Urine C-peptide immunoreactivity (UCPR) was measured by a double antibody radioimmunoassay method in normal and diabetic subjects who had no gross renal impairment. The total UCPR excreted in 24 hr in healthy subjects during ordinary life was 74.7 ± 26.3μg (mean ± SD). Individual total UCPR values showed a significant correlation with body height, weight index and urinary creatinine (Cr) excretion. Thus, the amount of UCPR per gram of creatinine excreted during 24 hr appears to represent a convenient index for quantifying pancreatic B cell secretory function. The 24-hr urine CPR/Cr was 53.5 ± 13.8μg/g in 20 healthy subjects.
In ketotic diabetics, the mean UCPR and UCPR/Cr were markedly decreased but some variation existed in their residual B cell function. In cases of post-pancreatitic, unstable, insulin-requiring diabetics, UCPR was reduced to undetectable levels, suggesting that B cell function was almost completely destroyed in these patients. On the other hand, urine CPR excretion was variable in adult-type diabetics. It was significantly decreased in non-obese patients. UCPR/Cr was also significantly decreased in insulin-treated, non-ketotic patients. The majority of patients treated on a diet and with sulfonylureas, however, excreted normal or higher than normal amounts of CPR in the urine. There was no correlation between UCPR and the duration of diabetes or the severity of retinopathy.
Meal-dependent diurnal variations were observed in urine CPR excretion rate in healthy subjects. In general, urine CPR changed in parallel with the changes in plasma CPR and insulin. Urine CPR excretion rate was the lowest from bedtime to breakfast (normal value: 1.14μg/hr). It increased promptly after each meal. The ratio of the UCPR excretion rate during daytime to that from bedtime to breakfast was 3.33 in normal subjects, 1.49 in ketotic diabetics, 1.84 in nonketotic diabetics on insulin treatment, 1.86 in non-ketotic diabetics on sulfonylurea drugs, and 2.57 in diabetics undergoing diet treatment. Although the basal CPR excretion rate was higher than normal in some adult-type diabetics, its relative increase during the daytime was not as high as in normal subjects, suggesting a decrease in extra-secretion of insulin relative to its basal secretion.
The results of the present study indicate that urine CPR is a useful index of the endocrine function of the pancreas, and measurements of divided urine samples appear to provide further information on the daily activity of B cells.

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Circulating plasma insulin is usually determined by radioimmunoassay (RIA) and very recently, in some institutes, it has also been determined by radioreceptor assay. Isotopically labeled insulin is necessary for both methods, so that a specially controlled laboratory is required for routine insulin determinations. Recently, a technique has been developed for coupling enzyme to plasma proteins including antibody.
This paper describes a study on the determination of plasma and serum insulin by enzyme immunoassay (EIA) using anti-insulin antibody labeled with horseradish peroxidase. The values obtained were compared with those obtained by RIA.
The intra- and interassay variances of the EIA method were 4.2% and 7.2% for plasma containing insulin at 31μU/m/, and 3.2% and 4.1% for plasma with 85μU/m/, respectively. Dilution tests using plasma with a very low concentration of insulin revealed a straight line starting from the 0 point. There were no differences between the values obtained for plasma and serum samples. Recovery tests also gave satisfactory results: the mean recovery was 99.5%.
A 100 g oral glucose tolerance test was performed in 60 subjects. Plasma samples obtained at 0 time and at 30, 60, 90. 120 and 180 min after glucose load were determined for insulin concentration by both EIA and RIA. The regression line and correlation coefficient between EIA (y) and RIA (x) were calculated as y=0.918x+ 3.60, and r=0.955, respectively.
This EIA method is simple and reliable, and does not require any specially-controlled room such as an isotope laboratory. The method is thus expected to have wide use in clinical medicine in the near future.

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The purpose of this report is to clarify the properties of insulin binding antibody to endogenous and exogenous insulin and to discuss its clinical significance.
1) The affinity of insulin binding antibodies in commercial insulin-treated diabetics (69 cases) and insulin autoimmune syndrome (3 cases of hyperthyroidism with methimazole treatment and no insulin treatment) to non-iodinated human, porcine, bovine and iodinated porcine insulin was evaluated. As a result, the insulin binding antibodies could be divided into the following 3 types.
Type I: showing almost the same affinities to various non-iodinated insulins and iodinated porcine insulin (8 cases, and the 3 cases of the autoimmune syndrome).
Type II: showing different affinities to various insulins (29 cases).
Type III: showing a high affinity only to iodinated insulin (9 cases). The sera of the remaining 23 cases could not be assigned to any types due to the low titers of insulin binding antibodies.
2) Among the patients of types I, II, III and the unclassified subjects, the percentage of chronic inflammations or chronic diseases such as chronic hepatitis, chronic pancreatitis or nephritis combined with diabetes, was 25.0%, 48.8%, 88.9% and 34.7%, respectively.
3) In insulin tolerance tests, antibodies classified as type I or II exerted a blocking effect on the hypoglycemic action of injected insulin (Regular and Actrapid insulin) but antibodies of type III did not show such an effect.
4) The affinity of the insulin binding antibodies to endogenous human insulin during 50g OGTT in commercial insulin-treated diabetics (24 cases) and insulin autoimmune syndrome (3 cases) was evaluated. It was found that the insulin antibodies in 50% of the commercial insulin treated diabetics and insulin autoimmune syndrome had an affinity for the endogenous human insulin.

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Sex differences in the incidence of diabetic retinopathy were examined in a total of 2962 diabetics.
1) The frequency of diabetic retinopathy was significantly higher in females than males among all the diabetic patients, and also in diabetic patients under the age of 39 yrs on ocular fundus examination at the first visit.
2) The incidence of diabetic retinopathy in the group with initial fasting blood sugar levels of more than 140 mg/dl was significantly higher in females than males which increased with duration of diabetics.
3) In both male and female patients, the incidence of diabetic retinopathy of grade III in Scott classification or severer increased with the duration of diabetes. However, the correlation between the frequency of diabetic retinopathy grade III (Scott) or severer and the duration of the illness was more marked in the females than males In particular, in diabetic patients under the age of 39 yrs the frequency of diabetic retinopathy grade IV or severer was significantly higher in females than in males among cases with duration less than 4 yrs.
4) The difference in rates of progression of diabetic retinopathy between the sexes was not statistically significant over 1 and 5-yrs observation periods irrespective of the control of diabetes.
Although the difference between males and femails at the first visit appears to support the previous reports, no significant differences during the follow-up period 1 and 5 yrs. might suggest that the observations at the first visit may not necessarily be sex linked.

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To determine whether ovarian steroids have any causal relationship to the development of diabetic retinopathy, serum total estrogens and estradiol levels were measured by radioimmunoassay in diabetic patients.
The serum total estrogens and estradiol levels among diabetics with or without retinopathy were not significantly different from those of age-sex matched healthy individuals. No significant differences in serum total estrogens or estradiol levels were found among groups of diabetics classified according to the stages of retinopathy. Furthermore, no significant changes in serum estrogen levels were observed even in diabetics with progressive retinopathy.
The above observations suggest that there is no obvious correlation between serum estrogen levels and the development of diabetic retinopathy.

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A case of diabetes mellitus and coexistent hypothyroidism is reported.
The patient, a 65-yr-old male farmer, had been diagnosed as having diabetes mellitus from the age of 56. During the first year after diagnosis, he received 1.5 g of tolbutamide per day, followed by 150 mg of butylbiguanide per day for the next 3 yr. Thereafter his diabetes was regulated by diet alone. At the age of 65, hypothyroidism was suspected because of slight anemia and edema.
The following laboratory data were obtained. Serum cholesterol was 204 mg per 100 ml, BMR-34%, triiodothyronine 65 ng per 100 ml thyroxine 0.3 μg per 100 ml and the triosorbtest gave 19.2%. Diagnosis of primary hypothyroidism was confirmed by an elevated TSH value of 460 pU per ml. The microsome test and thyroid test gave positive results. A 50 g oral glucose tolerance test showed FBS values of 100 mg per 100 ml; 1 hr, 190 mg per 100 ml; and 2 hr, 220 mg per 100 ml. The serum IRI levels during GTT were low, with delayed response to glucose. Fundoscopic examination revealed diabetic retinopathy (Scott II_b).
After months of treatment with desiccated thyroid the patient lost 10 kg in weight and remarkable improvement in the T wave on ECG was observed.

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Macular amyloidosis, a type of localized cutaneous amyloidosis, was observed at a relatively high frequency among diabetics. The skin lesions were generally located on the shoulders and extensors of the forearm. The characteristic manifestation was brown or darkbrown spotty pigmentation which corresponded to the dermal papillae. Itching was rare or only slight.
Histological examination revealed foci of amyloid material situated in the dermal papillae. The amyloid deposits were not distinguishable with haematoxylin-eosin stain, but favorable results were obtained with Van Gieson stain (light yellow), crystal violet stain, and methyl violet stain (light reddish violet). In particutar, by using the technique of thioflavine-T staining, the amyloid substance was easily recognized from its characteristic yellow fluorescence.
Neither the pathogenesis of localized cutaneous amyloidosis nor the significance of relatively high amounts of amyloid substance in diabetics has yet been explained.
Diabetic patients usually display various forms of dermatopathy, and such chronic inflammation and irritation of the skin may possibly lead to deposition of amyloid substance in the skin.
On the other hand, there are many reports of considerable amyloid deposition in the islets of Langerhans in diabetic patients, so the possibility exists that metabolic disturbance in the diabetic state may have a close relation to amyloid deposition in the skin. Further investigations are required.

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