Yukihito Fukumura

Type IV collagen as an early marker for diabetic nephropathy in non-insulin-dependent diabetes mellitus.

We measured urinary albumin (U-Alb) and type IV collagen (uIV.C) in spot urine collected from 82 patients with non-insulin-dependent diabetes mellitus (NIDDM) and 205 controls. Eighty-two NIDDM patients that had no increased excretion of either U-Alb or uIV.C were observed for 6 months. Prevalence of increased excretion of U-Alb and uIV.C at 6 months in these patients were 32.9%, and 62.2%, respectively. Increased excretion of uIV.C was detected in 27 patients without microalbuminuria. chi(2) analysis suggested that uIV.C was more sensitive than U-Alb, and that hypertension enhanced increased excretion of both U-Alb and uIV.C. uIV.C was significantly correlated (P<0.01) with U-Alb but not glycosylated hemoglobin A1C (HbA1C) in NIDDM patients. Taken together, uIV.C may be a useful marker for early diabetic nephropathy.

Determination of antipituitary antibody in patients with endocrine disorders by enzyme-linked immunosorbent assay and Western blot analysis☆

The identification of pituitary antigens recognized by human antipituitary antibodies (APAs) is important in evaluating the pathophysiology of multiendocrine disorders linked to autoimmune factors. However, there is no convenient method for the quantitative analysis of circulating APAs. This study reports the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of APAs. APAs were measured by ELISA and confirmed by Western blot analysis in sera from patients with endocrine disorders. APAs were detected frequently in patients with autoimmune thyroiditis, insulin-dependent diabetes mellitus (IDDM), or pituitary dwarfism. Circulating APAs were detected in 18% of patients with autoimmune thyroiditis. Confirmation by Western blot revealed positivity for APAs in the serum of 36% of patients with Hashimoto disease and in 29% of patients with Graves disease. Notably, 39% of patients with IDDM were also positive for APAs by ELISA. The identification of APAs by ELISA may be useful in evaluating autoimmune mechanisms involved in patients with multiendocrine disorders.

Determination of d-Mannose in Plasma by HPLC

d-Mannose is an essential monosaccharide in the structure of glycoproteins, cell-surface glycoconjugates, and glycosylated phosphatidylinositide anchors. Several stud-ies have focused on plasma or serum mannose concentrations in patients with invasive candidiasis (1)(2), diabetes mellitus (3)(4)(5)(6)(7)(8), or congenital disorders of glycosylation type 1 (9). There are various methods to determine mannose concentrations in human plasma or serum: enzymatic methods (4)(5)(7)(10), gas-liquid chromatography (2)(11), high-resolution liquid chromatography (12), gas-liquid chromatography–mass spectrometry (6), and capillary electrophoresis (13). None of these methods is fully suitable for routine use for various reasons, e.g., the incomplete or time-consuming elimination of the ∼100-fold excess of blood glucose, the use of instruments with limited availability, and the need for a large sample volume. We investigated whether a HPLC assay using an anion-exchange column would be appropriate for plasma mannose determinations. With the procedure described below, mannose could be rapidly and accurately determined in small amounts of plasma. Boric acid, guanidine hydrochloride, sodium metaperiodate, and acetonitrile were purchased from Wako Pure Chemicals Co. Ltd. d-Mannose was obtained from Sigma Chemical Co. We obtained blood samples from healthy individuals and from nondiabetic and diabetic patients after receiving their informed consent for this study. After collection of venous blood in tubes containing disodium EDTA (an anticoagulant) and NaF (a glycolysis inhibitor), plasma was separated by centrifugation. The plasma was mixed with an equal volume of 0.6 mol/L perchloric acid, a protein-precipitating reagent. The mixture was centrifuged at 8000 g for 10 min at 4 °C, and the protein-free supernatant was used for the analysis of mannose. We used a HPLC system (Gulliver system with a JASCO PU-980 pump) equipped …

Clinical significance of neutrophil apoptosis in peripheral blood of patients with type 2 diabetes mellitus.

UNLABELLED Although neutrophils are essential components of the natural immune system, they have also been implicated in the pathogenesis of tissue injuries. We assessed the clinical significance of neutrophil apoptosis in the peripheral blood of patients with type 2 diabetes mellitus (T2DM). PATIENTS AND METHODS The study included 52 patients with T2DM (30 men, 22 women). Control subjects were 16 healthy volunteers without diabetes (7 men, 9 women). Neutrophil apoptosis levels were measured active caspase-3 positive rate by flow cytometry. RESULTS The mean rate of neutrophil apoptosis in patients with T2DM was 15.0% (95% confidence interval [CI]: 9.5% approximately 20.5%), while that in the control group was 5.8% (95% CI: 1.6% approximately 10.0%). There were significant negative correlations between neutrophil apoptosis rate and hemoglobin (Hb) A1c levels (r = -0.352, P < .01). The mean rate of neutrophil apoptosis in the patient group with the 3 major complications (diabetic retinopathy, nephropathy, and neuropathy) was 11.1% (95% CI: 5.5%-16.7%, n = 36) and that of another group without complications was 23.8% (95% CI: 11.4%-36.2%, n = 16). There was a significant difference between these 2 groups (P < .05). CONCLUSIONS The neutrophil apoptosis rate in patients with T2DM was significantly correlated with HbA1C levels. The mean rate of neutrophil apoptosis in the patient group with 3 major diabetic complications remained lower than that in another patient group without complications. The inhibition of neutrophil apoptosis by chronic hyperglycemia is thought to promote tissue injury and to enhance the risk of microangiopathy.

Circulating interleukin-6 significantly correlates to thyroid hormone in acute myocardial infarction but not in chronic heart failure

To investigate relationships between thyroid states and the cardiac endocrine system, we analyzed thyrotropin (TSH), thyroid hormone, plasma levels of interleukin-6 (IL-6) and brain natriuretic peptide (BNP) in 50 patients with chronic heart failure (CHF), in 30 patients with heart failure from acute myocardial infarction (AMI), and in 15 controls. Plasma levels of IL-6 and BNP in both CHF and AMI were significantly elevated, while free triiodothyronine (FT3) was significantly decreased compared to controls. FT3/ free thyroxine (FT4) ratio was significantly decreased in CHF but not in AMI compared to controls. In CHF, diuretic treatment diminished circulating BNP but not IL-6, while diuretic treatment increased FT3/FT4 ratio. In AMI, FT3/ FT4 ratio was significantly decreased 72h compared to 12h after the onset of AMI, while BNP and IL-6 were significantly increased 72h compared to 12h after the onset of AMI. In both CHF and AMI, BNP significantly correlated with FT4. On the other hand, significant correlations between IL-6 and FT3, and between IL-6 and FT3/FT4 ratio were detected in AMI but not in CHF. This preliminary study suggests that IL-6, BNP and thyroid hormone reflect ventricular dysfunction in both acute and chronic heart failure, and that IL-6 significantly relates to circulating thyroid hormone in AMI but not in CHF.

Decreased Serum 1,5-Anhydroglucitol in Nondiabetic Subjects With a Family History of NIDDM

OBJECTIVE To investigate the effect of a family history of NIDDM on HbAlc and serum 1,5-anhydroglucitol (AG) in nondiabetic subjects. RESEARCH DESIGN AND METHODS A 75-g oral glucose tolerance test was performed; 258 subjects with normal glucose tolerance and 106 subjects with impaired glucose tolerance (IGT) were selected HbA1c and serum AG were compared between subjects with and without a family history of NIDDM. The relationships between age, BMI, HbA1c, serum AG, fasting and 2-h plasma glucose, and urinary glucose were also examined using principal component analysis with a varimax rotation. RESULTS In the normal group, only serum AG was lower in subjects with a positive family history than in those with no family history. On the other hand, in the IGT group, subjects with a positive family history were younger and had a higher 2-h plasma glucose, a higher urinary glucose, and a lower serum AG than those with no family history, whereas there was no difference in HbA1c. Principal component analysis identified three factors. The first factor, a linear combination of HbA1c and fasting plasma glucose, was labeled an average glycemic factor. The second factor, which included serum AG, 2-h plasma glucose, and urinary glucose, was labeled an oscillatory glycemic factor. The third factor, which contrasted age against BMI, was labeled an environmental factor. CONCLUSIONS Serum AG is related to glycosuria even among nondiabetic subjects, and its concentrations are decreased in those with a family history of NIDDM. Our results suggest that serum AG rather than HbA1c reflects early metabolic abnormalities in these subjects.

Behaviour of urinary dipeptidase in patients with chronic renal failure

Renal dipeptidase (EC 3.4.13.19) activity in serum and urine from healthy volunteers (n = 20), patients with diabetes (n = 18) and patients with chronic renal failure (n = 5) was measured using glycyl-d-alanine as substrate. The assay was highly specific for the enzyme and was not affected by the various aminopeptidases present in serum and urine. No difference in serum renal dipeptidase activity was observed between the groups. The enzyme activity (U/L) in urine was higher than that in serum, irrespective of the group, suggesting the urine concentration was not affected by the serum concentration. The mean renal dipeptidase activities in urine were 2·56, 2·46 and 0·78 U/mol creatinine for healthy subjects, patients with diabetes and patients with chronic renal failure, respectively. The renal dipeptidase activity was significantly lower in the chronic renal failure group. The urinary excretion of dipeptidase (U/mmol creatinine) showed significant inverse correlations with that of β2-microglobulin, albumin and α1-microglobulin, and with serum concentrations of creatinine, β2-microglobulin and α1-microglobulin. We suggest that urine dipeptidase may be a useful marker of renal diseases.

Type IV Collagen Serum and Vitreous Fluid Levels in Patients with Diabetic Retinopathy

We compared the levels of type IV collagen (IV-C) in vitreous fluid and serum and the levels of glycosylated haemoglobin in 47 patients with proliferative diabetic retinopathy (DR) and 21 patients with non-inflammatory retinopathies. Levels of IV-C were found to be higher in the vitreous fluid in patients with DR than in patients with non-inflammatory retinopathy (53.2 ± 14.9 μg/l versus 14.7 ± 4.5 μg/l). Serum levels were likewise higher in patients with DR (349.7 ± 106.2 μg/l versus 97.7 ± 13.1 μg/l) as were glycosylated haemoglobin levels (8.3 ± 0.3% versus 5.2 ± 0.4%). In addition, levels of type IV collagen in the vitreous fluid were found to be higher in the patients who had been diabetic for ≥ 10 years than in patients who had been diabetic for < 10 years (54.8 ± 15.5 μg/l versus 16.8 ± 4.6 μg/l). We conclude that accumulation of vitreous fluid IV-C may relate to high levels of glycosylated haemoglobin and long duration of diabetes. This suggests that the concentration of IV-C in vitreous fluid, and possibly also the serum levels of IV-C, reflects the progression of DR. Further investigation is needed to verify this and to investigate whether or not measuring IV-C levels is a useful means to assess therapeutic effects and/or prognosis of diabetic microangiopathy.

Occurrence of serum M-protein species in Japanese patients older than 50 years based on relative mobility in cellulose acetate membrane electrophoresis.

We investigated the occurrence of serum M‐protein species in 2,007 Japanese patients older than 50 years of age. All sera samples were analyzed by cellulose acetate membrane electrophoresis. The relative mobility of an M‐protein band was calculated by dividing the migration distance of M protein by that of albumin. M proteins were found to be present in 71 of 2,007 cases (3.5%). Men 80–89 years old showed the highest occurrence of M proteins, 11.0%. The relative mobility of M‐protein bands, especially the band of the IgA‐type M protein, increased as the patients age advanced. The patients had higher levels of the IgG‐type M protein than healthy Japanese subjects. We found that the occurrence of M‐protein species in Japanese patients increases with their age. The IgG‐type M protein was most frequently expressed among other types. The mobility of the M protein was greater in older patients probably because of aging‐related changes in the carbohydrate chain of immunoglobulins composing an M‐protein molecule. J. Clin. Lab. Anal. 14:64–69, 2000.

Simultaneous analysis of serum immunoglobulins in patients with M protein using cellulose acetate membrane isoelectric focusing.

We developed a method for the simultaneous analysis of microheterogeneity of human serum IgG, IgA, IgM, IgD, and IgE, and serum protein pattern using cellulose acetate membrane isoelectric focusing, and analyzed in 11 healthy subjects and 67 patients with M protein (17 cases of multiple myeloma [MM] and 50 cases of monoclonal gammopathy of undetermined significance [MGUS]). Using this method, bands indicating the microheterogeneity of each immunoglobulin could clearly be detected. Among healthy subjects, the detected IgG, IgA, and IgM bands did not vary, but the detected IgE and IgD bands did vary. Therefore, IgA, IgM, and IgG were selected for comparison of serum immunoglobulins in MM and in MGUS. In the IgA‐type M protein group, normal IgM and IgG bands were decreased in MM patients compared to MGUS patients, while the M band and other bands were increased in MM patients compared to MGUS patients, but the differences between the two groups were not significant. In the IgG‐type M protein group, normal IgM, IgA, and IgG were significantly decreased in MM patients compared to MGUS patients. We examined the changes in electrophoretic pattern in six MM patients and eight MGUS patients with IgA‐type M protein after neuraminidase treatment. The width of the M band in MM patients with IgA‐type M protein decreased with neuraminidase treatment. On the other hand, the width of the M band in MGUS patients with IgA‐type M protein increased with neuraminidase treatment. We concluded that the decrease of the normal immunoglobulins in MM patients with IgG type M protein could be detected by this method, and IgA type of M protein binding sugar chain were different between MM and MGUS patients. J. Clin. Lab. Anal. 13:145–150, 1999.