In the measurement of glycosylated protein (G-P), the presence of free glucose in the plasma or serum results in falsely high values. To avoid such interference of free glucose, sample dialysis before assay has been used. The dialysis, however, requires many hours and the possibility of partial release of protein-bound glucose during the dialysis has been suggested.
In the present study, plasma G-P was measured by an improved method, treating the plasma with trichloroacetic acid (TCA) before assay.
The G-P values of normal plasma samples, with various amounts of previously added glucose, showed a linear increase in absorbance. However, pretreatment of these plasmas with TCA completely abolished the interference of glucose. The lowest G-P values were obtained in dialyzed samples, while nontreated samples showed the highest values. The difference among the nontreated, TCA treated and dialyzed samples was found to be highly significant (p<0.001). Further studies on the effects of TCA on the hydrolysis were carried out. Dialyzed plasmas were divided into three groups according to their pretreatment before assay (i.e. no treatment, TCA treatment or addition of a small dose of TCA after TCA treatment). The levels of G-P were proportional to the dose of TCA. This result suggests that the TCA enhanced the hydrolysis since TCA did not affect the thiobarbituric acid (TBA) reaction.
The G-P values of normal subjects and diabetic patients were 1.32±0.13 and 2.0±0.66n mol HMF/mg of protein (mean±SD), respectively. When the diabetic group was classified into four groups according to the mean levels of plasma glucose obtained during the previous 1 week, three groups, i. e. except for that with a mean glucose level of below 100mg/dl, revealed significantly high G-P values as compared to those of normal subjects. The difference among the three groups with a mean glucose level of below 150mg/dl, however, was not significant. The correlation coefficients between the G-P values and plasma glucose levels on the same day or HbAi values were 0.64 and 0.57, respectively.

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Diabetic retinopathy worsens during pregnancy. To elucidate the worsening factor, plasma β-thromboglobulin (β-TG) concentrations were determined in 19 pregnant diabetic women, 76 pregnant nondiabetic women, 57 nonpregnant diabetic women, and 35 nonpregnant nondiabetic women. The results were analyzed in relation to the stage of retinopathy.
The plasma jS-TG concentrations in the pregnant nondiabetic women and nonpregnant nondiabetic women were 33.4ng/ml (95% confidence interval 24.7-45.2ng/ml) and 24.4 ng/ml (20.7-28.7ng/ml), respectively. The plasma β-TG concentrations in the pregnant nondiabetic women were significantly higher than those in the nonpregnant nondiabetic women. The plasma β-TG concentrations in the pregnant diabetic women and nonpregnant diabetic women were 54.9 ng/ml (44.7-67.4 ng/ml) and 71.2 ng/ml (57.8-87.9 ng/ml), respectively. The plasma β-TG concentrations in both these types of women were significantly higher than those in the nondiabetic women and nonpregnant nondiabetic women. There was no significant difference between the plasma β-TG concentrations in the pregnant diabetic women and nonpregnant diabetic women, but there was a significant difference between the plasma β-TG concentrations in the pregnant diabetic women and nonpregnant diabetic women who had almost the same stage of diabetic retinopathy.
Throughout all stages of pregnancy and after delivery, there was no significant difference in the plasma β-TG concentrations in the nondiabetic women and pregnant diabetic women. The plasma β-TG concentrations in the nonpregnant diabetic women tended to be the highest in the group with proliferative retinopathy and to be higher in the group with simple retinopathy compared to the group without retinopathy. The plasma β-TG concentratios in the nonpregnant diabetic women with proliferative retinopathy were significantly higher than those in the pregnant nondiabetic women without retinopathy. During pregnancy, there was no significant difference between the plasma β-TG concentrations in the pregnant diabetic women with progressive retinopathy and those in the pregnant diabetic women without progressive retinopathy.

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Hyperinsulinemia is currently a focus of attention because it is a probable risk factor in coronary atherosclerosis. In this study, the relation between coronary arteriographic findings and the IRI response in OGTT was investigated.
1) Forty-six non-fatty patients with significant changes on coronary arteriograms, including 36 with myocardial infarction and 10 with angina pectoris, were studied in comparison with 9 healthy controls.
2) IRI secretion was estimated from ΣIRI, ΣBS, ΣIRI/BS, and ΣIRI/ΣBS.ΣIRI was high in patients with angina pectoris or myocardial infarction involving three vessels. EBS was high in all. As a result, ΣIRI/ΣBS in the patients was equal to or slightly lower than the value in controls.ΣIRI/ΣBS was significantly low in all of the patients.ΣIRI/ΣBS and ΣIRI/ΣBS were low in most of the patients, especially in those with multiple vessel involvement. In general, OGTT type D showed a low ΣIRI/ΣBS and often accompanied more coronary risk factors.
3) The IRI response curve in the patients showed a later peak and high values, and these features were remarkable in patients with the B or D type of multiple vessel involvement. However, there was no significant difference between myocardial infarction and angina pectoris in the response curves. The major differences between the patients and healthy controls were seen in the data at 30 and 120 min, when low (at 30 min) or high (at 120 min) values were recorded.

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An attempt was made to investigate the effect of age on the plasma BTG levels in diabetics and also the relationship between plasma BTG levels and diabetic retinopathy. For this purpose, the plasma BTG levels were determined using a radio-immunoassay kit (Radio-Chemical Center, Amersham) in 84 normal subjects, 140 diabetics with retinopathy and 139 diabetics without retinopathy. The above patients were arbitrarily divided into four age groups, as follows: group A, younger than 30; group B, 31-50; group C, 51-70; and group D, older than 71.
The results obtained may be summarized as follows. (1) The mean plasma BTG level in normal subjects increased significantly with increasing age. (2) The mean plasma BTG level in diabetics without retinopathy was always higher than that in normal subjects of the same age group, and in particular in groups A and B the difference was highly significant. (3) The mean plasma BTG level in diabetics with retinopathy decreased with increasing age, being higher than that in diabetics without retinopathy in groups A and B. However in groups C and D there was no significant difference in mean plasma BTG level between diabetics with retinopthy and diabetics without retinopathy or normal subjects. (4) No relationship was observed between plasma BTG level and the severity of retinopathy or type of therapy or level of fasting blood sugar averaged over the previous 3 months, although the mean plasma BTG level was higher in patients with a duration of diabetes of less than 5 years compared to that in patients with a duration of longer than 5 years. A significant increase in plasma BTG level in younger diabetics was thus clearly observed, and this may be involved in the genesis of diabetic retinopathy.

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The organic acids in the urine and serum of diabetic patients with ketoacidosis were analyzed by gas chromatography-mass spectrometry. The sample preparation consisted of acidification, extraction, evaporation and methoxime-trimethylsilylation.
3-Hydroxyvaleric acid, 5-hydroxyhexanoic acid, and 2-hydroxy-2-methyllevulinic acid were identified in the urine of diabetic ketotic patients for the first time. These acids became undetectable in their urine after insulin therapy. The acids were not detected in the urine and serum of healthy subjects or of diabetic non-ketotic patients.
The occurrence of 3-hydroxyvaleric acid appears to be due to condensation of propionyl-Co A and acetyl-Co A. The formation of 5-hydroxyhexanoic acid may be due to w-1 oxidation of long chain fatty acids followed by p-oxidation of the resultant co-1 hydroxy fatty acids. 2-Hydroxy-2-methyllevulinic acid appears to be formed by condensation of pyruvic acid and acetone.
The diabetic patients with ketoacidosis showed increased urinary excretion of lactic acid, 2-hydroxybutyric acid, 3-hydroxyisovaleric acid, adipic acid, and 2, 3-dideoxypentonic acid.

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Ventilatory responses to CO₂ were investigated in order to assess the chemosensitivity and to clarify the mechanisms of respiratory arrest in diabetics with autonomic neuropathy using a hyperoxic rebreathing method.
The examinees comprised 5 insulin-dependent diabetics and 10 healthy individuals in their 20's and 30's. They lay comfortably on a bed and breathed from a closed spirometer containing pure O₂. The end tidal CO₂ (PA, co₂) and tidal volumes were monitored. They continued to breath until PA, co₂ reached 60 torr. Respiratory functions (vital capacity, forced expiratory volume, and maximum voluntary ventilation) were examined in advance. ECGs were obtained while the subjects were in the resting supine position, and the degree of autonomic neuropathy was expressed as the coefficient of variation of the R-R intervals (CV_R-R).
The ventilation increased as PA, CO₂ rose, and the slope of the linear regression line between the minute ventilation and PA, co2 was calculated. During hyperoxic rebreathing, the slope for the diabetic group (0.73±0.47l/min/torr) was significantly reduced compared to that of the control group (1.79±0.57l/min/torr)(p<0.01). The minute ventilation at a PA, co₂ level of 50 torr was 9.14±4.02l/min in the diabetic group, which was also significantly reduced compared to that of the control group (17.65 8.97l/min). (p<0.10). The CV_R-R values of the diabetic subjects were less than 2%, indicating advanced autonomic neuropathy.
These results suggest that not only the peripheral but also the central chemosensitivities are impaired in advanced autonomic neuropathy. This could represent one of the mechanisms of respiratory arrest, since it has been found that elimination of the central chemosensitive area on the ventral surface of the medulla oblongata results in disappearance of the respiratory rhythm in anesthetized cats.

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The etiology of vascular impairments, especially arteriosclerotic vascular impairment, in diabetics was studied based on clinical observations. The subjects comprised 526 out-patients with diabetes mellitus attending the Third Department of Internal Medicine, Nagoya University Hospital, for whom the progress of the disease could be followed up for more than 5 years. The subjects were divided into obese and non-obese groups, and the frequency of vascular impairments was compared by further classifying them according to therapeutic agents and the degree of control of diabetes. The results obtained were as follows.
(1) ECG abnormality and retinopathy were noted at a high frequency in the obese group. However, the frequency of ECG abnormality was related to age and that of retinopathy to the duration of the disease. A comparison was then made of the same conditions for a duration of the disease of 11 to 15 years, at ages ranging from 41 to 60. No difference in the progress of retinopathy was observed between the obese and non-obese groups, while ECG abnormality was noted at a high frequency in the obese group. A similar comparison was made among those showing normal cholesterol levels and normal blood pressure, and a similar tendency was observed.
(2) When comparisons were made according to therapeutic agents, the results revealed that ECG abnormality was encountered at a high frequency in the obese group undergoing therapy with insulin or oral hypoglycemic agents, whereas in the non-obese group no difference existed from those undergoing diet therapy. In both groups, retinopathy occurred at a higher frequency among those being treated with insulin or oral hypoglycemic agents than those on diet therapy.
(3) Comparisons according to the degree of control of diabetes showed that ECG abnormality was observed at a high frequency in the obese group regardless of good or poor control On the other hand, retinopathy was observed in patients with poor control among both the obese and non-obese groups.
Based on these results, it might be suggested that the etiology of arteriosclerotic vascular impairment in diabetics is not attributable to hyperglycemia due to insufficient insulin action, but may be associated largely with a specific state of being obese, or with obesity coupled with a state assisting endogenous and exogenous hyperinsulinemia.

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The relationship between gall-bladder function and the clinical features of diabetes mellitus, including autonomic neuropathy, was studied. Gall-bladder kinetics as determined by real-time ultrasonography, scanned at 10-min intervals for 60 min after administration of two yolks, were investigated in 56 diabetics and 42 normal controls.
There was no significant difference between the two groups as regards the initial area of the gall-bladder. The contractility of the gall-bladder in diabetics was lower on average at each interval compared to controls. It was classified by its kinetics into 3 types of patterns, which showed normal contraction (Type I: N=30), mild impairment of contraction (Type II: N=13), and severe impairment of contraction and/or transitory enlargement in size (Type III: N=13). Type III was most frequently found in diabetics with some neurological abnormalities including signs of autonomic neuropathy such as postural hypotension, impotence and abnormal sweating. Furthermore most diabetics with Type III showed pancretic exocrine dysfunction and in some of these cases recurrence of diarrhea and constipation was observed.
These results suggest that some inter-relationship exists between gall-bladder function and pancreatic exocrine function in diabetics with autonomic neuropathy.

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Insulin binding was studied in erythrocytes and liver plasma membranes from rats with acute hyperinsulinemia caused by intraperitoneal injection of insulin. The specific ¹²⁵I-insulin binding to liver plasma membranes fell to 59.6% of the control as early as 10 min after the insulin injection, and the binding remained decreased for 60 min. Based on Scatchard analysis, the decreased insulin binding to the liver plasma membranes observed after the insulin injection was attributed primarily to a decrease in the number of receptors. Results for insulin extraction and ¹²⁵I-insulin dissociation from these plasma membranes indicated that the decreased insulin binding was not due to simple occupancy of insulin receptors by exogenous insulin. In contrast, the ¹²⁵I-insulin binding to erythrocytes remained unchanged after insulin injection. In control rats, a good correlation was observed between the ¹²⁵I-insulin binding to erythrocytes and to liver plasma membranes [r=0.6465, p<0.01].
The following conclusions were drawn.[1] In the acute hyperinsulinemic state caused by high dose insulin injection, a decrease in the receptor number can rapidly occur in liver plasma membranes whereas the erythrocyte insulin receptors are unaffected.[2] The insulin receptors of erythrocytes reflect those of liver plasma membranes in a state where there is no rapid change in the insulin concentrations.[3] Under certain conditions, the erythrocyte insulin receptors do not reflect changes in the insulin receptors of other insulin target cell membranes such as liver plasma membranes.

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Since menstrual disorder represents a very serious problem in mature women, the incidence of abnormal menorrhea was studied in 35 female patients with diabetes mellitus, aged less tan 34 years, and the clinical findings were also investigated. Moreover, in 11 patients who were confirmed to be anovulatory, estrogen-progesterone tests as well as LH-RH tests, were performed. Their prognosis was followed up subsequently.
(1) Menstrual disorder was found in 8 (8%) out of 100 healthy women, but in 22 (62.9%) out of 35 diabetic women.
(2) There were no significant differences in the onset of diabetes mellitus in age or menarche between abnormal menorrhea and normomenorrhea in diabetic patients. The former showed a fasting blood sugar level of 239.1±80.9mg/dl, which was significantly higher than that in the latter (164.3±44.5mg/dl).
(3) By means of the estrogen-progesterone test, 6 cases of hypo-estrogenic amenorrhea, who had associated severe emaciation, and 5 cases of normo-estrogenic amenorrhea were found.
(4) By the LH-RH test, all of 11 cases were presumed to be suffering from hypothalamic disturbance. The peak blood level of LH was reduced in diabetic patients with severe emaciation.
(5) Spontaneous ovulation was recognized in 3 patients with diabetic amenorrhea. Patients in whom the period of ovulation disturbance was relatively short, showed spontaeous ovulation. Patients, in whom ovulation disturbance had not been treated for a long time, have not been found to show spontaneous ovulation so far. Thus, diabetic patients who wish pregnancy should be kept under treatment for not only diabetes mellitus but also ovulation disturbance as quickly as possible.

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