In order to evaluate the oxygen release from erythrocyte hemoglobin in diabetics with nephropathy, especially in hemodialyzed cases, P₅₀ and other parameters of oxygen release were investigated in 43 diabetics without nephropathy, 46 diabetics with nephropthy and 20 cases of hemodialysis caused by diabetic nephropathy. Hcparinized blood samples were drawn from the cubital vein before breakfast in nonhemodialyzed cases and from the arterial site of the hemodialyzer before and after hemodialysis in 20 hemodialyzed cases. The results obtained were as follows.
1) In 46 diabetics with nephropathy, P₅₀ in vivo pH (26.0±2.3mmHg) was significantly higher than in diabetics without nephropathy (p<0.01).
2) In 89 diabetics with or without nephropathy, except for hemodialyzed cases, there were significant inverse correlations between P₅₀ in vivo pH and blood pH, and blood hemoglobin concentration (Hb)(p<0.001, p<0.005). 2, 3-diphosphoglycerate (2, 3-DPG) had a similar inverse correlation with Hb (p<0.001), and P₅₀ pH 7.4 had a positive correlation with 2, 3-DPG (p<0.001).
3) In 20 cases of diabetic nephropathy where hemodialysis was carried out, P₅₀ in vivo pH and plasma inorganic phosphate (which were at high levels) and 2, 3-DPG were almost all the in the normal range before dialysis and they all decreased significantly after dialysis (p<0.001, p<0.05, p<0.001). However, blood pH and Hb increased significantly (p<0.001, p<0.005). In the course of hemodialysis, P₅₀ in vivo pH also correlated inversely with blood pH (p<0.001) and positively with Pi (p<0.05). 2, 3-DPG had a significant inverse correlation with Hb (p<0.001).
4) The oxygen release from erythrocyte hemoglobin decreased relatively (0.5-37.3%) after hemodialysis in 18 out of the 20 hemodialyzed patients.
Accordingly, the elevation of P₅₀ in vivo pH in 46 diabetics with nephropathy depended not only on the Bohr effect caused by renal acidosis, but also on the compensatory increase of 2, 3-DPG associated with renal anemia. It was also suggested that the decrease of P₅₀ in vivo pH following hemodialysis was caused mainly by the rapid improvement in renal acidosis, partly by the normalization of hyperphosphatemia, and partly by a decrease of 2, 3-DPG accompanied by water exclusion.

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In our previous retrospective studies on diabetic patients, we found that hyerinsulinism plays an important role in the development of diabetic macroangiopathy.
In the present experiment, we examined the metabolic effects of insulin upon the arterial wall of experimental animals.
Forty young adult male Wistar rats weighing 200 g were divided into two groups. One group was subjected to daily subcutaneous injection of insulin zinc suspension (group I, N=25), whereas the other was treated with saline (group S, N=15). After one year, all animals were sacrificed and the lipid contents in the intima-media of their arota were biochemically measured. For morphological studies, further parts of the ascending aorta were dissected out and examined by light and electron microscopy.
1) The biochemical data obtained showed significant increases in triglyceride (TG) content in the aorta tissues of group I as compared with those in the tissues of control group S (p<0.05).
The TG/phospholipid (p<0.05) and TG/total cholesterol (p<0.01) ratios were also significantly increased in the experimental group.
2) The light microscopic examination revealed that the intima of the aorta in group I rats was apparently thickened and the subendothelial tissue consisted of eosinophilic fiber bundles, amorphous ground substances and irregularly arranged cells containig a long oval nucleus. Electron micoscopically, the majority of fibers in the intima of the aorta of group I rats were found to show collagenous and elastic fiber-like structures, and the cells contained microfilaments, dense bodies and sub-plasmamembranous vesicles in the cytoplasm. From these electron microscopic features, the cells were identified as being of smooth muscle cell origin.
In conclusion, arteriosclerosis-like lesions can be induced by long-term insulin injection in the aorta of rats. The present results suggest that hyperinsulinism plays an important role in both the occurrence and development of diabetic macroangiopathy.

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Urinary proteins of 55 patients with diabetes mellitus consisting of 41 with no proteinuria (group A), 5 with intermittent proteinuria (group B) and 9 with persistent proteinuria (group C) were analyzed by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic pattern of urinary proteins was compared with renal histology in biopsy specimens and the urinary albumin excretion rate (AER) at rest. In addition, urinary protein were traced for three years at the longest by “Albustix.”
The following results were obtained:
1) In short-duration diabetics in group A, T (tubular) pattern was predominant with an accompanying diffuse glomerular lesion of grade 1-2. AER remained normal in these cases when the blood glucose was well controlled.
2) In long-duration diabetics in group A, M (mixed) and G (glomerular) patterns were frequently demonstrated with a concomittant diffuse glomerular lesion of grade 2-3 or grade 3. AER was elevated in most of the cases with M and G patterns even when the blood glucose was well controlled. More than 50% of the cases with these patterns were complicated by proliferative retinopathy as well as simple retinopathy.
3) Intermittent proteinuria was demonstrated within two years in three of the five cases with M pattern in group A, and persistent proteinuria was noted within a year in one of three cases with G pattern in group A.
4) Progression of diabetic renal disease seemed to be delayed when HbA5 was controlled below 8.0%.
From these results, it was concluded that 1) T pattern is an early sign of diabetic glomerulosclerosis, and M and G patterns are predictive signs of persistent proteinuria, and 2) analysis of urinary prteins by SDS-PAGE is very useful as one of the indices for the evaluation of diabetic renal disease.

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A method for quantitative analysis of urinary proteins by SDS-polyacrylamide disc gel electrophoresis, using a certain amount of cytochrome-c as an internal standard, was established to reveal microproteinuria, and the characteristic features of urinary proteins in diabetic nephropathy.
Correlation between the urinary albumin content measured by radioimmunoassay (y) and by quantitative polyacrylamide disc gel electrophoresis (x) was shown to be represented by the equation: y=0.040+0.664x (r=0.959. p<0.01).
Microproteinuria, containing more than 176.8μg/ml of protein, was detected in 53.6% of diabetics whose urine was negative for protein by albustix.
Duration of diabetes was longer and incidence of hypertension was higher in diabetics with microproteinuria than in diabetics with normoproteinuria.
The ratio of albumin to total protein was larger and the ratio of low-molecular-weight protein was smaller in diabetics with microproteinuria than in diabetics with normoproteinuria. The ratio of high-molecular-weight protein to total protein tended to be increased in diabetics with microproteinuria and more so in diabetics with macroproteinuria compared with diabetics with normoproteinuria.

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The amounts of insulin and C-peptide immunoreactivity in the tail of the pancreas of 35 diabetic and 21 nondiabetic autopsied subjects were determined. The 28 diabetic patients among them who were able to be followed for more than 6 months in the hospital were analyzed from the viewpoint of the relationship between the amount of insulin or C-peptide immunoreactivity in the tail of pancreas and the stability of the fasting serum glucose levels during diabetic life before death.
Furthermore the relationship between the C-peptide immunoreactivity response in the breakfast meal tolerance test before death and insulin content of the pancreas was investigated. The standard deviation of the mean of 15 determinations of the fasting serum glucose level was used an index of instability of the blood sugar level. The results are as follows:
1. Not only insulin but C-peptide immunoreactivity contents of the tail of the pancreas was significantly decreased in diabetics compared with those of nondiabetics. The IRI content of the pancreas of diabetics and nondiabetics was 1.371±0.197U/g and 2.737±0.342U/g (p<0.01) respectively, and the CPR content was 14.07±2.45μg/g and 22.0±6.24μg/g, respectively.
2. Standard deviation of the mean of the fasting serum glucose level and insulin or C-peptide immunoreactivity content of the tail of the pancreas were in significant inverse correlation on a logarithmic scale (p<0.01, r=-0.704; p<0.01, r=-0.757, respectively). The relationships are expressed as y (mg/dl) =151x^-0.203 (mU/g) and y=728x^-0.325 (ng/g), respectively.
3. The serum X C-peptide immunoreactivity value during the breakfast meal tolerance test was significantly correlated with insulin content in the pancreas of diabetic subjects (p<0.01, r=0.953).
4. Severe renal dysfunction was considered to play some role in the stability of the blood sugar level, but not a significant one.
These findings suggest that the instability of the fasting serum glucose level is mainly due to the destruction of pancreatic B cells, and that the pancreas insulin content is logarithmically and inversely related to the fluctuation of fasting serum glucose level.

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We experienced three diabetic patients, whose ST-T abnormalities disappeared in a few months with blood-sugar control.
The first patient was a 73-year-old female with a 2-year history of diabetes. She was admitted on July 16, 1981, for poor control of blood glucose. The blood glucose and HbA₁ levels were 195 mg/dl and 10.3%, respectively, on admission. She had been receving glibenclamide drugs. ECG showed ST segment depression in I, II, aV_L, V_3-6 and T wave inversion in V_1-3 on admission, but these abnor malities disappeared with diabetic therapy and the ST-T segment returned to normal by August 21, 1981.
The second patient was a 44-year-old male with a 1-year history of diabetes. He was admitted on April 19, 1981. The blood glucose and HbA₁ levels were 214mg/dl and 10.9%, respectively on admission. He had been receiving insulin therapy. ECG showed ST segment depression in II, III, aV_F, V₆ on admission, but with diabetic therapy the abnormalities disappeared by June 20, 1981.
The third patient was a 56-year-old female with thirst starting 10 days earlier. She was admitted on May 5, 1981. The blood glucose and HbA₁ levels were 302mg/dl and 12.2% respectively, on admission. She had been receiving insulin therapy. ECG showed ST segment depression in I, II, III, aV_L, V_4-6 and T wave inversion in II, III, aV_F, V_1-6 on admission, but with diabetic therapy the abnormalities disappeared by May 16, 1981.
This report suggests that ECG findings in diabetics are influenced by the control of blood sugar and it is necessary to evaluate ST-T changes in diabetic patients cautiously.

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Islet-cell antibodies (ICA) were measured in 198 patients with insulin-dependent diabetes mellitus (IDDM) and 177 non-diabetic controls using a standard indirect immunofluorescence method (IF method) and a newly established three-layer immunofluorescence method applying the biotinavidin system (BAS method) as described below.
All sera were diluted 1: 2 in 10mM phosphate-buffered saline (PBS), pH7.2, applied to unfixed human pancreatic sections (5μm), and incubated at room temperature for 30min in a moist chamber. The slides were washed over 5-min intervals in PBS 3 times. Biotinated goat anti-human IgG (H+L) serum (Vector Labs., Burlingame, California) diluted 1: 150 in PBS, was then applied to the tissue sections which were then incubated at room temperature for 30min. The sections were then washed for 5-min intervals 3 times in PBS. Fluorescein-isothiocyanate (FITC)-conjugated avidin D (Vector Labs., Burlingame, California) diluted 1: 150 in PBS was then applied to the sections which were incubated at room temperature for 30 min. The sections were then washed for 5min 3 times in PBS.
ICA titers detected by the BAS method correlated well with those determined by the standard IF method (r_s=0.987, p<0.01). The BAS method had a sensitivity for ICA about eight times higher than that of the IF method.
The overall prevalence of ICA detected by the BAS method (ICA-BAS) vs that by the IF method (ICA-IF) was 41%(82/198) vs 28%(56/198) in patients with IDDM. All 177 non-diabetic controls except one showed negative for ICA-BAS. ICA-IF was detected in none of these non-diabetic controls.
In IDDM patients, ICA-BAS was positive in all cases less than 1 month after the onset of diabetes, while the prevalence of ICA-IF was 83%(20/24) during the same period. The prevalence of ICA-IF decreased rapidly with the duration of disease, reaching a value of 6%(3/55) in patients with a disease duration of 10 years or more. The incidence of ICA-BAS also decreased, although to a lesser extent than ICA-IF, with the duration of disease.
These results suggest that in almost all Japanese IDDM patients pancreatic autoimmune processes occur immediately after the onset of disease. The higher sensitivity of the BAS method compared with the standard IF method was of significant diagnostic value, especially in diabetic patients with a long duration of disease.

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DG-5128, a new α₂-adrenergic blocking agent, was successfully used for the diagnosis of insulinoma in a 61-year-old female who showed normal responses of insulin to intravenous glucagon and of C-peptide to insulin-induced hypoglycemia. As compared to normal controls, oral administration of 150mg DG-5128 induced marked and prolonged increase of serum insulin level, resulting in concomitant hypoglycemia. These abnormalities were corrected to normal postoperatively. These results suggest the usefulness of DG-5128 as an agent for provocation tests of insulinoma.

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