Study title: Demographic and Seasonal Evaluation (DACE-D) of 1,25-OH Vitamin D Levels in Canadians across Provinces and Territories

Participants: Canadians interested in participating in research to learn more about vitamin D concentrations in their demographic

Investigators: Dr. N.A. Karrow (PhD, Immunotoxicolgy and Nutritional Immunology)

Dr Byram Bridle (PhD, Viral Immunology and Cancer Biology) and Dr. B. Mallard (PhD, Immunogenetics)

Contact information: E-mail: customersupport@immunoceutica.ca

Sponsor name: ImmunoCeutica Inc. (ICI) - Labs and Technologies

Conflict of interest: Dr. Mallard is a founder, majority shareholder, and Chief Executive Officer of ImmunoCeutica Inc. Dr. Karrow is a founder, shareholder, and the Chief Science Officer of ImmunoCeutica Inc. Dr. Bridle is a founder and Chief Operations Officers of ImmunoCeutica Inc. All are also professors at the University of Guelph.

Introduction: Vitamin D has been shown to have a significant impact on overall health, beyond its role in calcium absorption and bone health. Vitamin D supports the immune system, dampens inflammation, promotes oral health, and helps prevent disorders such as diabetes and depression.^1,2,3,4,5,6

Recommended Dosage: The current recommended daily dose of vitamin D by Health Canada (1,000 IU) may not be sufficient to optimize protective immunity or to promote optimal levels of the biologically active form of vitamin D (1,25-OH Vitamin D)^{8,9,10,11,12,13,14}. Vitamin D (1,25-OH Vitamin D) is assessed by the presence of its precursor (25-OH vitamin D) in most studies. Individual vitamin D concentrations are dependent on various factors such as age, Body Mass Index (BMI), skin tone, genetic makeup, diet, and sunlight exposure.

Additional Factors: Many of the models used to access vitamin D do not account for other physiological factors and needs experimental validation. People who live in northern latitudes, such as Canada, are specifically prone to low vitamin D because of low-level light exposure, particularly in the winter months.

Summary of the Problem being Addressed: Most Canadians are unaware of their vitamin D concentrations and may or may not be taking vitamin D. People supplementing with vitamin D often do not test their blood vitamin D levels, so they have no way to establish if their supplementation is helpful or not. Adequate vitamin D is associated with improve health. Therefore, there is a crucial need to determine the levels of vitamin D in Canadians across various demographics and seasons.

References:

1. Ao T, Kikuta J, Ishii M. The Effects of Vitamin D on Immune System and Inflammatory Diseases. Biomolecules. 2021 Nov 3;11(11):1624.

2. Botelho J, Machado V, Proença L, Delgado AS, Mendes JJ. Vitamin D deficiency and oral health: a comprehensive review. Nutrients. 2020;12(5):1471.

3. Lemieux P, Weisnagel SJ, Caron AZ, et al. Effects of 6-month vitamin D supplementation on insulin sensitivity and secretion: a randomised, placebo-controlled trial. European Journal of Endocrinology. 2019;181(3):287-299.

4. Aless, Cuomo R, Giordano N, Goracci A, Fagiolini A. Depression and vitamin D deficiency: causality, assessment, and clinical practice implications. Neuropsychiatry. 2017;7(5):606–614.

5. Menon V, Kar SK, Suthar N, Nebhinani N. VitaminD and Depression: A Critical Appraisal of the Evidence and Future Directions. Indian J Psychol Med. 2020 Jan 6;42(1):11-21.

6. Lin L, Zhang L, Li C, Gai Z, Li Y. Vitamin D and vitamin D receptor: new insights in the treatment of hypertension. Curr Protein Pept Sci. 2019;20(10):984-995.

7. Huang Z, You T. Personalise vitamin D3 using physiologically based pharmacokinetic modelling. CPT Pharmacometrics Syst Pharmacol. 2021 Jul;10(7):723-734.

8.Shi JW, Wu JN, Zhu XY, Zhou WH, Yang JY, Li MQ. Association of serum 25-hydroxyvitamin D levels with all-cause and cause-specific mortality among postmenopausal females: results from NHANES. J Transl Med. 2023 Sep 16;21(1):629.

9. Ames, Bruce N., William B. Grant, and Walter C. Willett. 2021. "Does the High Prevalence of Vitamin D Deficiency in African Americans Contribute to Health Disparities?" Nutrients 13, no. 2: 499.

10. Chen T-B, Chang C-M, Yang C-C, Tsai I-J, Wei C-Y, Yang H-W, Yang C-P. Neuroimmunological Effect of Vitamin D on Neuropsychiatric Long COVID Syndrome: A Review. Nutrients. 2023; 15(17):3802.

11.  Groves NJ, John J. McGrath and Thomas H.J. Burne. Vitamin D as a Neurosteroid Affecting the Developing and Adult Brain. Annu Review Nutrition. 2014. 34:117.

12. Song D, Deng Y, Liu K, Zhou L, Li N, Zheng Y, Hao Q, Yang S, Wu Y, Zhai Z, Li H, Dai Z. Vitamin D intake, blood vitamin D levels, and the risk of breast cancer: a dose-response meta-analysis of observational studies. Aging (Albany NY). 2019 Dec 28;11(24):12708-12732.

13. Lowe, L.C. Michelle Guy, Janine L. Mansi, Clare Peckitt, Judith Bliss, Rosalind Given Wilson, Kay W. Colston, Plasma 25-hydroxy vitamin D concentrations, vitamin D receptor genotype and breast cancer risk in a UK Caucasian population, European Journal of Cancer, Volume 41, Issue 8,

2005, Pages 1164-1169.

14. McDonnell SL, Baggerly CA, French CB, Baggerly LL, Garland CF, Gorham ED, et al. Breast cancer risk markedly lower with serum 25-hydroxyvitamin D concentrations ≥60 vs <20 ng/ml (150 vs 50 nmol/L): Pooled analysis of two randomized trials and a prospective cohort. 2018. PLoS ONE 13(6).

Hypotheses: Canadians living at latitudes that are more northern will have a greater probability of having lower vitamin D, particularly in winter months. Appropriate seasonal testing and supplementation with vitamin D will bring vitamin D concentration to more optimal levels.

Objectives: The principal objective of this study is examine the impact of various factors such as BMI, skin tone, age and latitude on vitamin D concentrations of Canadians. The study will also seek to develop a personalized seasonal vitamin D3 dosage regimen based on a research study questionnaire, as well as individual 25-OH vitamin D baseline values.

Vitamin D will be measured in a blood droplet either: 1 - through a home-based dried blood spot collection, ship-in-mail and ELISA test results, or 2 – through an on-site lateral flow rapid vitamin D test kit.

The study will assess the impact of different daily oral doses of vitamin D3 on the improvement of 25-OH vitamin D levels in the circulation over time and correlate these results with factors such as BMI, skin tone, age and latitude where they are located. Additionally, ICI's proprietary Vitamin D serum improvement model will be tested experimentally based on vitamin D regimen, BMI and age.

Study Procedures: A small blood droplet obtained by self-capillary puncture from participants will either be: 1 - spotted at home on a specialized paper membrane, allowed to air dry and shipped to the Immunoceutica lab to determine vitamin D by an Enzyme-Linked Immunosorbent Assay (ELISA), or

2 - the small blood droplet will be mixed with a specialized buffer and applied directly to a lateral flow rapid test kit. The test utilized for each participant will be determined based on availability of the test and location within Canada.

Risk and Benefits: There are no expected risks to participants from the donation of a capillary drop of blood utilizing a small lancet designed and approved for this purpose. It is similar to the skin prick test used to determine insulin values at home or in a health practitioner’s office.

The identities of study participants will be entirely confidential and not revealed in any reports. However, the blinded results from the vitamin D questionnaire and the vitamin D test values will be analyzed to help inform future best practices for vitamin D supplementation of Canadians depending on age, BMI, and a variety of other predictive factors.

Unless required by law, any participant identifying information and blood sample will only be shared with a third party that has confidentiality agreements in place with ICI in a formalized collaboration, and they will further protect the identity of the participants. ICI confirms that the blood sample will not be used for any other form of analysis, including DNA sequencing or genetic testing. The participants have the right to withdraw consent, and their data, at any time without giving reasons and without any penalty.

Participants will be informed of the test results showing the range of vitamin D concentration in their sample.

Expected Outcomes: This research has the potential to contribute to a better understanding of the relationship between vitamin D and overall health of Canadians, as well as to inform personalized vitamin D supplementation for optimal health outcomes.

Compensation: There is no monetary compensation for being a participant in this study and there will be a cost applied to participants to cover the expenses associated with this research. This cost will be made known to the participant prior to enrolling in the study.

Independent Institutional Review Board Approval: This study has not been reviewed by a third party outside of ICI, but has been given thorough in-house review by the team of experienced researchers at ICI. The team of internal reviewers at ICI who have approved this study have designed, expedited and reported on hundreds of research studies over the course of their careers.