Yuzo Kayamori

Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes

In primary cultured adult rat hepatocytes stimulated by epidermal growth factor and insulin, dramatic changes in the subcellular distribution of metallothionein were clarified by indirect immunofluorescence using antisera specific for this protein. Metallothionein was detected only in the cytoplasm of cultured hepatocytes in the G0 and G1 phases, but was concentrated in the cell nuclei in the early S phase. The strongest staining pattern in the nuclei was observed 12 h after stimulation. Subsequently, the intensity of metallothionein staining in the nuclei decreased. These results suggests that primary cultured hepatocytes are suitable for examining the relation between subcellular localization of metallothionein and cell growth.

Effect of reduced alcohol consumption on blood pressure in untreated hypertensive men.

Fifty-four untreated, mildly hypertensive men whose daily alcohol consumption was > or = 28 ml ethanol and who drank at least 4 times per week took part in a randomized, controlled crossover trial. The purpose of the trial was to test the effects of alcohol reduction on blood pressure. After a 2-week familiarization period, the participants were assigned to either a reduced alcohol drinking group or a usual drinking group for 3 weeks (experimental period 1). The situation was then reversed for the next 3 weeks (experimental period 2). The participants were requested to limit their daily alcohol consumption to zero or reduce it as much as possible for the reduced alcohol consumption period. The self-reported alcohol consumption was 56.1 +/- 3.6 (SEM) ml/day during the usual alcohol drinking period and 26.1 +/- 3.0 ml/day during the period of reduced alcohol consumption. Systolic and diastolic blood pressures in the intervention group were found by analysis of variance to be significantly lower (2.6-4.8 and 2.2-3.0 mm Hg, respectively) than those in the control group during experimental period 2 for systolic blood pressure and experimental period 1 for diastolic blood pressure. Significant (3.6 mm Hg) and nonsignificant (1.9 mm Hg) decreases in systolic and diastolic blood pressure, respectively, were observed. The method of Hills and Armitage was used, reducing ethanol in daily alcohol consumption by 28 ml. The lowering effect of reduced alcohol consumption on blood pressure was independent of changes in salt consumption, which were estimated by 24-hour urine collection and body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Validation of homogeneous assays for HDL-cholesterol using fresh samples from healthy and diseased subjects

BACKGROUND High-density lipoprotein-cholesterol (HDL-C) is a negative risk factor for cardiovascular events. Although several homogeneous HDL-C assays are available, their accuracy has not been validated, particularly in subjects with disease. We aimed to clarify whether HDL-C concentrations measured by homogeneous assays [HDL-C (H)] agree with those determined by the reference measurement procedures [HDL-C (RMP)] using ultracentrifugation and precipitation with heparin-manganese reagent in fresh clinical samples. METHODS HDL-C concentrations in samples from 48 healthy subjects and 119 subjects with disease were determined using 12 homogeneous assays and RMPs. RESULTS All reagents showed excellent intra- and inter-assay CVs (<2.23%) for two pooled sera. Furthermore, the mean bias was within ± 1.0% in nine reagents using samples from healthy subjects and in eight reagents using samples from subjects with disease. In a single HDL-C (H) determination, the total error requirement of the National Cholesterol Education Program (95% of results < 13%) was fulfilled in nine reagents using samples from healthy subjects and six reagents in those from subjects with disease. Error component analysis revealed that only one reagent exceeded ± 10% total error in samples from healthy subjects, whereas four reagents exceeded this error in samples from subjects with disease. Correlations between HDL-C (H) and HDL-C (RMP) revealed that the slopes were within 1.00 ± 0.06 in six reagents in healthy subjects, and eight reagents in subjects with disease. CONCLUSIONS Except for three reagents, HDL-C (H) agrees well with HDL-C (RMP) in subjects with common disease, but not in those with extremely low HDL-C or abnormal HDL composition.

Paediatric presentation and outcome of congenital protein C deficiency in Japan

Severe heritable protein C (PC) deficiency is quite rare, although heterozygous PROC mutation is the second leading cause of genetic predisposition to thrombosis in Japanese adults. The aim of the study was to search the optimal management, the paediatric onset and outcomes of PC deficiency were characterized in Japan. The genetic study, postmarketing survey of activated PC (aPC) concentrate (Anact®C) and intensive review in Japan for 20 years enabled the analysis of the disease onset, genotype, treatment and prognosis. Symptomatic PC deficiency was determined in 27 Japanese children. All but two patients presented within 16 days after birth (three prenatal and six neonatal onsets). Postnatal‐onset cases had normal growth at full‐term delivery. Of the 27 patients, 19 suffered intracranial thrombosis or haemorrhage (ICTH) (three foetal hydrocephalies), 16 developed purpura fulminans (PF) and 10 had both at the first presentation. ICTH preceded PF in both affected cases. Low PC activities of 18 mothers and/or 12 fathers indicated 20 inherited PC deficiencies (2 homozygotes, 11 compound heterozygotes and 7 heterozygotes) and seven unidentified causes of PC deficiency. Nine of 11 patients studied had PROC mutations. Four unrelated patients (50%) carried PC nagoya (1362delG). No PC‐deficient parents had experienced thromboembolism. Of the 18 patients with aPC therapy, two died and eight evaluable survivors had neurological sequelae. This first comprehensive study of paediatric PC deficiency suggested that perinatal ICTH was the major presentation, occurring earlier than neonatal PF. PC nagoya was prevalent in paediatric, but not adult, patients in Japan. Early maternal screening and optimal PC therapy are required for newborns at risk of PC deficiency.

Platelet contamination causes large variation as well as overestimation of mitochondrial DNA content of peripheral blood mononuclear cells

Background Alterations in the copy number of mitochondrial DNA (mtDNA) play a role in the pathogenesis of mitochondrial diseases and other many common diseases. Recently, the copy number of leukocyte mtDNA has been considered to serve as a biomarker to monitor or chase such diseases. Therefore, reproducible mtDNA measurement is required. Methods Peripheral blood mononuclear cells were prepared by a density-based method. The mtDNA/cell was measured by quantitative realtime polymerase chain reaction. Results The degree of platelet contamination varied to a large extent among preparations. The mtDNA copy numbers per mononuclear cell were 269 ± 51 and 146 ± 14 in the samples before and after the platelet depletion, respectively. Conclusion A density-based mononuclear cell preparation causes heavy platelet contamination. The platelet depletion from a sample is particularly important for comparing the mtDNA contents between different dates or between different patients.

A sensitive determination of uric acid in serum using uricase/catalase/formaldehyde dehydrogenase coupled with formate dehydrogenase

We developed and evaluated an assay for serum uric acid based on the uricase (EC 1.7.3.3)-catalase (EC 1.11.1.6)-formaldehyde dehydrogenase (FADH, EC 1.2.1.46) method coupled with formate dehydrogenase (formate:NAD oxidoreductase, FDH, EC 1.2.1.2). Formate dehydrogenase from Pseudomonas oxalaticus catalyzes the formation of NADH from formate produced by FADH. Owing to the NADH and formate oxidase activity of the FDH itself, the full reaction curve is not linear, but gradually decreases. The formation of NADH is not stoichiometric with formate removal, but is strictly proportional to it. To overcome this decrease of extinction, we added hydroxylamine hydrochloride to the FDH. The sensitivity of the full reaction in the presence of FDH was about 1.8 times that without FDH. Analysis with a Cobas Bio centrifugal analyzer revealed a linearity of up to 3.56 mmol/L. The uricase-catalase-alcohol dehydrogenase method correlated well with the uricase-peroxidase-chromogen method. Our method is more sensitive than other methods.

Nonenzymatic elimination of ascorbic acid in clinical samples

OBJECTIVES Ascorbic acid interferes significantly in the oxidative reaction of chromogenic reagents by peroxidase and hydrogen peroxide. Currently, ascorbate oxidase is commonly utilized for eliminating the interference of ascorbic acid in the oxidative colorimetric reaction. This enzyme, however, displays several disadvantages, such as high cost, variation from lot to lot, and low stability. We applied a series of commercially available and stable radicals (ascorbic acid quenchers [AAQs]) for nonenzymatic quenching of ascorbic acid in the uricase-based uric acid determination in serum and urine. DESIGN AND METHODS In order to evaluate the quenching activity of AAQs, a commercially available uric acid detection kit was used. TBA-80FR.NEO biochemical analyzer was utilized for the assay. RESULTS 4-Hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy free radical (AAQ-2) was the most effective ascorbic acid quencher among the four stable radicals, and the uric acid assay suffered no interference by AAQ-2. The ascorbic acid quenching ability of 2 mmol/L of AAQ-2 in reagent solution (reagent-I) was > or = 2 U/ml ascorbate oxidase in reagent solution. CONCLUSIONS AAQ-2 was proven to be a suitable quencher of ascorbic acid in clinical samples.

Factor XII gene (F12) –4C/C polymorphism in combination with low protein S activity is associated with deep vein thrombosis

Factor XII gene (F12) –4C/C polymorphism in combination with low protein S activity is associated with deep vein thrombosis -

HDL cholesterol performance using an ultracentrifugation reference measurement procedure and the designated comparison method

BACKGROUND Accurate high-density lipoprotein cholesterol (HDL-C) measurements are important for management of cardiovascular diseases. The US Centers for Disease Control and Prevention (CDC) and Cholesterol Reference Method Laboratory Network (CRMLN) perform ultracentrifugation (UC) reference measurement procedure (RMP) to value assign HDL-C. Japanese CRMLN laboratory (Osaka) concurrently runs UC procedure and the designated comparison method (DCM). Osaka performance of UC and DCM was examined and compared with CDC RMP. METHODS CDC RMP involved UC, heparin-MnCl₂ precipitation, and cholesterol analysis. CRMLN DCM for samples containing <200 mg/dl triglycerides involved 50-kDa dextran sulfate-MgCl2 precipitation and cholesterol determination. RESULTS HDL-C regression equations obtained with CDC (x) and Osaka (y) were y=0.992x+0.542 (R(2)=0.996) for Osaka UC and y=1.004x-0.181 (R(2)=0.998) for DCM. Pass rates within ±1 mg/dl of the CDC target value were 91.9 and 92.1% for Osaka UC and DCM, respectively. Biases at 40 mg/dl HDL-C were +0.22 and -0.02 mg/dl for Osaka UC and DCM, respectively. CONCLUSIONS Osaka UC and DCM were highly accurate, precise, and stable for many years, assisting manufacturers to calibrate products for clinical laboratories to accurately measure HDL-C for patients, calculate non-HDL-C, and estimate low-density lipoprotein cholesterol with the Friedewald equation.

Japanese collaborative study to assess inter-laboratory variation in factor VII activity assays

Summary.  Background: The clinical phenotype manifest by patients with factor VII (FVII) deficiency correlates poorly with that predicted by laboratory tests. Despite its importance, there are no data on the variability of inter‐laboratory determinations of low to very low plasma FVII activity (FVII:C).Methods: We distributed three FVII‐deficient plasma samples, prepared by immunoaffinity chromatography, to 58 laboratories in Japan. All samples were assayed using standardized reference plasma as a calibrator. Recombinant thromboplastin was also supplied as a common reagent.Results: In the case of sample A, which had a very low FVII:C, the use of standardized reference plasma and thromboplastin, lowered the variability of inter‐laboratory measurements, when compared with the variability observed when samples were assayed using the respective laboratory’s routine method.Conclusions: The data obtained indicated that results for samples with a very low FVII:C were greatly influenced by the number of plasma dilutions used in constructing a standard activity curve, and also by the type of calibrator and thromboplastin. Such variability was not seen for samples with moderate FVII:C. We conclude that it is necessary to develop a more sensitive and accurate FVII:C measurement system for the diagnosis and treatment of FVII deficiency.