[Development of Rapid Assay of Transthyretin in Whole Blood].

Transthyretin (TTR), a rapid turnover protein with a half-life of 2 days, which is shorter than that of albumin, is considered to be a sensitive indicator of a patient's nutritional status. We have newly developed a rapid assay of TTR by immunochromatography (IC) using samples of whole blood as well as serum at volumes of 10 µL. The fundamental performance was evaluated using control sera. In addition, whole blood samples were measured by IC and the results were compared with measured levels of plasma samples by turbidimetry and those of sera by nephelometry. The limit of quantification was 8.3 mg/dL. The linearity was 8.7-35.4 mg/dL. The reproducibility was 6.1-8.6% coefficient of variation. Bilirubin, hemolysis, chyle, and rheumatoid factor did not influence the measured levels. An increase of 1% in hematocrit resulted in a decrease of 1% in the measured levels. The correlation between IC (y) and nephelometry (x) was determined to be y = 0.927x+2.62 (r = 0.935, n = 64). The newly developed IC showed good performance and it was suggested that IC would be useful for nutrition monitoring as point of care test.

[Usefulness of survivin/glyceraldehyde-3-phosphate dehydrogenase ratio in urine exfoliated cells for the detection of bladder tumor].

OBJECTIVE: Survivin is one of the apoptosis inhibitor proteins and is rarely expressed in adult normal tissues. However, survivin expression has been detected in various tumors. In this study, we evaluated the usefulness of urinary survivin/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio as a marker for bladder tumor. PATIENTS AND METHODS: Urine samples were obtained from 72 patients with bladder tumor, 36 with urinary tract inflammation as controls. Survivin and GAPDH mRNA expression was measured by quantitative real-time PCR assay in urine cells. The GAPDH housekeeping gene was used for normalization of survivin expression. We also analyzed survivin protein levels using urine samples and recombinant protein by western blotting. RESULTS: High expression of survivin was confirmed on the protein level using urine samples of bladder tumor by western blotting. Survivin/GAPDH mRNA ratios of bladder tumor quantified by real-time PCR was significantly higher than those of controls (p=0.001). In pathological stage of bladder tumor, survivin/ GAPDH mRNA ratio of pTis was significantly high compared with pTa and pT1 (p < 0.001, p=0.001, respectively). Grade3 tumors expressed high level of survivin/GAPDH mRNA ratio compared with Grade 1 and Grade 2 tumors (p=0.03). The sensitivity, the specificity and AUC(area under the curve) of survivin/ GAPDH mRNA ratio was 83.3%, 86.1% and 0.898, respectively. CONCLUSION: Measuring survivin/GAPDH mRNA ratio in urine is non-invasive and high sensitive examination. Therefore, survivin/GAPDH mRNA ratio is useful marker for the detection of bladder tumor, especially to detect carcinoma in situ.

C-514T polymorphism in hepatic lipase gene promoter is associated with elevated triglyceride levels and decreasing insulin sensitivity in nondiabetic Japanese subjects.

Hepatic lipase (HL) is synthesized in the liver and hydrolyses triglyceride and phospholipids. C-514T polymorphism in HL gene promoter was reported to associate with hepatic lipase activity and plasma lipid levels. We examined whether C-514T polymorphism affects glucose metabolism beyond its effect on plasma lipid levels in nondiabetic Japanese subjects. Gene frequencies of C/C homozygote, C/T heterozygote and T/T homozygote were 18, 51 and 31%, respectively. The allelic frequencies of C and T were 44 and 56%, respectively. T allele frequency was much higher than in Caucasian subjects. Moreover, -514T allele carriers had higher levels of triglyceride (P=0.027), fasting insulin (P=0.016) and HOMA-IR (P=0.033) than non-carriers. In contrast to some former studies, -514T allele affected triglyceride levels and insulin sensitivity. Taken together, HL gene might be one of the important susceptibility genes of type 2 diabetes and the high incidence of type 2 diabetes could be explained by high frequency of -514T allele in the Japanese population. Moreover, since HL and adiponectin showed an additive effect on insulin sensitivity, these genetic variations can be independently associated with insulin sensitivity.

Adiponectin gene variation associates with the increasing risk of type 2 diabetes in non-diabetic Japanese subjects.

Adiponectin is an adipocyte-secreted protein that is known to modulate insulin sensitivity and glucose homeostasis. A number of genetic variations have been studied. Among them, two single-nucleotide polymorphisms (SNP45T>G, SNP276G>T) showed an association with type 2 diabetes in the Japanese population. In this study, we examined the association between these SNPs and risk factors of type 2 diabetes in 194 non-diabetic Japanese subjects. SNP45 was associated with insulin sensitivity (determined by HOMA-IR, p=0.046) and obesity (body mass index; BMI, p=0.043). SNP276 showed a stronger association with HOMA-IR (p=0.018) and BMI (p=0.017). However, neither SNP had an association with insulin secretion (insulinogenic index) and plasma lipid levels. Moreover, a linkage dis-equilibrium was observed between SNP45 and SNP276. Carriers with SNP45G-SNP276G haplotype had higher BMI (p=0.034) and carriers with SNP45T-SNP276T haplotype had lower BMI (p=0.005) and HOMA-IR (p=0.037). Adiponectin gene variations showed an association with obesity and insulin sensitivity, and adiponectin genotypes may predict the increasing risk for type 2 diabetes in non-diabetic subjects.

Increased fibrinolysis increases bleeding in orthopedic patients receiving prophylactic fondaparinux.

We evaluated hemostatic markers in patients who underwent major orthopedic surgery, including total hip and total knee arthroplasty, and were treated for the prophylaxis of deep vein thrombosis (DVT) with or without fondaparinux (anti-Xa group, n = 98 and without anti-Xa group, n = 20). The frequency of DVT was significantly higher in the without anti-Xa group than in the anti-Xa group, but the reduction of hemoglobin and fibrinolytic marker levels was significantly lower in the without anti-Xa group than in the anti-Xa group. Eighteen patients in the anti-Xa group showed a reduction in hemoglobin of more than 2 g/dl, and those individuals were considered to be the increased bleeding (IB) group. The concentration of fibrinolytic markers in the anti-Xa group was significantly higher in the IB group than in the non-IB group. There were also no significant differences in the levels of anti-Xa activity, plasminogen activator inhibitor-I, soluble fibrin and antithrombin between the IB and non-IB groups. In conclusion, elevated fibrinolysis induced by increased bleeding may lead to further increases in bleeding in patients receiving thromboprophylaxis with fondaparinux following major orthopedic surgery.

Monitoring for anti-Xa activity for prophylactic administration of Fondaparinux in patients with artificial joint replacement.

The efficacy of measuring anti-Xa activity was evaluated in major orthopedic surgery patients receiving thrombo-prophylaxis with Fondaparinux. Although 98 orthopedic patients including those receiving total hip replacement (THR) and total knee replacement (TKR) were treated with 1.5 mg of Fondaparinux for prophylaxis of deep vein thrombosis (DVT). Sixteen patients developed DVT, but none was associated with a fatal pulmonary embolism. There was a wide range of anti-Xa activity, but there were no patients with less than 0.15 mg/l or more than 0.90 mg/l. Anti-Xa activity gradually increased from days 1 to 8 and showed no significant difference between patients with and without DVT. Anti-Xa activity was correlated with weight, height, body mass index, and antithrombin activity. Postoperative plasma levels of D: -dimer and soluble fibrin (SF) were markedly high, and those were significantly reduced at days 1 and 4 of treatment with Fondaparinux. Plasma levels of SF were significantly reduced at days 8 and 15, but D: -dimer was not. These findings suggested that there was continued thrombin generation after the injection of Fondaparinux until day 8 and secondary fibrinolysis occurred on day 8. In conclusion, 1.5 mg of Fondaparinux may not be sufficient for the prophylaxis of silent DVT, but it was found to be useful for that of fatal pulmonary embolism. Consequently, monitoring anti-Xa activity may be unnecessary for the administration of Fondaparinux at such doses.