The serum concentrations of Cu, Zn-superoxide dismutase (SOD) in 32 patients with primary diabetes were determined by Sandwich Enzyme Immunoassay (EIA) for application to clinical diagnosis. Furthermore, comparison was made between the biological activity by conventional negative assay and the immunological activity by EIA. Serum Cu, Zn-SOD in the diabetics showed a significantly high level of 64±45ng/ml compared with 33±9ng/ml in the controls (p<0.001). The immunological values of Cu, Zn-SOD in the diabetics tended to increase in patients with a prolonged duration of the disease, with insulin-dependent diabetes, with a high triglyceride level and with obesity. The increase was significantly high especially in patients with a high hemoglobin Al level (p<0.05). However, no correlation was noted between the serum Cu, Zn-SOD level and the blood sugar level in the glucose tolerance test.
In the group with microangiopathy, on the other hand, serum Cu, Zn-SOD was markedly elevated in all cases and demonstrated the highest value of 103±52ng/ml especially in those with nephropathy, which was a significant increase compared to those without nephropathy (p<0.01). The immunological and biological activities were well correlated on the whole.
The above results suggested that the Cu, Zn-SOD level determined by immunological assay is closely related to the pathological conditions of diabetes, reflecting the relative or absolute deficiency of insulin and that Cu, Zn-SOD plays a physiological defensive role as a scavenger of active oxygen against tissue injuries due to primary diabetes and its complications.

View full abstract

This study was designed to evaluate the systemic appearance of subcutaneously injected insulin.
The arterial serum immunoreactive insulin (IRI) responses after bolus subcutaneous or intravenous injection of MC-Actrapid porcine insulin (0.2 U/kg) in 4 rabbits were analyzed with a compartmental and a noncompartmental model. Each rabbit had 4 times undergone paired experiments composed of subcutaneous and intravenous insulin injection.
The kinetics of subcutaneous insulin injection could be described by the one-compartmental model in 16 of 16 injections.
The values estimated by one-compartmental model analysis were as follows: absorption rate constant (ka), 0.021±0.006/min (mean±SD); elimination rate constant (ke), 0.189±0.021/min; lag time, 4.7±2.4 min; absorption half-life, 37±14 min.
The values calculated by noncompartmental analysis were as follows: total clearance (CLtot), 17.5±3.0ml/min/kg, total apparent distribution volume (ADV), 95.2±25.0ml/kg; fraction of the dose absorbed (F), 0.430±0.132.
Intra-individual variations (CV, %) were as follows: F, 31.8±8.7; CLtot, 18.6±3.9; ka, 22.5±9.1; ke, 8.6±2.0.
The results suggest that a considerable amount of subcutaneously injected insulin is degraded at the injection site and that inter-and intra-individual variations should be taken into account, particularly when commencing pharmacokinetic challenge to insulin therapy.

View full abstract

The measurement of glycosylated hemoglobin is well recognized as a retrospective assessment of glycemic control in diabetic patients. This non-enzymatic glycosylation occurs with other proteins of the body.
In the present study, we determined the levels of glycosylated nail protein (GNP) by measuring furosine, winch is a specific degradation product of fructose-lysine, upon acid hydrolysis. Levels of GNP arc expressed as a furosine value, (peak area of furosine/peak area of tyrosine) x 100%. The levels were 4.5±1.3%(Mean±S.D., n=31) for normal subjects and 10.5±4.6%(n=83) for diabetic patients. The elevation was highly significant (p<0.001). The levels of GNP were correlated to the levels of HbA₁ determined at nail sampling. The levels of GNP were correlated well with fasting blood glucose levels obtained 3 to 5 months before nail sampling. These results show that glucose is bound to the ε-amino group of nail protein lysine and that GNP may be clinically more useful in the assessment of longer-term blood glucose control than HbA₁.

View full abstract

A quantitative method to measure islet cell surface antibodies (ICSA) in human patients, using the enzyme-linked immunosorbent assay (ELISA), has been developed. Heat-inactivated human sera were incubated with monolayer cultures of a human pancreatic B cell clone (JHPI-1, 10⁴ cells/well) for 60 min at 37°C. The cells were then washed and incubated with peroxidaseconjugated anti-human IgG rabbit serum for 30 min at room temperature. After incubation, the optical density was measured with a microplate calorimeter at a wavelength of 492 nm. The intraassay and interassay coefficients of variation were 6.6% and 13.0%, respectively. There was a significant relationship between the results of ELISA and immunofluorescence assay (IF)(p<0.005). With this method, ICSA were detected in 34.7% of insulin-dependent diabetes mellitus patients. We developed a useful method for measurement of ICSA quantitatively.

View full abstract

Prostacyclin (PGI₂) mainly produced from vascular walls has an inhibitory effect on platelet aggregation. It has been reported that, in diabetes mellitus, PGI₂ production is decreased and platelet thromboxane B2 (TXB₂: a stable metabolite of thromboxane A₂) production is increased, leading to vascular complications. In this study, the relationship between plasma 6-keto-prostaglandin F_1α (6 KF: a stable metabolite of PGI₂) level and platelet function in diabetes mellitus was examined.
Plasma 6 KF level was significantly decreased in diabetics with proliferative retinopathy (DP) compared with age-matched controls (247.1±41.8 vs. 378.4±45.5pg/ml). On the other hand, platelet aggregation rate (78±4%) and TXB2 production during aggregation (8.34±1.70 nmol/10¹⁰ platelets) were significantly increased in DP compared with controls (37±9, 2.60±0.56), respectively. It was observed that plasma 6 KF level was significantly inversely correlated with platelet aggregation rate and TXB₂ production during aggregation. On the other hand, samples of aorta obtained from streptozotocin-induced diabetic rats produced less PGI₂-like substance than samples from controls.
These results suggest that decreased plasma PGI₂, which would be derived from decreased aortic production, may be one of the causes of abnormal platelet function in diabetes mellitus.

View full abstract

We investigated the effect of cyclic AMP on glucose metabolism in 5 non-diabetics and 10 diabetics using dibutyryl cyclic AMP (DBcAMP) which is a derivative of cyclic AMP thought to enter cells easily. Blood glucose (BG), serum immunoreactive insulin (IRI), serum C-peptide reactivity (CPR) and plasma immunoreactive glucagon (IRG) were observed at 15, 30, 60, 90 and 120 minutes during intra-venous drip infusion of 600 mg DBcAMP.
These data were compared with those obtained by 75 g oral glucose tolerance test (75g OGTT) and glucagon loading test (GT), and the following observations were obtained:
1) BG increased gradually after DBcAMP infusion. The peak level of BG was significantly highcr in diabetics (252.1± 57.0mg/dl, mean ± S.D.) than in non-diabetics (189.2±28.5mg/dl), (p<0.05). The increases of BG (difference between peak level of BG and fasting BG) were almost similar in these two groups. The increases of BG during DBcAMP infusion were significantly correlated with the increases of BG in GT (r=0.54, p<0.05). 2) Serum IRI and CPR also increased gradually during DBcAMP infusion. The peak level of serum IRI was significantly higher in non-diabetics (77.2±57.0μU/ml) than in diabetics (12.9±4.2μU/mi), (p<0.01). The peak level of serum CPR was also significantly higher in non-diabetics (7.0±2.6ng/ml) than in diabetics (2.6±1.2 ng/ml), (p<0.01). The peak level of serum IRI during DB cAMP infusion showed significant correlation with the peak level of serum IRI in 75g OGTT (r=0.72, p<0.05) and in GT (r= 0.75, p<0.01). Also the peak level of serum CPR during DBcAMP infusion showed significant correlation between the peak level of serum CPR in 75g OGTT (r=0.86, p<0.01) and in GT (r=0.77, p<0.001), respectively. 3) During DBcAMP infusion plasma IRG did not increase or decrease. The data may suggest the usefulness of DBcAMP infusion as a test of insulin secreting capacity.

View full abstract

Measurement of 24-h urine C-peptide immunoreactivity is a useful means of distinguishing patients with insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). However, 24-h complete urine collection is not always easy especially for ambulant patients. Therefore, amounts of C-peptide in fasting and postprandial (2h after breakfast) urine were measured and correlated with 24-h urine C-peptide excretion and serum C-peptide concentration.
The amount of postprandial urine C-peptide excretion (spot urine CPR), expressed as an urine CPR: urine creatinine ratio (μg) in normal subjects (n=7), those with IDDM (n=14) and those with NIDDM (n=29) were 55.1±32.6, 3.7±7.3 and 64.7±37.6 (mean±SD), respectively. The spot urine CPR in IDDM was significantly lower than that in NIDDM (p<0.001). In patients with IDDM spot urine CPR was less than 15μg/g in most cases.
There were highly significant correlations between fasting or postprandial spot urine CPR and 24-h urine CPR (r=0.66, r=0.67, p<0.001). There were also significant correlations between fasting or postprandial spot urine CPR and fasting or postprandial serum CPR (r=0.53, r=0.59, p<0.001). Correlation coefficients between fasting or postprandial serum CPR and 24-h urine CPR were also significant but they were lower than those between spot urine CPR and 24-h urine CPR.
These results suggest that, despite some limitations, the spot urine CPR can be used as an additional index of insulin dependency in diabetic patients. A postprandial spot urine CPR value of less than 15μg/g would suggest that the patient is likely to be insulin-dependent.

View full abstract

For the detection of ICSA, the indirect immunofluorescence method has been mainly used. In this report, an indirect rosette assay using protein A-labelled SRBC was compared with original indirect immunoflorescent assay for measuring the titer of ICSA in the sera of IDDM patients. Rat islet cells and a human pancreatic B-cell clone (JHPI-1) were employed as a target of ICSA in these assays.
Using this rosette formation assay, there was a good correlation between rat and JHPI-1 cells in islet cell antigen (s)(p<0.01). However, in comparing the rosette assay and indirect immunofluorescent assay, the percentage of rosette-forming cells showed no correlation with the percentage of fluorescence-positive cells both in rat islet cells and JHPI-1 cells. The reason for this discrepancy should be clarified in subsequent studies.

View full abstract

We report a high-aged case of IDDM associated with elevation of the levels of pancreatic enzymes and diabetic ketoacidosis at onset.
A 78-year-old man was admitted to our hospital because of fatigue and thirst. His family history and past history were non-contributory and he had not received any medication or insulin therapy. Some days before admission, he had been irritated by the problem of the construction of a building in his neighborhood. Thirst and polyuria continued for a few days and his body weight decreased by about 5kg. Laboratory data revealed that blood sugar was 699mg/dl and acetone positive. Arterial blood gas was as follows; pH 7.20, PaCO₂ 32.0mmHg, PaO₂ 92.3mmHg, HCO^-₃ 12.2mM/L, base excess-15.0mEq/L. On examination heart rate was 90/min and his skin and tongue were dry. Heart and lung auscultation was normal and his abdomen revealed no tenderness on palpation. Regular insulin and lactic saline were given by drip infusion and both hyperglycemia and dehydration improved. Although ICA (anti-islet cell antibody) and anti-insulin antibody were negative, ICSA (anti-islet cell surface antibody) was positive. HbAic was 7.2% in spite of hyperglycemia and one of the HL-A DR loci was DR 4. From these findings the patient was diagnosed as IDDM in spite of his advanced age.
Pancreatic enzymes including amylase, elastase-I, trypsin and lipase were also increased remarkably. However, neither enlargement of the pancreas nor dilatation of the pancreatic duct were observed using ultrasonograhy and computed tomography. A month later ERCP (endoscopic retrograde cholangiopancreatography) showed that the pancreatic duct was dilatated and irreglar in some portions. Although it was not possible to evaluate the pathological changes in the pancreas in this case, some relationship between the onset of IDDM and elevation of the levels of pancreatic enzymes was suggested.

View full abstract

Diabetic osteo-arthropathy with a high degree of peripheral nervous disturbance is not always rare. The authors report an interesting case who developed stress fractures of the bilateral calcaneus during kinesitherapy. The patient was a 27-year old female with insulin-dependent diabetes mellitus accompanied by remarkable dysfunction of the autonomic nervous system.
She had a rapid onset of diabetes mellitus and received insulin therapy for 12 years. She had obesity (severity of obesity; 26.5%), retinopathy (right; Scott IIIa left; Scott Vb) and neuropathy. She was admitted in a hypoglycemic coma because of poor control of blood sugar. She started gait exercises of 6 km per day after recovery from hypoglycemia, and developed avulsion stress fractures of the bilateral calcaneus in the course of approximately 3 weeks. Calcaneal pain was diminished by rest, use of plantar arch supports and loss of body weight. After one year and 5 months, X-ray photographs showed that the fractures had healed with some residual of deformity. The motor nerve conduction velocity was delayed, and remarkable dysfunctions of the vagus and sympathetic nervous systems were revealed in various autonomic nervous function tests. X-ray revealed slight osteoporosis, and analytical results by the MD method of hand X-ray showed the initial stage of osteopeny.
It was supposed that, based on her slight osteopeny and dysfunction of autonomic and motor nervous systems, the stress fractures of the calcaneus were induced by obesity and the sudden start of kinesitherapy, in spite of her comparatively young age.

View full abstract

High doses of atropine sulfate have a parasympatholytic effect, but low doses have a parasympathomimetic effect. Variations in the R-R interval in electrocardiogram were studied before and after low-dose atropine injection in 16 healthy controls and 24 diabetic patients.
The R-R interval variations during deep breathing were significantly prolonged by low-dose atropine administration in diabetics without sensory or autonomic neuropathy. The variations were also prolonged in diabetics without sensory neuropathy, but with autonomic nerve dysfunction.
However in diabetics with sensory neuropathy, there was no response to low-dose atropine injection.
These findings suggest that a reserve capacity of the parasympathetic nerve function is retained in diabetics without sensory neuropathy.

View full abstract

This report describes the variation in glucose content in sweat following changes in blood glucose. In order to change the level of glucose in blood in normal subjects, a 50 g Glucose Tolerance Test (GTT) was conducted. GTTs for sampling were put done several times on separate occasions. Blood or sweat was sampled at intervals of 30 minutes for two hours during a GTT. Glucose in sweat, sampled from the head of the subject in a steam bath, was measured by High Performance Liquid Chromatography (HPLC).
In the fasting condition the average values of sweat and blood glucose were 0.25mg/dl and 89mg/dl, respectively. At 60 minutes after oral administration of glucose, the sweat value increased to 0.45mg/dl, and the blood value rose to 149mg/dl. These sweat values were by one to two orders of magnitude lower than those reported elsewhere. This discrepancy may be ascribed to the differences between classical and modern techniques. The previously reported values may include some erroneous factors such as coucomitant reducing substances. HPLC enabled us to measure minute changes of glucose content in sweat following blood glucose level. Blood glucose may be estimated transcutaneously using a correlation between blood and sweat, if a statistically significant relationship between the two can be established.

View full abstract