Hidetaka Nakayama

A Simple Method for the Determination of Serum Free Insulin Levels in Insulin-treated Patients

A method for the determination of free, active insulin in the sera of insulin-treated diabetics is described. This involved radioimmunoassay after extraction of free insulin with polyethylene glycol. Recovery tests with cold insulin showed 73 per cent recovery of free insulin and no recovery of bound insulin. The fasting free insulin levels were slightly lower in the patients than in normal persons; exceptions were some patients with complications expected to cause insulin resistance at the peripheral tissue level. Free insulin levels did not correlate with total insulin levels, antibody titers, insulin requirements or conditions of insulin treatment. A very slight increase of insulin was observed after glucose loading in insulin-treated patients, but a marked increase of the free insulin level followed by an exaggerated increase in the total insulin level was observed in a patient with the insulin autoimmune syndrome. The diurnal changes of the free insulin suggested the dynamic states of this fraction and its usefulness for determining control of diabetes with insulin.

Immunochemical Detection of Advanced Glycation End Products in Lens Crystallins From Streptozocin-Induced Diabetic Rat

To reassess the significance of AGEs in cataract formation in diabetic animals, we measured amounts of AGEs in lens crystallins from STZ-induced diabetic animals with a newly developed ELISA. Lenses were removed at 5 and 20 wk after STZ injection. In 20-wk diabetic rats, all lenses were cataractous but not in control rats. In 20-wk diabetic compared with control rats, significant increases were observed in AGEs (172.3 ± 18.3 vs. 14.3 ± 1.7 All, P < 0.01) and fluorescence (2.04 ± 0.22 vs. 1.27 ± 0.10 AU, P < 0.05). The amounts of AGEs in lens crystallins, measured by the ELISA, were > 12-fold higher in diabetic rats. In agreement with earlier studies, we found that fluorescence in lens crystallins increased by 61% in diabetic rats. In 5-wk diabetic rats, all lenses were noncataractous. In 5-wk diabetic compared with control rats, significant increases were observed in AGEs (84.1 ± 7.7 vs. 9.4 ± 1.5 AU, P < 0.01) and fluorescence (1.45 ± 0.06 vs. 1.05 ± 0.06 AU, P < 0.01). Analysis of the AGE content by ELISA showed that accumulation of AGEs in diabetic lens crystallins does markedly occur with time, and a large amount of AGEs exists in the diabetic (cataractous) lens crystallins. The disproportionate elevation of AGEs, measured by the ELISA, compared with fluorescence suggests that the actual levels of AGEs in cataractous lens crystallins from diabetic animals are higher than previously anticipated, and nonfluorescent AGEs may exist in diabetic lens crystallins. With this premise, our data suggest that a newly developed ELISA for the detection of AGEs in tissue proteins may be a powerful tool for investigating the role of the advanced Maillard reaction in complications of diabetes and aging.

Immunochemical Detection of Advanced Glycation End Products in Renal Cortex From STZ-Induced Diabetic Rat

To reassess the accumulation of advanced glycation end products in diabetic renal cortex, we used a newly developed enzyme-linked immunosorbent assay to measure AGEs in renal cortex from STZ-induced diabetic and age-matched control rats. Kidneys and aortas were obtained from rats after 5 and 20 wk of STZ injection. At 5 wk of diabetes, the mean AGE content in collagenase-digested materials of renal cortex was > 16-fold higher in diabetic animals compared with controls (1044.4 ± 151.8 vs. 64.3 ± 5.7 arbitrary units, P < 0.01). At 20 wk of diabetes, it was > 45-fold higher in diabetic compared with control animals (3841.0 ± 1077.3 vs. 83.8 ± 12.8 AUs, P < 0.01). These increases were surprisingly large compared with the < 1.5-fold increase in the fluorescence levels both after 5 and 20 wk of diabetes. In control animals, neither the AGE content nor the fluorescence level increased during this period. Moreover, at 20 wk of diabetes, the AGE content was 39-fold higher in renal cortex compared with aorta. This study provided the first immunochemical evidence that collagenase-digested materials of renal cortex, as well as aorta, contained AGE products and that these products were present in much higher levels in diabetic animals than in control animals. With duration of diabetes, the AGE contents increased significantly both in renal cortex and aorta. The excessive accumulation of AGEs was most apparent in the diabetic kidney. These findings suggest that the actual level of AGEs, in particular, in diabetic renal cortex is much higher than previously anticipated, and a newly developed enzyme-linked immunosorbent assay may be a powerful tool for investigating the role of the advanced Maillard reaction in the development of diabetic nephropathy.

Production and characterization of antibodies to advanced glycation products on proteins.

Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.

Immunochemical approaches to AGE-structures: characterization of anti-AGE antibodies.

Recent immunological approaches have greatly helped broaden our understanding of the biomedical significance of advanced glycation end products (AGEs) in aging and age-enhanced disease processes. Recently, Nepsilon-(carboxymethyl) lysine (CML), one of the glycoxidation products of AGEs, was demonstrated to be a major immunological epitope among AGEs. In the subsequent study, we characterized 13 different polyclonal anti-AGE antibodies and showed that these antibodies could be classified into three groups (Groups I, II and III). Group I was specific for CML and both Group II and Group III were specific for other epitopes (non-CML). Time-course study suggested that the epitope of Group II was formed earlier than that of Group III. In the present study, we prepared two monoclonal anti-AGE antibodies (2A2 and 3A3) whose epitope structures appeared to be closely related to Group III and Group II, respectively. The result indicates that AGE-proteins express at least two major non-CML epitopes.

Influence of Streptozotocin Diabetes on Intestinal 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in the Rat

Studies were undertaken to examine cholesterogenesis in the intestine of streptozotocin-diabetic rats by measuring incorporation of [214C] acetate into cholesterol and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) activity. In these diabetic rats, the intestinal mucosal weight and food consumption were markedly high. The incorporation of [214 C] acetate into cholesterol was significantly increased in all diabetic intestinal segments. However, the rates of production of fatty acids and carbon dioxide were not affected. Hepatic H MG-CoA reductase activities were markedly reduced during both the diurnal high and low periods in these diabetic rats, and there was no diurnal variation. In contrast, the specific activities of this enzyme in jejunal crypt cells during both the diurnal high and low periods were significantly higher in these diabetic rats without loss of diurnal variation. Total reductase activity per segment of intestine in jejunal and ileal mucosa (villi + crypt cells) was increased in these diabetic rats. Control rats had higher total and specific activity of ileal mucosal (villi + crypt cells) reductase than of jejunal mucosal reductase during the diurnal high period. The jejunal-ileal gradient in reductase activity and the incorporation of [214 C] acetate into cholesterol did not change significantly with streptozotocin-diabetic rats. The results indicate that in streptozotocin-diabetic rats, hepatic cholesterogenesis decreases but intestinal synthesis increases.

Characterization of antibodies to advanced glycosylation end products on protein.

Antibodies directed against advanced glycosylation end products (AGEs) formed during a Maillard reaction have been generated and characterized. Since protein-bound AGEs recognized by the antibodies were labile to acid hydrolysis, the antibodies were further characterized by using the AGE-alpha-acetyl-L-lysine methyl ester (AGE-ALME) with a brown and fluorescent property as well as the AGE-proteins. The antibodies reacted with fluorescent compounds, rather than brown pigment compounds, in the AGE-ALME. The fluorescent compounds in the AGE-ALME were separated into four fluorescent compounds by reversed-phase thin layer chromatography (TLC). Of the fluorescent compounds tested, compound 3 (Rf = 0.63), as designated on a TLC plate, showed the highest affinity for the antibodies. In addition, the antibody recognition to the cross-linked oligomers with fluorescence in the AGE-protein was investigated by using bovine pancreatic ribonuclease A (RNase), which is known as a model protein for studying AGE-induced cross-linking. Fluorescence in the AGE-RNase existed in both of the oligomers and the monomer. The cross-linked oligomers exhibited higher affinity to the antibodies than did the monomer, which has a similar degree of fluorescent intensity. These results indicate that our antibodies against cross-linked protein-bound AGEs may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.

Quantitative enzyme-linked immunosorbent assay (ELISA) for non-enzymatically glycated serum protein

A competitive ELISA for quantitative determination of glucitollysine, the reduced hexose alcohol form of glucose conjugated to the epsilon amino group of lysine was developed. We applied it to measure non-enzymatically glycated serum proteins. The antiserum obtained by immunizing guinea pigs with reductively glycated human albumin was capable of identifying and quantitating glucitollysine residues of serum proteins in normal and diabetic subjects after reduction of the proteins with sodium borohydride. The ELISA assay developed here had satisfactory reproducibility as judged by the intra-assay precision of 2.3-7.6% and the interassay precision of 6.7-9.8%. Results from this assay procedure correlated well with those from the radioimmunoassay and the boronate affinity chromatography procedure. The data suggested that diabetic serum proteins contained at least three times as much immunochemically detectable glucitollysine residues as normal serum proteins after reduction of the proteins with sodium borohydride. This method allows to quantitate glucitollysine residues on any of the proteins that have been implicated in the pathological sequelae of diabetes.

Influence of Diet on Intestinal Cell DNA Synthesis in the Diabetic Rat

The incorporation of thymidine methyl-3H (3H-TdR) into deoxyribonucleic acid (DNA) was measured in the intestine of diabetic rats. Animals were allowed food ad libitum or were pair-fed. Diabetes was induced with streptozotocin. In the pair-feeding experiment, there was a decrease in 3H-TdR incorporation into DNA in the intestine of diabetic rats, and the mucosal weight did not differ significantly from controls. However, when diabetic rats were allowed food ad libitum, there was a significant increase in DNA synthesis in the intestine. The mucosal weight of the diabetic animal was 62 per cent greater in the jejunum and 31 per cent greater in the ileum than in the control segments. It is concluded that a decrease in proliferative cellular activity occurs in the intestinal mucosa of diabetic rats but hyperphagia leads to a compensatory increase in DNA synthesis in these animals.

Radioimmunoassay for the determination of glycated haemoglobin

SummaryA competitive radioimmunoassay for the quantitative determination of glycated haemoglobin was developed. The antiserum, obtained by immunizing guinea pigs with reduced glycated human albumin, was capable of identifying and quantitating the glucitollysine residues of glycated Hb after reduction with sodium borohydride. To simplify the sample preparation we introduced trichloroacetic acid precipitation to remove unreacted sodium borohydride instead of using dialysis or gel filtration. Using this procedure, our radioimmunoassay became relatively simple and provided satisfactory within- and between-run (1.3–2.8% and 1.9–5.4% coefficient of variation, respectively). The radioimmunoassay method was compared to the measurement of HbA1c by high performance liquid chromatography which is the most widely used method for quantitating glycated Hb. For this purpose glycated Hb was measured in normal glucose tolerance, impaired glucose tolerance, and diabetes mellitus groups based on WHO criteria. Both assays were able to discriminate between the normal and diabetic groups. In addition, while the determination of glycated Hb by the radioimmunoassay method was able to clearly discriminate between the normal and impaired glucose tolerance groups, the determination of HbA1c by the high performance liquid chromatography method failed to discriminate between these two groups. Moreover, 15 of the 20 impaired glucose tolerance patients exceeded the upper normal range (mean normal values + 2 SD) in radioimmunoassay. But all 20 patients with impaired glucose tolerance were within the upper normal range in HbA1c values.These results demonstrate that the measurement of glycated Hb by radioimmunoassay is more sensitive than the measurement of HbA1c by high performance liquid chromatography since it can discriminate between the normal and impaired glucose tolerance groups.