Quantitation of Cell Membrane Permeability of Cyclic Peptides by Single-Cell Cytoplasm Mass Spectrometry.

Cyclic peptides (CPs) have attracted attention as next-generation drugs because they possess both cell-permeable potential as small molecules and specific affinity similar to antibodies. As intracellular molecules are important targets of CPs, quantitation of the intracellular retention and transmembrane permeability of CPs is necessary for drug development. However, permeated CPs within cells cannot be directly assessed by conventional permeability assays using methods such as artificial membranes and cell monolayers. Here, we propose a new approach using single-cell cytoplasm mass spectrometry (SCC-MS). After cells were incubated with CPs, the cytoplasm was directly collected from a single cell using a microneedle followed by nanoelectrospray ionization mass spectrometry detection of the CPs. The height of the CP peak was plotted against time and fitted with a simple function, y = a(1 - e-bx), to calculate the apparent permeability coefficient (Papp) for both the influx and efflux directions. MCF-7 cells were selected as model cancer cells and cultured with cyclosporin A (CsA) and its demethylated analogs (dmCsA-1, -2, and -3) as model CPs. Papp values (10-6 cm/s) obtained from cells incubated with 50 µM CPs ranged from 0.017 to 0.121 for influx and 0.20 to 1.48 for efflux. The higher efflux ratio was possibly caused by efflux transporters such as P-glycoprotein, a well-known receptor of CsA. The equilibrated intracellular concentration of CPs was estimated to be as low as 4.1-6.8 µM, which showed good consistency with the high efflux ratio. SCC-MS is promising as a reliable permeability assay for next-generation CP-based pharmaceuticals.

Ultrasensitive Single Cell Metabolomics by Capillary Electrophoresis-Mass Spectrometry with a Thin-Walled Tapered Emitter and Large-Volume Dual Sample Preconcentration.

Single cell metabolome analysis is essential for studying microscale life phenomena such as neuronal networks and tumor microenvironments. Capillary electrophoresis-mass spectrometry (CE-MS) is one of the most sensitive technologies; however, its sensitivity is still not enough for single cell analysis on general human cells such as HeLa. To address these issues, we first developed an efficient ionization emitter, named as a "nanoCESI" emitter, that had a thin-walled (∼10 µm) and tapered (5-10 µm) end. The thin conductive wall enabled sheathless ionization and minimized the flow rate of ionizing sample, and the tapered end efficiently ionized analytes via an electrospray ionization mechanism, providing up to 3.5-fold increase in sensitivity compared with a conventional sheathless emitter. Fifty repetitive analyses on 20 amino acids were successfully achieved with a nanoCESI emitter. Relative standard deviations of 50 analyses were 1.5%, 4.4%, and 6.8% for migration time, peak height, and peak area, respectively, where a limit of detection (LOD) of 170 pM (850 zmol) was achieved. Second, a sample enrichment method, large-volume dual preconcentration by isotachophoresis and stacking (LDIS), was applied to a newly designed protocol of nanoCESI-MS. This approach achieved up to 380-fold enhanced sensitivity and LOD of 450 fM. Compared with normal sheathless CE-MS, coupling of nanoCESI and LDIS provided up to 800-fold increase of sensitivity in total. Finally, metabolome analyses of single HeLa cells were performed, where 20 amino acids were successfully quantified with triple-quadrupole MS and 40 metabolites were identified with quadrupole-time-of-flight MS, as a promising analytical platform for microscale bioanalysis for the next generation.

Clinical assessment of the GNAS mutation status in patients with intraductal papillary mucinous neoplasm of the pancreas.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is characterized by cystic dilation of the pancreatic duct, caused by mucin hypersecretion, with slow progression via the adenoma-carcinoma sequence mechanism. Mutation of GNAS at codon 201 is found exclusively in IPMNs, occurring at a rate of 41-75%. Recent advances in molecular biological techniques have demonstrated that GNAS mutation might play a role in the transformation of IPMNs after the appearance of neoplastic cells, rather than in the tumorigenesis of IPMNs. GNAS mutation is observed frequently in the intestinal subtype of IPMNs with MUC2 expression, and less frequently in IPMNs with concomitant pancreatic ductal adenocarcinoma (PDAC). Research has focused on assessing GNAS mutation status in clinical practice using various samples. In this review, we discuss the clinical application of GNAS mutation assessment to differentiate invasive IPMNs from concomitant PDAC, examine the clonality of recurrent IPMNs in the remnant pancreas using resected specimens, and differentiate pancreatic cystic lesions using cystic fluid collected by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), duodenal fluid, and serum liquid biopsy samples.

Role of SpyGlass-DStm in the preoperative assessment of pancreatic intraductal papillary mucinous neoplasm involving the main pancreatic duct.

BACKGROUND/OBJECTIVES: It is often difficult to determine an adequate resection line during pancreatectomy for intraductal papillary mucinous neoplasm involving the main pancreatic duct during partial pancreatectomy. The aim of this study was to evaluate the usefulness of improved peroral pancreatoscopy using SpyGlass-DStm in the preoperative assessment of intraductal papillary mucinous neoplasm involving the main pancreatic duct. METHODS: We collected and retrospectively analyzed clinicopathological data from seven consecutive patients who underwent preoperative assessment of intraductal papillary mucinous neoplasm involving the main duct using SpyGlass-DStm. RESULTS: Good imaging quality of the intraductal protruding lesion was obtained in all seven patients, and only one adverse event was noted wherein a patient had mild pancreatitis. Six patients underwent pancreatectomy. In one patient, masked-type concomitant pancreatic ductal adenocarcinoma and low-length dysplastic lesion was found near the surgical margin, which was not detected by preoperative imaging modalities including SpyGlass-DStm. The sensitivity of targeting biopsy during SpyGlass-DStm to diagnose high-grade dysplasia was 0%. CONCLUSIONS: SpyGlass-DStm can be safely performed in patients with intraductal papillary mucinous neoplasm involving the main duct, and has excellent visualization of the target lesion. However, challenges include poor diagnostic ability of targeting biopsy, and, therefore, intraoperative frozen section is still needed to obtain negative surgical margins.

Nucleus accumbens dopamine D2-receptor expressing neurons control behavioral flexibility in a place discrimination task in the IntelliCage.

Considerable evidence has demonstrated a critical role for the nucleus accumbens (NAc) in the acquisition and flexibility of behavioral strategies. These processes are guided by the activity of two discrete neuron types, dopamine D1- or D2-receptor expressing medium spiny neurons (D1-/D2-MSNs). Here we used the IntelliCage, an automated group-housing experimental cage apparatus, in combination with a reversible neurotransmission blocking technique to examine the role of NAc D1- and D2-MSNs in the acquisition and reversal learning of a place discrimination task. We demonstrated that NAc D1- and D2-MSNs do not mediate the acquisition of the task, but that suppression of activity in D2-MSNs impairs reversal learning and increased perseverative errors. Additionally, global knockout of the dopamine D2L receptor isoform produced a similar behavioral phenotype to D2-MSN-blocked mice. These results suggest that D2L receptors and NAc D2-MSNs act to suppress the influence of previously correct behavioral strategies allowing transfer of behavioral control to new strategies.

[Basal Ganglia Circuit Mechanisms in Cognitive Learning].

The nucleus accumbens (NAc), the ventral part of the striatum, plays a critical role in motivation, learning, and cognition in the basal ganglia circuit. Outputs of the NAc are transmitted through two parallel direct and indirect pathways. We have developed a reversible neurotransmission blocking (RNB) technique, in which neurotransmission of each pathway in the NAc is selectively blocked by specific expression of a transmission-blocking tetanus toxin (D-RNB or I-RNB). In visual cue and reversal tasks in the cross-maze, the NAc direct pathway was critical for learning acquisition. In contrast, the NAc indirect pathway was essential not only for learning flexibility, but also for subsequent acquisition of a new strategy. In place discrimination and serial reversal learning tasks in the IntelliCage, we showed that the NAc indirect pathway controls behavioral flexibility by suppressing the influence of previously correct behavioral strategies during the reversal stage. These basal ganglia circuit mechanisms provide new insight into pathophysiologies associated with compulsive behaviors, including addiction and obesity.

ALDH2 polymorphism is associated with fasting blood glucose through alcohol consumption in Japanese men.

Associations between alcohol consumption and type 2 diabetes risk are inconsistent in epidemiologic studies. This study investigated the associations of ADH1B and ALDH2 polymorphisms with fasting blood glucose levels, and the impact of the associations of alcohol consumption with fasting blood glucose levels in Japanese individuals. This cross-sectional study included 907 men and 912 women, aged 35-69 years. The subjects were selected from among the Japan Multi-institutional Collaborative Cohort study across six areas of Japan. The ADH1B and ALDH2 polymorphisms were genotyped by Invader Assays. The ALDH2 Glu504Lys genotypes were associated with different levels of fasting blood glucose in men (P = 0.04). Mean fasting glucose level was positively associated with alcohol consumption in men with the ALDH2 504 Lys allele (P trend = 0.02), but not in men with the ALDH2 504Glu/Glu genotype (P trend = 0.45), resulting in no statistically significant interaction (P = 0.38). Alcohol consumption was associated with elevated fasting blood glucose levels compared with non-consumers in men (P trend = 0.002). The ADH1B Arg48His polymorphism was not associated with FBG levels overall or after stratification for alcohol consumption. These findings suggest that the ALDH2 polymorphism is associated with different levels of fasting blood glucose through alcohol consumption in Japanese men. The interaction of ALDH2 polymorphisms in the association between alcohol consumption and fasting blood glucose warrants further investigation.

[Distinct roles of the direct and indirect pathways in the basal ganglia circuit mechanism].

The basal ganglia are key neural substrates that control not only motor balance but also emotion, motivation, cognition, learning, and decision-making. Dysfunction of the basal ganglia leads to neurodegenerative diseases (e.g. Parkinson's disease and Huntington's disease) and psychiatric disorders (e.g. drug addiction, schizophrenia, and depression). In the basal ganglia circuit, there are two important pathways: the direct and indirect striatal pathways. Recently, new molecular techniques that activate or inactive selectively the direct or indirect pathway neurons have revealed the function of each pathway. Here we review the distinct roles of the direct and indirect striatal pathways in brain function and drug addiction. We have developed a reversible neurotransmission blocking technique, in which transmission of each pathway is selectively blocked by specific expression of transmission-blocking tetanus toxin, and revealed that the activation of D1 receptors in the direct pathway is critical for reward learning/cocaine addiction, and that the inactivation of D2 receptors is critical for aversive learning/learning flexibility. We propose a new circuit mechanism by which the dopaminergic input from the ventral tegmental area can switch the direct and indirect pathways in the nucleus accumbens. These basal ganglia circuit mechanisms will give us insights into the pathophysiology of mental diseases.

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia.

Sugars, sucrose and colorectal cancer risk: the Fukuoka colorectal cancer study.

OBJECTIVE: A diet high in sugars may promote colorectal carcinogenesis, but it remains uncertain whether high intake of sugars or sucrose confers increased risk of colorectal cancer. The authors investigated the associations of sugars and sucrose intake with colorectal cancer risk in a community-based case-control study in Japan. METHODS: The study subjects comprised 816 incident cases of colorectal cancer and 815 community controls. Consumption frequencies and portion sizes of 148 food and beverage items were ascertained by a computer-assisted interview. The authors used the consumption of 29 food items to estimate sugars and sucrose intake. The odds ratios of colorectal cancer risk according to intake categories were obtained using a logistic regression model with adjustment for potential confounding variables. RESULTS: Overall, intakes of sugars and sucrose were not related to colorectal cancer risk either in men or women. The association between sugars intake and colorectal cancer risk differed by smoking status and alcohol use in men, but not in women. In men, sugars intake tended to be associated with colorectal cancer risk inversely among never-smokers and positively among male ever-smokers (interaction p=0.01). Sugars intake was associated with an increased risk among men with no alcohol consumption, but was unrelated to the risk among male alcohol drinkers (interaction p=0.02). Body mass index did not modify the association with sugars intake in either men or women. CONCLUSION: Sugars intake was associated with increased risk of colorectal cancer among smokers and non-alcohol drinkers in men selectively.

Potential treatment option of rivaroxaban for breastfeeding women: A case series.

The use of direct oral anticoagulants (DOACs) in breastfeeding women is currently challenging due to limited safety data for breastfeeding infants, and there have been no previous studies on the drug concentration in breastfeeding infants. We treated 2 patients (one case was twin pregnancy) with venous thromboembolisms in breastfeeding women administered rivaroxaban at our institution. Blood samples from the mothers and breastmilk samples were collected at time 0 and 2 h after the rivaroxaban administration, breastfeeding was conducted 2 h after the rivaroxaban administration, and blood samples from the infants were collected 2 h after breastfeeding (4 h after maternal rivaroxaban administration). The milk-to-plasma (M:P) ratios were 0.27 in Case 1 and 0.32 in Case 2. The estimated relative infant dose (RID) was 0.82 % in Case 1 Children 1 and 2, and 1.27 % in Case 2. The rivaroxaban concentration in the infant plasma was below the lower limit of quantification in all infants. In addition, even in the high-exposure case simulation based on 5 days of breastfeeding in Case 2, the infant plasma concentration level was below the lower limit of quantification. At 3 months of follow-up, breastfeeding was continued, and all infants grew and developed without any health problems including bleeding events. The current case series showed that there were no pharmacokinetic or clinical concerns for breastfeeding women or breastfed infants, and provides support for rivaroxaban as a safe treatment option for these patients.

Identification and association study with lung cancer for novel insertion polymorphisms of human endogenous retrovirus.

Sequences of human endogenous retroviruses (HERVs) are members of the long terminal repeat (LTR) retrotransposon family. Although the expression of HERV has long been a topic of investigation, HERV-insertion polymorphisms are not well known, and a genetic association between HERV-insertion polymorphisms and cancer has never been reported. To identify novel HERV loci in the genome from cancer tissues, we carried out the inverse PCR method targeting a conserved LTR region of HML-2, which is the most recently acquired HERV group. Novel two insertions, HML-2_sLTR(1p13.2) and HML-2_sLTR(19q12), were identified as insertionally polymorphic solo LTRs. Furthermore, a significant prevalence of HML-2_sLTR(1p13.2) homozygosity was detected in female never-smoking patients aged 60 years and over who had lung adenocarcinoma [versus the other genotyping; odds ratio (OR): 1.97; 95% confidence interval (CI): 1.01-3.81]. In another cohort consisting of female never-smoking patients with lung adenocarcinoma, a prevalence of HML-2_sLTR(1p13.2) homozygosity tended to be high in patients aged 60 years and over (versus the other genotyping; OR: 2.03; 95% CI: 0.96-4.29), whereas a low prevalence of HML-2_sLTR(1p13.2) homozygosity was detected in patients <60 years old (versus the other genotyping; OR: 0.31; 95% CI: 0.11-0.94). Our results suggest that HML-2_sLTR(1p13.2) is involved with the susceptibility to lung adenocarcinoma in female never-smokers in an age-dependent manner and that other HERV polymorphisms related to human diseases might remain to be identified in the human genome.

Estrogen receptor-ß gene polymorphism and colorectal cancer risk: effect modified by body mass index and isoflavone intake.

Estrogen receptor (ER)-ß signaling has generally been implicated in protection against colorectal cancer. The ER-ß gene cytosine-adenine (ESR2 CA) repeat polymorphism was reported to be associated with colorectal cancer, although showing contradicting results probably caused by ethnicity or age distribution of the subjects. We investigated the association between this polymorphism and the colorectal cancer risk in a community-based case-control study in Japan (685 cases/778 controls), including only subjects younger than 75. The effect modifications of the body mass index (BMI) and isoflavone intake were also examined. ESR2 CA repeat polymorphism was determined by polymerase chain reaction using fluorescein-labeled primers. CA repeat alleles were classified into short (S) allele (<22 repeats) and long (L) allele (≥ 22 repeats). Subjects were divided into three genotype groups (SS/SL/LL). The risk of colon cancer, but not of rectal cancer, was increased with an increasing number of L alleles among postmenopausal women; age-adjusted odds ratio (OR) for SL and LL genotypes compared with the SS genotype were 1.78 and 2.91, respectively (trend p = 0.002). Increased risks of colon cancer associated with the L allele were more evident among postmenopausal women with low BMI (<25 kg m(-2)) or with high isoflavone intake. Such associations were not observed among men or premenopausal women. Having longer ESR2 CA repeat increases colon cancer risk among postmenopausal women younger than 75, possibly with modification of BMI and isoflavone intake. Aging and estrogenic condition may be important in the colon cancer pathogenesis associated with ESR2 CA repeat polymorphism.

Microsomal epoxide hydrolase polymorphisms, cigarette smoking, and risk of colorectal cancer: the Fukuoka Colorectal Cancer Study.

Microsomal epoxide hydrolase (EPHX1) plays an important role in the activation and detoxification of polycyclic aromatic hydrocarbons, carcinogens found in cigarette smoke. Polymorphisms in exon 3 (Y113H) and exon 4 (H139R) of the EPHX1 have been associated with enzyme activity. We investigated the risk of colorectal cancer in relation to the EPHX1 Y113H and H139R polymorphisms and assessed effect modifications of cigarette smoking and the other covariates. The interaction between the EPHX1 polymorphisms and selected genetic polymorphisms was also examined. We used data from Fukuoka Colorectal Cancer Study, a community-based case-control study, including 685 cases and 778 controls. In-person interviews were conducted to assess lifestyle factors. The EPHX1 Y113H and H139R polymorphisms were determined by the TaqMan assay and the polymerase chain reaction-restriction fragment length polymorphism, respectively. Neither of the two polymorphisms nor the imputed EPHX1 phenotype was associated with colorectal cancer risk. Cigarette smoking and alcohol intake showed no effect modification on the association with the EPHX1 polymorphisms or the imputed EPHX1 phenotype. Increased risks of colorectal cancer associated with the 113Y allele and imputed EPHX1 phenotype were observed among individuals with high body mass index (BMI; ≥25.0 kg/m(2)), but not among those with low BMI (<25.0 kg/m(2)). The risk decreased with an increasing number of the 139R allele in the null genotypes of GSTM1/GSTT1. It is unlikely that the EPHX1 polymorphisms play an important role in colorectal carcinogenesis. The observed interactions of the EPHX1 polymorphisms with BMI and the GSTM1/GSTT1 genotypes warrant further investigation.

Behavioral and clinical correlates of serum bilirubin concentrations in Japanese men and women.

BACKGROUND: A considerable interest has been drawn to potential protective effects of bilirubin against oxidative stress-related diseases. Smoking is known to be associated with lower concentrations of serum bilirubin, but other behavioral correlates of serum bilirubin have not been well studied. In this cross-sectional study, we examined the associations of behavioral and clinical factors with serum total bilirubin in Japanese men and women. METHOD: The study subjects comprised of 4802 men and 6414 women aged 49-76 years who participated in the baseline survey of an ongoing cohort study on lifestyle-related diseases in Fukuoka, Japan. With consideration to time of the day of blood sampling and fasting hours, the associations with smoking, alcohol intake, body mass index, physical activity, coffee, tea, blood pressure, glycated hemoglobin (HbA1c), HDL cholesterol and non-HDL cholesterol with serum bilirubin were evaluated by analysis of covariance and multiple linear regression analysis. RESULTS: While smoking was negatively associated with serum bilirubin, alcohol consumption was positively associated with serum bilirubin in both men and women. Coffee consumption was associated with lower bilirubin concentrations in both sexes. In the multiple linear regression analysis, HDL cholesterol was positively and HbA1c was negatively associated with bilirubin in both men and women, and the associations were more evident in women. CONCLUSION: Smoking, alcohol use and coffee consumption were important behavioral correlates of serum bilirubin in Japanese men and women. Serum HDL cholesterol was a measurable clinical correlate of bilirubin in women.

Methionine synthase and thymidylate synthase gene polymorphisms and colorectal adenoma risk: the self defense forces study.

Folate-mediated one-carbon metabolism has been implicated in colorectal carcinogenesis. We investigated associations of functional genetic polymorphisms of methionine synthase (MTR), MTR reductase (MTRR), and thymidylate synthase (TS) with colorectal adenomas. The study subjects were 455 cases of colorectal adenomas and 1052 controls with no polyp at colonoscopy. Genotypes were determined for MTR A2756G, MTRR A66G and two polymorphisms in the TS gene, 28-bp tandem repeat polymorphism in the promoter enhancer region (TSER) and 6-bp deletion polymorphism at position 1494 in the 3' untranslated region (TS 1494del6). We also examined the alcohol-genotype and gene-gene interactions on adenoma risk. The GG genotype of MTR A2756G was associated with an increased risk of colorectal adenomas; odds ratios for AG and GG versus AA genotype were 0.99 (95% confidence interval 0.78-1.26) and 1.72 (1.04-2.82), respectively. The increase in the risk associated with MTR 2756GG genotype was evident in men with high alcohol consumption (≥30 mL/d), but not in those with low alcohol consumption (interaction P = 0.03). Men who were homozygous for the TSER double-repeat allele had a slightly decreased risk of colorectal adenomas as compared with those homozygous for the TSER triple-repeat allele. Neither MTRR A66G nor TS 1494del6 was associated with colorectal adenomas. There was no measurable interaction either between MTR A2756G and MTRR A66G or between TSER and TS 1494del6. MTR A2756G appears to be associated with colorectal adenoma risk differently according to alcohol consumption. The MTR-catalyzed reaction may play an important role in the development of colorectal adenomas.

Dietary intakes of retinol, carotenes, vitamin C, and vitamin E and colorectal cancer risk: the Fukuoka colorectal cancer study.

It has long been a matter of interest whether antioxidant vitamins are protective against colorectal cancer as well as human cancers in general, but epidemiological evidence is inconclusive. We investigated associations of dietary intakes of retinol and antioxidant vitamins with colorectal cancer risk in 816 incident cases of histologically confirmed colorectal cancer and 815 controls randomly selected for the Fukuoka colorectal cancer study in Japan. Dietary intakes were assessed by a PC-assisted interview regarding 148 food items. Statistical adjustment was made for body mass index, physical activity, calcium, and n-3 fatty acid intake and other factors. Retinol intake was significantly, inversely associated with colorectal cancer risk; the odds ratio for the highest vs. lowest was 0.55 (95% CI: 0.35, 0.88; P (trend) = 0.01) in women, but a modest increase in the risk was observed among men with the highest intake of retinol. Liver was the major source of retinol intake and showed similar associations with colorectal cancer risk in men and women. Intake of carotenes, vitamin C, and vitamin E were not related to colorectal cancer risk in either men or women. The study did not support a hypothesis that dietary intake of antioxidant vitamins is protective in the development of colorectal cancer.

Behavioral and clinical correlates of high-sensitivity C-reactive protein in Japanese men and women.

BACKGROUND: Inflammation has been implicated in the pathogenesis of cardiovascular disease, type 2 diabetes mellitus and cancer. Serum concentration of high-sensitivity C-reactive protein is a good biomarker of chronic low-grade inflammation. Few studies have evaluated relative importance of behavioral and clinical covariates of high-sensitivity C-reactive protein in Japanese population. METHODS: The study subjects were men and women aged 49-76 years from the cohort study of lifestyle-related diseases between February 2004 and July 2006. Analysis of covariance and multiple linear regression analysis were used to estimate geometric means of high-sensitivity C-reactive protein and trends of association. RESULTS: Smoking, body mass index, hypertension, type 2 diabetes mellitus, elevated non-high density lipoprotein cholesterol, prudent dietary pattern were independently associated with serum high-sensitivity C-reactive protein in both men and women. High-sensitivity C-reactive protein concentrations were lowest in men with a moderate intake of alcohol (<30 mL/day). In men, smoking and body mass index accounted for 28% and 26% of the variation in high-sensitivity C-reactive protein, respectively, while body mass index accounted for 60% of the variation of high-sensitivity C-reactive protein in women. CONCLUSIONS: Smoking and body mass index in men, and body mass index in women, were major correlates of serum high-sensitivity C-reactive protein in Japanese people.

No effect modification of serum bilirubin or coffee consumption on the association of gamma-glutamyltransferase with glycated hemoglobin in a cross-sectional study of Japanese men and women.

BACKGROUND: Oxidative stress has been implicated in the development of type 2 diabetes mellitus. Bilirubin is a potent endogenous antioxidant, and coffee is a major source of exogenous antioxidants. Serum gamma-glutamyltransferase (GGT), a marker of oxidative stress, is a strong predictor of the risk of type 2 diabetes mellitus. This study evaluated the effect modification of bilirubin and coffee consumption on the association of serum GGT with glycated hemoglobin (HbA1c) and the combined effect of bilirubin and coffee on HbA1c concentrations. METHODS: The subjects were 4492 men and 6242 women aged 49-76 years who participated in the baseline survey of an on-going cohort study on lifestyle-related diseases in Fukuoka, Japan. Geometric means of HbA1c were examined according to quartile categories of GGT, with stratification by serum total bilirubin (≥ 0.6 mg/dL versus less in men and ≥ 0.5 mg/dL versus less in women) and coffee consumption (< 1, 1-3 and ≥ 4 cups of per day). Statistical adjustment was made for age, smoking, alcohol use and body mass index by using analysis of covariance. RESULTS: HbA1 concentrations increased progressively with increasing levels of GGT in both men and women. The increasing trend of HbA1c concentrations associated with GGT did not differ by either bilirubin status or coffee consumption. Both men and women with high bilirubin had consistently lower concentrations of HbA1c across the GGT quartiles. Higher coffee consumption was associated with lower concentrations of HbA1c in women with low bilirubin (trend P = 0.04), but not with high bilirubin (trend P = 0.37). There was no such association between coffee and HbA1c in men with either low or high bilirubin levels. CONCLUSIONS: Bilirubin is possibly protective against deterioration of glucose metabolism. Further studies are needed regarding the combined effect of bilirubin and coffee on glucose metabolism.