The experiments in mice were approved by the Tsurumi University Animal Experiment Committee (approval number: 20A020) and conducted according to the related guidelines, laws, and regulations. The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Male Balb/c mice aged 11 to 14 weeks were reared in a husbandry facility with controlled temperature and humidity under a 12-hour light cycle. The mice had free access to feed and water. Randomization was not used to allocate animals to groups. No data were obtained from animals that died during the experiment. Confounders were not controlled, and all study investigators were aware of group allocation during the experiments, assessment of outcome, and data analysis.
Sodium bicarbonate was diluted in ultrapure water at 3.8 mg/mL, and citric acid was added to the solution at 0.5 mg/mL to obtain a solution that had a neutral pH and 2500 mg/L of bicarbonate ions. Ultrapure water and SBW at 3.8 mg/mL were used as controls. The bicarbonate ion concentration was determined by titration with sulfuric acid. The Balb/c mice were anesthetized with 3 types of mixed anesthetic agents (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol)33 and bathed in NBIW (n=6) or control solution at 37 °C for 20 minutes. For the NOS inhibitory experiment, L-NAME 100 mg/kg was injected intraperitoneally into 4 mice 1 hour before bathing. After bathing, blood flow in the mice was determined by the laser doppler blood flowmeter RBF-101 (Pioneer Corporation).
The other 2 mice were bathed under anesthesia and euthanized by cutting the carotid vein with a 5-mm Glodenrod Animal Lancet (MEDIpoint, Inc.), and the released blood was collected in a 1.5-mL centrifuge tube. The blood gas analyzer GASTAT-navi (Techno Medica Co., Ltd.) was used to measure the partial pressure of oxygen, partial pressure of carbon dioxide, pH, and bicarbonate levels of the collected blood. The femoral arteries and veins of the mice were also isolated and stored at -80 °C. The isolated vessels were homogenized in purified water, and the amount of NO was measured by the NO2/NO3 Assay kit-FX (Fluorometric) 2,3-Diaminonaphthalene Kit (Dojindo Laboratories).
Western blot analysis
The vessels isolated from the mice were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor and phosphatase inhibitor, and the tissue lysate was used for Western blot analysis with anti-eNOS antibody (M221; ab76198, 1:1000, Abcam plc) and anti-eNOS (phospho S1177) antibody (EPR20991; ab230158, 1:1000, Abcam plc). Both eNOS and p-eNOS were detected in the samples by C-DiGit® Blot Scanner (LI-COR Inc.) on the basis of the chemiluminescence intensity generated by the ECL Prime Western Blotting Detection Reagent (Cytiva) and were quantitatively assessed by Image StudioTM (LI-COR Inc.).
In vitro experiments in HUVEC
HUVEC were starved for 6 hours in a medium not containing serum or VEGF supplement (KBM VEC-1; Kohjin Bio Co., Ltd). NBIW was diluted 1,000,000-fold in the culture medium so that the bicarbonate ion content became 2.5 ppb. After 5 minutes, cell lysate was obtained in ultrapure water or RIPA buffer, and the amount of NO and eNOS phosphorylation activity was evaluated as described above.
The ROS scavenging activity of NBIW was determined by ESR. Superoxide generated by xanthine/xanthine oxidase, a major ROS, and a hydroxy radical generated by UV-irradiated hydrogen peroxide solution were trapped by 5-(2,2- dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) and determined as a CYPMPO adduct34. NBIW at a concentration 100-fold higher than that used for the mouse bathing experiment was prepared, and its ROS scavenging activity was compared with that of SBW at a concentration 100-fold higher than that used in the mouse bathing experiment and that of ultrapure water. Each condition was repeated 3 times, and the data were analyzed.
Randomized, double-blind, parallel-group comparison study
The clinical study was performed in compliance with the Declaration of Helsinki and CONSORT statement and was approved by the Chiyoda Paramedical Care Clinic Ethics Review Committee (IRB number: KRK171C1; approval number: HTT18C1) and registered with the UMIN Clinical Trials Registry (UMIN000031026) on 29/06/2018. All participants provided written informed consent to participate in the study. The study was conducted between December 2016 and April 2017, and no major changes were made to the protocol after the start of the study. Data were collected at the Chiyoda Oral Healthcare Clinic and Chiyoda Paramedic Care Clinic, and statistical analysis of the data was performed by CPCC Company Limited, a contract research organization.
Participants consisted of male and female volunteers between 30 and 59 years of age with a subjective symptom of cold intolerance who fulfilled the inclusion criteria and did not meet the exclusion criteria. Inclusion criteria were subjective symptoms of cold intolerance or consistently low body temperature, menopause or a stable menstrual cycle in women, and ability to bathe daily during the study period and to provide written informed consent. Exclusion criteria included regular intake of medicine, regular consumption of foods that may improve blood flow and body temperature, history of serious illness or skin conditions, and pregnancy or breastfeeding. The participants were divided into 2 groups by block randomization on the basis of sex and body temperature on waking. The person responsible for allocating assigned participants to be given either NBIW tablets containing bicarbonate ions as the main ingredient or control tablets containing magnesium sulphate and sodium sulphate. Table 1 shows the composition of NBIW and control tablets. Both tablets were designed as white tablets of 15 g each that were indistinguishable. The randomization code was not disclosed to participants or the people responsible for collecting and analyzing trial data until the end of analysis. Participants were tested during a 1-week pre-observation period and a 4-week intervention trial. They were instructed to dissolve 4 tablets in a bath with hot water at a temperature below 41 °C once daily for 4 weeks and to soak in the water for at least for 15 minutes. They were also told to not make significant changes to their lifestyle during the study period. Primary outcome endpoints were body temperature on waking, sleep quality assessed by the Japanese version of Pittsburgh Sleep Quality Index (PSQI-J)35,36, and score on the simplified POMS-2 for adults37,38. Secondary outcomes were body temperature before lunch, before supper, before bathing, 1 hour after bathing, and before going to bed and the presence or absence of a subjective symptom of cold intolerance. Participants were given a logbook and instructed to use it daily to record the items specified in the protocol, such as use of bath tablets, time of bathing, and body temperature upon waking and at bedtime, from one week before to the end of the intervention.
Statistical analyses were performed with Mac statistical analysis (ESUMI Co., Ltd.) and StatPlus:mac (AnalystSoft Inc.). For the animal experiments, comparisons of 2 independent groups were performed by Student’s t test for parametric data and the Mann-Whitney U test for non-parametric data. For the clinical study, Wilcoxon rank sum test was used for comparisons of 2 independent groups and Wilcoxon signed rank test, for intragroup comparisons before and after the study. Hedge’s g with the 95% CI was calculated as an index of impact of the effect39.