Vitamin D deficiency and inadequate calcium intake are supposed to be potentially related to cardiovascular outcomes, however, their combined association with hypertension remains unclear. In this cross-sectional study among 2,352 subjects, dietary calcium intake was assessed by using a valid food frequency questionnaire, and serum 25-hydroxyvitamin D (25OHD) was measured by the Ultra Performance Liquid Chromatography system. Hypertension was defined as a level of systolic pressure ≥140 mmHg or diastolic pressure ≥90 mmHg, or both, or administration of antihypertensive medications. Vitamin D status was classified into deficiency (25OHD<20 ng/mL), insufficiency (20 ng/mL≤25OHD<30 ng/mL) and sufficiency (25OHD≥30 ng/mL), while dietary calcium intake was divided into tertiles as low, medium, and high. Two-way analysis of variance (ANOVA) and multivariable logistic regression models were adopted. A significant interaction between vitamin D status and dietary calcium intake in relations to systolic blood pressure (p=0.042) and hypertension (p=0.029) indicates the associations of dietary calcium intake with systolic blood pressure and hypertension depend on the vitamin D status, and vice versa. Only in the vitamin D deficiency group, dietary calcium intake was significantly associated with systolic blood pressure level (β=−0.162, p<0.001) and prevalence of hypertension (odd ratio=2.20, p<0.001). The significance was not substantially compromised after further adjustment for confounding factors. In conclusion, the combination of vitamin D deficiency and low dietary calcium intake, rather than alone, is associated with hypertension.

Rat Cyp27b1 was successfully expressed in HepG2 cells using an adenovirus vector. High vitamin D 1α-hydroxylation activity was detected in them, whereas no activity was observed in non-infected cells. Similarly, vitamin D 1α-hydroxylation activity was also observed in HepG2 cells expressing Cyp27b1-Flag, which is tagged with a Flag at the C-terminus of Cyp27b1. Western blot analysis using an anti-Flag antibody showed a clear band of Cyp27b1-Flag. Next, we screened three types of anti-Cyp27b1 antibodies, which consist of two commercially available antibodies and our self-made antibody using Cyp27b1- or Cyp27b1-Flag expressing HepG2 cell lysate as a positive control. Surprisingly, Western blot analysis revealed that two commercially available antibodies did not detect Cyp27b1 but specifically detect other proteins. In contrast, our self-made antisera specifically detected Cyp27b1 and Cyp27b1-Flag in the HepG2 cells expressing Cyp27b1 or Cyp27b1-Flag. These commercially available antibodies have been used for the detection of Cyp27b1 by Western blotting and immunohistochemistry. Our results suggest that those data should be reanalyzed.

We evaluated the α-glucosidase inhibitory activity of acidic polysaccharides (APs) extracted from seaweeds in vitro and their antidiabetic effects in KK-A^y mice. The α-glucosidase inhibitory activity of APs was differed among various seaweed species. Some APs showed higher inhibitory activity in the high-molecular-weight range, whereas others showed higher inhibitory activity in the low-molecular-weight range. Mice were fed low-molecular-weight APs from hijiki (LMWAPsH), which showed higher α-glucosidase inhibitory activity. Fasting blood glucose and HbA1c levels were significantly lower in the LMWAPsH group than in the control group (p<0.01). The calculated homeostasis model assessment-insulin resistance in the LMWAPsH group was significantly lower than that in the control group (p<0.05). These results suggest that α-glucosidase inhibitory activity differ among APs from different seaweed species, and each have an optimum molecular-weight range, and that LMWAPsH prevents the hyperglycemia in KK-A^y mice.

The effects of folic acid on body weight gain in obesity and gut microbiota-associated branched-chain amino acids (BCAAs) and mitochondrial function were investigated. Three- to four-wk-old male C57BL/6J conventional (CV) and germ-free (GF) mice were fed a high-fat diet (HD), folic acid-supplemented HD (FSHD) and a normal-fat diet (ND) for 25 wk. In CV mice, the HD-induced increases in body weight and plasma BCAA concentrations, downregulated expression of genes related to BCAA catabolism (Bcat2, Bckdha, or Ppm1k), mitochondrial biogenesis (Pgc-1α, Cox1, Nd1 or Nd6), fusion (Mfn1, Mfn2 or Opa1) and mitophagy (Pink1 or Park2), and upregulated expression of the fission-associated gene Drp1 in epididymal fat were reversely corrected with FSHD feeding. In contrast, the expression of these genes in the liver was the opposite under HD feeding or folic acid supplementation. In GF mice, plasma BCAA concentrations were much less affected by HD feeding and were reduced by FSHD feeding, with almost no alterations in the expression of genes associated with BCAA catabolism and mitochondrial function. Further analysis indicated a correlation between adipose and hepatic Mt C/N and plasma BCAA concentrations, and the latter had a close association with specific gut bacteria. Therefore, dietary folic acid supplementation differentially affected body weight gain, BCAA catabolism, and mitochondrial dynamics and metabolism under HD feeding between CV and GF mice, suggesting that gut bacteria-altered BCAAs and mitochondria might partially share the responsibility for the beneficial effects of dietary folic acid on obesity.

Currently, there is considerable interest in ketone metabolism owing to the benefits for human health. Conventionally, strict dietary restrictions on carbohydrates are required to increase plasma ketone levels, while supplementation with D-β-hydroxybutyric acid (D-BHB) can easily increase plasma ketone levels. We hypothesized that a daily intake of D-BHB could promote weight loss, especially through fat reduction. Herein, D-BHB (OKETOA^TM) was produced via a proprietary fermentation process from sugar. In this randomized, double-blind, placebo-controlled study, we assessed the safety and fat-reduction effects after 12 wk of daily ingestion of D-BHB (2.9 g) in 22 healthy Japanese adults and 22 control participants. Blood samples were collected pre- and post-treatment. Blood chemistry, anthropometric variables, and the body composition of the participants were investigated. Data analysis revealed that visceral fat at 12 wk significantly decreased by 9.0 cm² (p=0.037), as evidenced by analysis of covariance. Blood parameters and body condition showed no significant differences between the two groups, and the participants reported no adverse effects or discomfort. Furthermore, data were analyzed by regrouping the participants. After removing one suspicious diabetes participant, all others showed significant decreases in visceral fat, body weight, BMI, and fat weight. Additionally, those aged under 50 y old had significantly decreased abdominal circumference and body fat percentage, in addition to visceral fat, body weight, BMI, and fat weight. Overall, our findings indicate that daily D-BHB intake may reduce body fat without dieting or exercise intervention. This study was registered with the UMIN Clinical Trials Registry as UMIN000045322.

We have previously reported that lipocalin-type prostaglandin D synthase (L-PGDS) in egg white reacts with IgE antibodies from children with egg allergies. However, antibodies against chicken L-PGDS are not commercially available, and the amount of L-PGDS in egg white is unclear. In this study, we prepared four monoclonal antibodies against chicken L-PGDS and developed a sandwich enzyme-linked immunosorbent assay (ELISA) and a highly sensitive immune complex transfer enzyme immunoassay (ICT-EIA) to quantify L-PGDS in hen egg whites. The detection sensitivity of ICT-EIA for L-PGDS (0.01 ng/mL) was 2,000-fold higher than that of ELISA, which could not be adapted to determine the amount of L-PGDS in egg white. Thus, ICT-EIA is a better method for quantification of trace allergens and expected to be applied to the quantification of other food allergens. Hen eggs (white-shelled eggs from Julia Lite hens, brown-shelled eggs, and iodine-enriched eggs from Boris Brown hens) were purchased from markets in Kochi City, Japan, and the amounts of L-PGDS in them were determined by ICT-EIA. The amounts of L-PGDS per hen egg white were: brown-shelled eggs, 1,179.3±214.3 μg/egg; iodine-enriched eggs, 607.7±126.1 μg/egg; and white-shelled eggs, 350.0±74.1 μg/egg. These results show that the amount of L-PGDS in hen eggs varies depending on the hen lineage; it could also be affected to some extent by other factors, such as feeds and breeding environment.

Senile cataract has become the leading cause of visual impairment and even blindness in the world, but there are few reports on its relationship with methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms. This study is aimed to investigate the correlation between MTHFR gene polymorphisms or its enzyme metabolites and senile cataract. From January 2019 to June 2020, 663 patients with senile cataract at the Mianyang Central Hospital were enrolled as the observation group, and 646 healthy subjects were randomly selected as the control group. MTHFR gene polymorphisms (i.e., CC, CT, or TT genotypes) and serum homocysteine (HCY), folic acid (FOL), vitamin B₁₂ (VitB₁₂), and vitamin B₆ (VitB₆) levels were detected. The mutation rate of MTHFR C677T and HCY levels in the observation group were significantly higher than those in the control group, whereas FOL, VitB₁₂, and VitB₆ were significantly lower. With an increase in the MTHFR C677T mutation, HCY showed an upward trend, whereas FOL and VitB₁₂ showed a decreasing trend in both the observation and control groups. Multiple logistic regression analysis showed that HCY and FOL were associated with senile cataract and MTHFR mutations; VitB₁₂ was only associated with senile cataract. Compared to that with the CC genotype, CT and TT genotypes were associated with an increased senile cataract risk. Monitoring MTHFR gene polymorphisms and changes in serum HCY, FOL, and VitB₁₂ levels could provide references in predicting senile cataract.

Management of chronic psychological stress is important for the prevention of depression, mood disorders, and other related ailments. Recent studies have shown that dietary methylxanthines, such as caffeine and theobromine, exert preventive effects on these ailments. Although the psychological effects of caffeine are well-investigated, those of theobromine are not fully understood. In the present study, the effects of theobromine were evaluated and compared with those of caffeine using a mouse stress model based on confrontational housing. Male mice were kept separately in partition cages (two per cage) to allow the establishment of territories for confrontational housing. The mice were administered caffeine or theobromine daily via oral gavage (6 mg/kg). Thereafter, the partition was removed to induce confrontational stress. We found that theobromine, but not caffeine, suppressed adrenal hypertrophy caused by confrontational stress. Moreover, sociability tests revealed that caffeine and theobromine had different effects on the behavioral changes caused by confrontational stress. Our results suggest that orally administered theobromine suppresses adrenal hypertrophy caused by psychosocial stress and induces different behavioral changes than dose caffeine.

By comparing germ-free mice and specific pathogen-free mice, we recently demonstrated that the presence of gut commensals upregulates microRNA-200 family members in lamina propria leukocytes (LPL) of the murine large intestine. The present study tested whether the consumption of 1-kestose (KES), an indigestible oligosaccharide that alters gut microbiota composition, influences the microRNA expression in the LPL. Supplementation of KES (4%) in drinking water for 2 wk increased the levels of miR-182-5p, -205-5p, -290a-5p, miR-200 family members (miR-141-3p, -200a-3p, -200b-3p, -200c-3p, and -429-3p) as well as miR-192/215 family members (miR-192-5p, -194-5p, and -215-5p) as determined by microarray analysis in large intestinal LPL of C57BL/6 mice. Quantitative reverse transcription-PCR further confirmed the increase in miR-192-5p, -194-5p, -200a-3p, -200b-3p, -200c-3p, -205-5p, and 215-5p. KES consumption significantly increased Bifidobacterium pseudolongum in the cecal contents. In a separate experiment, intragastric administration of B. pseudolongum (10⁹ CFU/d) for 7 d increased the levels of miR-182-5p, -194-5p, and -200a-3p and tended to increase the levels of miR-200b-3p, -215-5p, and -429-3p. These results suggest that dietary KES influences miRNA expression in the large intestinal LPL, which may be associated with the increased population of B. pseudolongum.