. 16 males and 17 females). There were both significantly raised HbAlc levels (4.6+/-0.8 vs. 8.3+/-2.4%, P<0.001) and significantly increased ratios of 3-cholesten-6-one to cholesterol (0.2+/-0.4 vs. 21+/-1.8, P<0.001) in diabetic patients compared to control subjects. A good correlation between HbAlc levels and ratios of 3-cholesten-6-one to cholesterol was found in participants (r = 0.75, P<0.001, y = 0.46x-1.8). This suggests that an oxidative stress exists in diabetes and the link between glycation and lipoxidation is found in diabetic red blood cell.

Formation of 9-hydroxy linoleic acid as a product of phospholipid peroxidation in diabetic erythrocyte membranes.

The increased production of oxygen-derived free radicals (OFR) and lipid peroxidation may contribute to vascular complications in diabetes. Some lipid peroxidation products have already been reported to be formed via glucose-induced oxidative stress. We have identified 9-hydroxy linoleic acid (9-OH-C18:2) in the red cell membrane phospholipid of diabetic subjects. We hypothesized that 9-OH-C18:2 would be formed in hydroxyl radical reactions to linoleic acid (C18:2) during glucose-induced oxidative stress, and confirmed that the formation of 9-OH-C18:2 was induced by ultraviolet (UV)-C irradiation to the synthetic C18:2. UV-C light generates highly reactive hydroxy radicals. C18:2 is confirmed to be the precursor of 9-OH-C18:2. To estimate the degree of oxidative damage to red cell membrane phospholipids, we developed a selective ion monitoring gas chromatography-mass spectrometric measurement for C18:2 and 9-OH-C18:2, following methanolysis of red cell membrane phospholipids. The relative peak height ratio of C18:2 to 9-OH-C18:2 (9-OH-C18:2/C18:2) was measured in phospholipid extracts of red cell membranes from healthy (n=29, 3.1+/-1.9%) and diabetic (n=27, 20. 9+/-16.1%) subjects. It was confirmed that 9-OH-C18:2/C18:2 is significantly (P<0.001) elevated in patients with diabetes. The measurement of 9-OH-C18:2/C18:2 in red cell membranes should be useful for assessing oxidative damage to membrane phospholipids in diabetes.

Comparative analysis of plasma and erythrocyte 7-ketocholesterol as a marker for oxidative stress in patients with diabetes mellitus

OBJECTIVES To reveal increased lipid peroxidation in diabetics by quantification of cholesterol oxidation products (COPs) not only in plasma, but also in erythrocytes. DESIGN AND METHODS We quantified 7-ketocholesterol (7-kCho) by gas chromatography-mass spectrometry as a surrogate measure for COPs. These assays were performed on both plasma and erythrocytes in 20 control subjects and 20 treated patients with relatively poorly controlled Type 2 diabetes. RESULTS Both plasma and erythrocyte 7-kCho levels in diabetics were significantly higher than those in control subjects. Although neither plasma nor erythrocyte 7-kCho levels were associated with markers for glucose tolerance in diabetics, a negative correlation of serum HDL-cholesterol levels with erythrocyte, but not plasma, 7-kCho levels was found. CONCLUSION Increased oxidative stress in diabetics affects oxidation of cholesterol. Assays of COPs not only in plasma, but also in erythrocytes, may yield complementary information in lipid peroxidation.

Nilvadipine protects low-density lipoprotein cholesterol from in vivo oxidation in hypertensive patients with risk factors for atherosclerosis

AbstractObjective: Nilvadipine, a calcium antagonist, has been shown to have antioxidant activity in vitro, but its effect on in vivo oxidation has not been assessed. The aim of this study was to investigate the antioxidant effect of this agent in vivo. The ratios of 7-keto cholestadien to cholesterol are believed to be an available marker of lipid peroxidation. Using these ratios, we have assessed the antioxidant effect of nilvadipine on low-density lipoprotein (LDL) in hypertensive patients with high risk of atherosclerosis. The risk factors of atherosclerosis may involve oxidation of LDL. Methods: Fifteen healthy subjects (seven females and eight males aged 35–72 years, mean ± SD=55.3 ± 13.8 years) and fifteen hypertensive patients (seven females and eight males aged 45–80 years, mean ± SD = 60.2 ± 11.8 years) were recruited. Patients were treated orally with nilvadipine (4 mg b.i.d.) for 4 weeks. Cholesterol oxidation levels of LDL in patients before and after nilvadipine therapy and healthy subjects were studied. Results: The ratios of 7-keto cholestadien to cholesterol in LDL of hypertensive patients before and 4 weeks after nilvadipine treatment and in healthy subjects were 6.5 ± 1.6% (mean ± SD), 3.8 ± 1.2%, and 0.2 ± 0.1%, respectively. There were significantly (P < 0.001) increased levels of cholesterol oxidation in LDL in patients with hypertension both before and after nilvadipine treatment compared with healthy subjects. However, there was a significantly (P < 0.001) decreased level of cholesterol oxidation in LDL in patients after nilvadipine treatment compared with patients before nilvadipine treatment. Conclusion: Our data showed that nilvadipine may protect LDL cholesterol from in vivo oxidation in hypertensive patients with high risk of atherosclerosis.

Lipid abnormalities of erythrocyte membranes in hemodialysis patients with chronic renal failure.

Lipids in erythrocyte membranes from 16 hemodialysis patients and 16 healthy volunteers were studied using gas chromatographic mass spectrometry. 7-keto cholestadiene was first reported in this study. The ratios of 7-keto cholestadiene to cholesterol, the ratios of arachidonate to cholesterol and the ratios of dochosahexanate to cholesterol in peak heights of chromatograms were measured in both groups as the markers of lipid peroxidation. Higher 7-keto cholestadiene/cholesterol ratios and lower arachidonate/cholesterol and dochosahexanate/cholesterol ratios were significantly observed in hemodialysis patients compared with healthy subjects. Our results are evidence that hemodialysis patients are exposed to much oxidative stress. It has been suggested that, during hemodialysis, leukocytes are activated by contract with non-physiological surfaces of the blood line tubing and produce oxygen free radicals. Oxygen free radicals attach cholesterol, arachidonate and dochosahexanate to produce lipid peroxides. In this study, this cell activation may be responsible for the increased lipid peroxidation of hemodialysis patients, 7-Keto cholestadiene, arachidonate and dochosahexanate can be used as markers of lipid peroxidation in hemodialysis patients.

Dicarboxylic acids as markers of fatty acid peroxidation in diabetes.

Increased urinary excretion of dicarboxylic acids (DAs) has been well known in patients with diabetic ketoacidosis (DKA). It was known that small amounts of such DAs were also detected in urine from healthy humans. Upon chemical, radiation-induced or enzymatic oxidation, cis-polyunsaturated fatty acids (PUFA) have previously been shown to generate saturated short- and medium-chain length DAs. In diabetes, it was confirmed that the imbalance between the generation of free radicals and antioxidant defense systems increases oxidative stress and leads to the damage of lipid, which contains PUFA. Some peroxidation products of PUFA, such as malondialdehyde and conjugated diene, are generally known to be elevated in patients with diabetes. The present study was undertaken to determine if urinary excretion of DAs is elevated in diabetic patients without DKA. Urine samples from ten non-ketoacidotic patients with type 2 diabetes and ten healthy subjects were examined for DAs by combined gas chromatography and mass spectrometry with selected ion monitoring. The diabetic subjects had significantly (Psebacic acid. Being stable and easily detectable compounds, DAs may be considered potential markers of oxidative attack on PUFA in diabetes.

A simple method to diagnose adrenoleukodystrophy using a dried blood spot on filter paper

A new, simple method for the diagnosis of adrenoleukodystrophy (ALD) using a dried blood spot sample, is described. Fatty acid from the dried blood spot was extracted and methylated simultaneously with HCl-methanol. Fatty acid methyl esters were analyzed by gas chromatography-mass spectrometry. Fatty acid compositions of the blood spot from four patients with ALD and five healthy controls were determined from the mass chromatograms of the m/z 143 ion, [(CH2)6 COOCH3]+. The ratios of tetracosanoic acid to docosanoic acid (C24:0/C22:0) and hexacosanoic acid to docosanoic acid (C26:0/C22:0) were significantly greater in ALD patients than in the controls. The fatty acid composition of the dried blood spot did not change at room temperature within a week. Since the specimens can be sent by mail, this method could be applied to the screening of ALD.