Study implementation: This study was conducted after it was reviewed and approved (approval date: December 11, 2015) by the HUMA R&D Ethical Review Committee consisting of third parties who have no involvement in the study. In accordance with the spirit of the Declaration of Helsinki (originally adopted in June 1964, and revised in October 2013), the study was implemented within the Ethical Guidelines for Medical and Health Research involving Human Subjects (Public Notice of the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare No. 3 of 2014). (UMIN ID: UMIN000020169)
The purpose and content of the study were fully explained by the principal investigator to the eligible subjects, and the study was performed only on those who gave consent in writing on their own free will.
For this study, the assessment of physical findings, physical measurements, physical examinations, and clinical examinations were conducted at Nihonbashi Egawa Clinic, Kei Medical Office TOC Building Medical Clinic, and Medical Corporation Kaiseikai Kita-Shinyokohama Medical Clinic, where the study subjects were managed and the study implementation system was maintained. The principal investigator controlled the operations related to the study; instructions and explanations to the study subjects, acquisition of written informed consent from the subjects, medical interviews, confirmation and assessment of adverse events, and maintenance of the study implementation system were all performed by the principal investigator. We also planned to give treatment to adverse events as needed. This study was implemented between December 2015 and September 2016.
Background surveys including medical history, drinking habit, and eating habit, physical condition checkup, measurements, and clinical examinations were performed as a screening test on the 428 subject candidates who gave written consent, and 252 cases, who met the selection requirements and did not meet the exclusion criteria, were selected as the study subjects.
For the selection of study subjects, those who had the capacity to consent to the study, who met the following: I. Healthy Japanese between 45 and 74 years of age at the time of consent, II. Subject whose serum 25-hydroxyvitamin D level is 30 ng/mL (= 75nmol/L) or less, and III. Subject whose BMI is between 18.5 kg/m2 and 24.9 kg/m2, and who did not meet the following exclusion criteria, were selected. I. Current smoker, II. Subject with severe disease (e.g., liver disease, renal disease, infectious disease, cancer), III. Subject with disease that would affect the study results (e.g., hypercalcemia), IV. Subject who has a plan to be exposed excessively to the sun (e.g., farm work, vacation in resorts) during the study, V. Subject whose fasting glucose level is 110 mg/dL or more, VI. Subject who received hormone replacement therapy for the past six months, VII. Subject who would take other supplements than multivitamins during the study; this excludes person who will stop taking them before the study and will not take any until the study ends, VIII. Subject who took vitamin D supplements for the past three months, IX. Subject who took 600 mg/day or more of calcium supplement for the past three months, X. Subject with hypertension: the systolic blood pressure at rest is 145 mmHg or higher, or the diastolic blood pressure is 95 mmHg or higher, XI. Subject being treated for hypertension, XII. Subject with history of mental illness, person in a state where it is difficult to understand the study content, XIII. Subject who does a highintensity exercise continuously (who does a high-intensity exercise more than three times a week), XIV. Subject in a state that would affect the absorption of the trial supplement: impairment of intestinal absorption, sprue (syndrome with impairment of small-intestinal absorption), colitis, gastroenterological surgery, M. Whipple disease (respiratory insufficiency syndrome due to systemic bacterial infection), XV. Subject with disease that carries a risk of hypercalcemia: multiple organ granulomatous disease (sarcoidosis), tuberculosis, lymphoma, primary hyperparathyroidism, XVI. Subject with kidney stones, XVII. Subject whose creatinine clearance is 30 mL/min or less (severe renal failure), XVIII. Subject who is sensitive or allergic to dairy products, XIX. Subject using drugs: anticoagulants, steroids, parathyroid hormone, thiazide diuretics, anticonvulsants, antipsychotics, drugs for schizophrenia, drugs that affect fat absorption (peripheral anti-obesity drugs such as Xenical and Alli, fat absorption inhibitors), XX. Subject who used drugs that could affect bone metabolism (Bisphosphonate, hormone replacement therapy drugs, estrogen-receptor regulators, calcitonin) for the past one year, XXI. Subject who consumes a non-fat diet or has other extreme dietary habits, XXII. Subject who is on a weightloss program or whose diet is under the supervision of a doctor, XXIII. Subject with symptoms of acute or severe diseases: unintended weight loss, night sweats, and cancer treatment, XXIV. Subject whose alcohol consumption is 140 g/week or more, and XXV. Subject who was judged to be unsuitable for the study by the principal investigator or other investigators.
For the trial supplement, a hard capsule formulation containing 10 μg of 25-hydroxivitamin D3 (25OHD) was used. Placebo was prepared by replacing 25OHD with crystalline cellulose so that it would not be distinguished from the trial supplement by the color. 25OHD was provided for free by DSM Nutritional Products, Ltd. (Heerlen, Netherlands).
Upper respiratory tract symptom survey
For the assessments of the incidence of URTI, the physical severity, and the quality-of-life (QOL), the Japanese version of Wisconsin Upper Respiratory Symptom Survey-21 (WURSS- 21) was used (26). WURSS-21 consists of 21 questions, and the first question (0-7 Likert scale: 0 = Not sick, 1 = Very mildly, 3 = Mildly, 5 = Moderately, 7 = Severely) was answered by the study subjects every day during the study to determine the onset and the duration of URTI.
The next 10 questions (the second to the 11th) are questions about 10 physical symptoms of URTI (Runny nose, Plugged nose, Sneezing, Sore throat, Scratchy throat, Cough, Hoarseness, Head congestion, Chest congestion, and Feeling tired), and they were answered on a 0-7 Likert scale (0 = Do not have this symptom, 1 = Very mild, 3 = Mild, 5 = Moderate, and 7 = Severe) to obtain the total score as the physical severity score. The next nine questions (the 12th to the 20th) are questions about nine events associated with QOL (Think clearly, Sleep well, Breathe easily, Walk, climb stairs, exercise, Accomplish daily activities, Work outside the home, Work inside the home, Interact with others, and Live your personal life), and they were answered on a 0-7 Likert scale (0 = Not at all, 1 = Very mildly, 3 = Mildly, 5 = Moderately, and 7 = Severely) to obtain the total score as the QOL score which is reflected mental severity.
The date of occurrence of URTI was the day on which the subject selected 1-7 after selecting 0 two days in a row for the first question, and the date of disappearance was the day, which was two days prior to the time when the subject selected 0 two days in a row after the onset (the day 1-7 was selected last). The events of URTI in this study were the ones whose duration (the period from the date of occurrence to the date of disappearance) was two days or more, and whose highest physical severity score when suffering from URTI was over 10. For the duration of URTI, the physical severity score, and the QOL score, the highest values during the study were evaluated.
The study was a randomized, double-blind, placebocontrolled, parallel group comparison study. Hence, a staff who has no involvement in the study prepared an assignment list using random numbers, and after the study subjects were randomly divided into the 25OHD group and the placebo group, 25OHD and placebo were given to the two groups respectively. The subjects for blinding were all those who were involved in the study, and the assignment was unblinded after securing analysis subjects
During the study, the subjects were required to fill in the WURSS-21 and to record subjective symptoms and whether they made a deviation from instruction such as intake of medical products and health food in their journals. The duration of the supplement intake of this study was from winter (January 2016) to spring (May 2016).
A body measurement, a blood pressure measurement, a pulse measurement, clinical examinations, and the food frequency questionnaire (FFQg ver. 4.0 (Kenpakusha, Tokyo, Japan)) were performed at baseline and 16-weeks tests (27). For the clinical examinations, as a blood test, 25-hydroxyvitamin D, 1α,25 (OH)2 vitamin D, serum Ca, intact parathyroid hormone (PTH) were measured, and as a urine test, urine calcium, and urine creatinine were measured. The clinical examinations were performed by a standard method at SRL Inc. (Tokyo, Japan).
Clinical laboratory tests were conducted using a γ-counter, ARC 950 (Hitachi, Ltd., Tokyo, Japan), Modular Analytics (Hitachi, Ltd., Tokyo, Japan), and clinical chemistry analyzer, BioMajesty ™ series JCA-BM 8060 (JEOL Ltd., Tokyo, Japan). For 25-hydroxyvitamin D, the 25-hydroxy-vitamin D 125I RIA Kit (DiaSorin S. P. A, Saluggia, Italy) based on the RIA 2 antibody method was used. The CV% of the 25-hydroxyvitamin D measurement during the test period was in the range of 8.8% to 12.1%.
Primary outcome measure: The primary outcome measure was the incidence proportion of URTI for the period of 16 weeks of supplement intake. The incidence proportion of URTI was calculated by dividing the number of study subjects who developed URTI in each group by the number of study subjects in each group.
Secondary outcome measures: The secondary outcome measures were the physical severity score, the QOL score, the duration of URTI, and the incidence of new URTI every four weeks. For the physical severity score, the QOL, and the duration of URTI, the study subjects who developed URTI were assessed.
The guideline by the task team of Michael F. Holick et al. says that the risk of osteomalacia and rickets is increased in a deficiency state where the serum 25-hydroxyvitamin D level is 50 nmol/L (= 20 ng/mL) or lower, and that the state where the serum 25-hydroxyvitamin D level is between 50 nmol/mL and 75 nmol/L (= 30 ng/mL) is an insufficiency state where the risk of falling is increased (28, 29). Based on the report that a decrease in the serum 25-hydroxyvitamin D level increases the incidence of URTI, the incidence of URTI, the physical severity score, the QOL score, the duration of URTI, and the incidence of new URTI every four weeks were assessed in the study subjects whose serum 25-hydroxyvitamin D levels at the test before the supplement intake were in a deficiency state and in an insufficiency state respectively.
Exploratory efficacy analysis
As an exploratory efficacy analysis not listed in the protocol, the total physical severity score and the total QOL score were assessed. Each total score was calculated by adding all the scores when suffering from URTI during the supplement intake.
The safety assessment was conducted based on changes in laboratory values, complaints of symptoms from the study subjects, and conventional assessments for adverse events by a doctor.
The efficacy analysis was conducted on the per-protocol set (PPS) who fulfilled the protocol. The PPS was a group that excluded out of the full analysis set (FAS): I. Person whose intake rate of the trial supplement was below 80%, II. Person who had developed URTI on the start date of supplement intake, and III. Person who was judged by the principal investigator to be appropriate for being excluded from the study.
The safety assessment was conducted on the FAS.
The primary outcome measure, the incidence proportion of URTI during the period of 16 weeks of supplement intake, and a secondary outcome measure, the incidence proportion of new URTI every four weeks were assessed by Fisher’s exact test. For assessments of the other secondary outcome measures, the generalized Wilcoxon test was used for the duration of URTI, Student’s t test was used for the physical severity score and the QOL score.
The significance level of the study was set to 5% on both sides, and 10% on both sides were marginally significant. JMP® 12 (SAS Institute Inc., Cary, North Carolina, USA) was used for all the analyses.