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We report a method for suppression of lactate production by lactic acid bacteria (LAB) in culture. LAB produce lactate to regenerate NAD+ that is consumed during glycolysis. Glucose suppresses NAD+ regeneration pathways other than lactate dehydrogenase and non-glycolytic ATP production pathways. Therefore, the carbon source was changed to sucrose, and fed-batch culture was performed to limit the glycolytic flux and thus suppress lactate production. As a result, lactate productivity (i.e., the amount of lactate produced per amount of grown cell) in the sucrose/fed-batch culture was decreased compared to that in glucose/batch culture, in all five LAB strains examined. The productivity level decreased to 24% and 46% in Lactobacillus reuteri JCM 1112 and Lactococcus lactis JCM 7638, respectively. Metabolic flux analysis of Lactobacillus reuteri JCM 1112 revealed increased contributions of the mannitol production pathway to NAD+ regeneration and the arginine deiminase pathway to ATP production in the sucrose/fed-batch culture.

Objectives: We aimed to examine the prevalence of orthostatic dysregulation among newly graduated female nurses after employment and its associations with autonomic nervous function, stress, and depressive symptoms. Methods: This follow-up study included 48 newly graduated female nurses (aged 22 ± 3 years) employed in acute care hospitals. The orthostatic dysregulation symptoms were evaluated using a screening checklist. A sit-to-stand test was conducted to assess the autonomic nervous function. Subjective stress and depressive symptoms were determined using a self-administered questionnaire. The data were collected at baseline on the first month and on the seventh month of employment. Statistical differences within groups were analyzed using paired t-test and McNemar’s test. The independent associations of orthostatic dysregulation status with stress and depressive symptoms were analyzed using a multivariate logistic regression model. Results: The percentage of individuals who were diagnosed with orthostatic dysregulation increased from 25.0% at baseline to 35.4% at follow-up. Logistic regression analyses revealed that stress and depressive symptoms were closely associated with orthostatic dysregulation status at follow-up, despite a weak association reported at baseline. The participants were categorized according to their orthostatic dysregulation status: among individuals without orthostatic dysregulation at baseline but with orthostatic dysregulation at follow-up, the increase in autonomic nervous activity, as assessed by the coefficient of variation of the R-R intervals, in response to the postural changes was significantly attenuated at follow-up. Furthermore, this group exhibited a significant increase in stress and depressive symptoms. Conclusions: At 7 months after employment, newly graduated nurses showed a higher prevalence of orthostatic dysregulation in combination with autonomic nervous system modulation, which was accompanied by an increase in stress and depressive symptoms. These observations suggest that the orthostatic dysregulation is associated with poor mental and physical health among newly graduated nurses in the early phase of employment.

Aerobic fed-batch cultures were studied as a means of suppressing the production of lactate, which inhibits the growth of lactic acid bacteria (LAB). LAB produce lactate via lactate dehydrogenase (LDH), regenerating nicotinamide adenine dinucleotide (NAD+) consumed during glycolysis. Therefore, we focused on NADH oxidase (NOX), employing oxygen as an electron acceptor, as an alternative pathway to LDH for NAD+ regeneration. To avoid glucose repression of NOX and NAD+ consumption by glycolysis exceeding NAD+ regeneration by NOX, glucose was fed gradually. When Lactococcus lactis MG 1363 was aerobically fed at a specific growth rate of 0.2 h-1, the amount of lactate produced per amount of grown cell was reduced to 12% of that in anaerobic batch cultures. Metabolic flux analysis revealed that in addition to NAD+ regeneration by NOX, ATP acquisition by production of acetate and NAD+ regeneration by production of acetoin and 2,3-butanediol contributed to suppression of lactate production.

Lactic acid bacteria (LAB) grow by producing lactate from sugar. However, the accumulation of lactate inhibits their growth. Here, the lactate productivity per cell in a semi-solid medium prepared with a chlorella powder in several LAB strains was much lower than that in the conventional MRS medium. Furthermore, the lactate production was suppressed not only in semi-solid medium, but also in chlorella liquid medium. The lactate productivity by Lactococcus lactis subsp. lactis NBRC 12007 in the chlorella liquid medium and MRS medium was 3.0 and 6.9 g-lactate·g-cell−1, respectively. The productivity of lactate in the chlorella liquid medium decreased to 44% of that in MRS medium. Gas chromatography/mass spectrometry (GC/MS) analysis of the culture supernatants revealed that the utilization of sucrose in the chlorella powder led to the suppression of lactate production. Comparison of the metabolites extracted from the cells indicated that the two ATP generating pathways, the arginine deiminase pathway and the decarboxylation reaction of glutamate and GABA, which are usually repressed by glucose, are activated in chlorella medium. It was considered that these pathways which do not require NAD+ for generation of ATP are not repressed when sucrose is used as a carbon source. Thus, the utilization of these pathways results in the suppression of the lactate production.

Polycyclic aromatic hydrocarbon quinones (PAHQs) are components in airborne particulate matter (PM) and generate reactive oxygen species (ROS) in a redox cycling process. 9,10-Phenanthrenequinone (9,10-PQ) is a PAHQ found in diesel exhaust particulates and PM. When inhaled, it produces much more ROS than other PAHQs. We hypothesized that urinary metabolites of 9,10-PQ could serve as biomarkers of PAHQ exposure. Here, we describe methods for pretreating urine samples and analyzing 9,10-PQ metabolites by liquid chromatography with tandem mass spectrometry (LC-MS/MS). In urine from rats intraperitoneally injected with 9,10-PQ, the monoglucuronide of 9,10-dihydroxyphenanthrene (9,10-PQHG) was found to be a major metabolite of 9,10-PQ. 9,10-PQHG was also identified in the urine of a nonoccupationally exposed human by its retention time and MS/MS spectra. Furthermore, the urine contained hardly any free (unmetabolized) 9,10-PQ, but treating it with hydrolytic enzymes released 9,10-PQ from conjugated metabolites such as 9,10-PQHG. The concentrations of 9,10-PQHG in urine samples from nonoccupationally exposed subjects who lived in a suburban area were 2.04-19.08 nmol/mol creatinine. This study is the first to demonstrate the presence of 9,10-PQHG in human urine. Determination of urinary 9,10-PQHG should be useful for determining 9,10-PQ exposure.

To understand the risk of hypoglycemia associated with urinary glucose excretion (UGE) induced by sodium-glucose cotransporter (SGLT) inhibitors, it is necessary to know the relationship between the ratio of contribution of SGLT2 vs. SGLT1 to renal glucose reabsorption (RGR) and the glycemic levels in vivo. To examine the contributions of SGLT2 and SGLT1 in normal rats, we compared the RGR inhibition by tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1 and SGLT2 (SGLT1/2) inhibitor, at plasma concentrations sufficient to completely inhibit rat SGLT2 (rSGLT2) while inhibiting rSGLT1 to different degrees. Under hyperglycemic conditions by glucose titration, tofogliflozin and phlorizin achieved ≥50% inhibition of RGR. Under hypoglycemic conditions by hyperinsulinemic clamp, RGR was reduced by 20–50% with phlorizin and by 1–5% with tofogliflozin, suggesting the smaller contribution of rSGLT2 to RGR under hypoglycemic conditions than under hyperglycemic conditions. Next, to evaluate the hypoglycemic potentials of SGLT1/2 inhibition, we measured the plasma glucose (PG) and endogenous glucose production (EGP) simultaneously after UGE induction by SGLT inhibitors. Tofogliflozin (400 ng/ml) induced UGE of about 2 mg·kg−1·min−1 and increased EGP by 1–2 mg·kg−1·min−1, resulting in PG in the normal range. Phlorizin (1,333 ng/ml) induced UGE of about 6 mg·kg−1·min−1 and increased EGP by about 4 mg·kg−1·min−1; this was more than with tofogliflozin, but the minimum PG was lower. These results suggest that the contribution of SGLT1 to RGR is greater under lower glycemic conditions than under hyperglycemic conditions and that SGLT2-selective inhibitors pose a lower risk of hypoglycemia than SGLT1/2 inhibitors.

A sensitive and variable-wavelength optical rotatory (OR) detector for high-performance liquid chromatography is presented. This design is entirely different from that of conventional OR detectors consisting of a crossed polarizer pair. By placing a polarizing prism and a retardation plate into a commercial circular dichroism (CD) detector, the OR signal was obtained. The Mueller matrix approach was used to prove the principle of the OR signal appearance. Sugars and 4-androstene-3,17-dione were chosen as test compounds. The limit of detection was below 0.5 μg of injected sucrose at 260 nm, which was superior to that obtained with a conventional OR detector. For 4-androstene-3,17-dione, which is CD active, and shows a large anomalous OR dispersion curve, our detector gave a large OR signal with approximately half the intensity of the CD signal at 340 nm. 2008 © The Japan Society for Analytical Chemistry.

Sodium/glucose cotransporter 2 (SGLT2) is the predominant mediator of renal glucose reabsorption and is an emerging molecular target for the treatment of diabetes. We identified a novel potent and selective SGLT2 inhibitor, tofogliflozin (CSG452), and examined its efficacy and pharmacological properties as an antidiabetic drug. Tofogliflozin competitively inhibited SGLT2 in cells overexpressing SGLT2, and K(i) values for human, rat, and mouse SGLT2 inhibition were 2.9, 14.9, and 6.4 nM, respectively. The selectivity of tofogliflozin toward human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 was the highest among the tested SGLT2 inhibitors under clinical development. Furthermore, no interaction with tofogliflozin was observed in any of a battery of tests examining glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. A single oral gavage of tofogliflozin increased renal glucose clearance and lowered the blood glucose level in Zucker diabetic fatty rats. Tofogliflozin also improved postprandial glucose excursion in a meal tolerance test with GK rats. In db/db mice, 4-week tofogliflozin treatment reduced glycated hemoglobin and improved glucose tolerance in the oral glucose tolerance test 4 days after the final administration. No blood glucose reduction was observed in normoglycemic SD rats treated with tofogliflozin. These findings demonstrate that tofogliflozin inhibits SGLT2 in a specific manner, lowers blood glucose levels by increasing renal glucose clearance, and improves pathological conditions of type 2 diabetes with a low hypoglycemic potential.

A new type of optical rotation (OR) detector based on the phase retardation of the first-order diffraction beam has been constructed. The proposed detector responded to only OR properties by eliminating the circular dichroism (CD) effect. Thus, it could measure the optical rotatory dispersion curves in the Cotton band. Although the optical system requires the incident beam in which the phase retardation is 0.5π, the actual retardation was estimated to be around 0.3π. It means that the OR signal intensity can be doubled. Since the proposed detector works over a range of UV wavelengths, there was a compound whose OR signal intensity was higher than the CD signal intensity. The OR detection was superior to CD in stability and in defining the baseline.