Effectiveness of Vitamin D Supplementation

This pilot study will examine the effectiveness of vitamin D supplementation on reducing
blood pressure and improving endothelial function. If results of the pilot study are
favorable, findings will be used to design a possibly larger study to confirm results.
Premenopausal African American women (n = 40) will be recruited through Houston Methodist
Hospital Internal Medicine Clinic and by flyers distributed at TWU. Potential participants
interested in the study will contact the Investigator (C. Moore) for further information.
All participant study visits will be held at the Houston Methodist Internal Medicine Clinic
or at the TWU Nutrition Laboratory. Interested individuals will be scheduled for the initial
screening visit. African American women will be recruited who are not taking hypertensive
medications, have average blood pressure measurements < 140/90 mmHg, are not pregnant or
planning on becoming pregnant in the next three months, or who are not lactating. To be
eligible, participants will have a physical completed within a year and will free of known
cardiovascular disease, diabetes mellitus, and latex allergies. They will be excluded with
known Crohn's, inflammatory bowel disease, previous bowel resections, bariatric surgeries,
or psoriasis. They will not be able to participate if they have artificial or long
fingernails they do not want to cut for testing, or are regularly taking medications that
might affect endothelial function such as anti-inflammatory medications, statins,
anti-hypertensive medication, daily aspirin, non-SSRI psychotropic medication, and dietary
supplements including vitamin D supplements.

Enrollment Visit The day prior to the enrollment visit potential subjects will fast
overnight and refrain from consuming any caffeinated products, vitamins, or medications that
may affect vascular tone and refrain from smoking. the investigators will explain the 10
week study design and obtain participant consent. Testing will be performed in the morning
with start times between 8 and 10:00 a.m. In a quiet and temperature-controlled room (72° to
75° F), initial systolic and diastolic blood pressure measurements following 5 minutes of
seated rest following the American Heart Association Blood Pressure Guidelines. The arm will
be supported at heart level and neither participant nor observer will talk during the
measurements. Three successive measurements will be collected with an automated
oscillometric sphygmomanometer and the average of the last two measurements will be
calculated. Any individual with an average blood pressure > 140/90 mmHg will be referred to
their personal physician for follow up.

Consented participants with blood pressure readings <140/90 mmHg will be instructed to
consume their usual diet for the next week and to complete a three-day diet record of two
week days and one weekend. Instructions on how to record the dietary diary will be given.
Food records will be used to estimate usual dietary intake for the baseline and 10 week time
points. An appointment will be scheduled for the participant to return with completed food
records to begin the intervention study. At the baseline clinic visit a 10 week supply of
vitamin D3 supplements (2,000 IU/day; all the same lot; Nature Made ®) will be given to
participants and log sheets provided to record supplement intake. In addition, participants
will be instructed to avoid traveling to sunny locations for 10 weeks.

Baseline and 10 week clinic visits

Nutrient intake:

Three-day diet records will be collected during the baseline and 10 week clinic visits. Food
records will be analyzed at TWU utilizing the University of Minnesota 2015 Nutrient Data
System for Research. Total energy intake; percent calories from fat, protein and
carbohydrates; and intake of vitamins A, C, D, E and beta carotene will be estimated from
food records.

Blood pressure measurements:

Participants will fast overnight and refrain from consuming any caffeinated products,
vitamins, medications that may affect vascular tone, or smoking. Testing will be performed
in the morning with start times between 8 and 10:00 a.m. In a quiet and
temperature-controlled room (72° to 75° F), initial systolic and diastolic blood pressure
measurements will be taken following 5 minutes of seated rest following the American Heart
Association Blood Pressure Guidelines. The arm will be supported at heart level and neither
participant nor observer will talk during the measurements. Three successive measurements
will be collected with an automated oscillometric sphygmomanometer and the average of the
last two measurements will be calculated.

Body fat and adiposity measures:

Anthropometric and body fat distribution assessments will be performed. Height and weight
will be measured. Percent body fat will be determined by bioelectric impedance (Tanita
BF-350). Body mass index (BMI) will be calculated as weight in kilograms (kg) divided by
height squared (m2).

Endothelial pulse amplitude measurements:

Pulse amplitude tonometry (PAT) will be measured with the EndoPAT 2000 (Itamar Medical Ltd)
which records digital pulse wave amplitude (PWA) using fingertip plethysmography (16). The
Endo-PAT finger probe consists of a thimble-shaped sensor cap that imparts a uniform
pressure field and exhibits a clamp-like effect on the entire surface of the distal phalanx
and measured pulsatile volume changes. PWA is measured continuously during three phases: a
quiet baseline period, 5-minute forearm occlusion, and reactive hyperemia following cuff
release. Occlusion of the brachial artery is performed on the non-dominant upper arm. The
continuous monitoring of blood volume reaching the finger tips allows the hyperemic response
to be quantified as a ratio, comparing PWA from pre to post occlusion. A reactive hyperemia
index (RHI) measures nitric-oxide dependent changes in vascular tone. The recordings from
the non-occluded arm serve as a control for changes in overall physiological state. The
Endo-PAT RHI index is defined as the ratio of the average PWS during the 1 minute period
beginning after exactly 90 second to reactive hyperemia compared with the average pulse
amplitude during the 210 second pre-occlusion baseline period. Endothelial dysfunction is
classified as a RHI < 1.67 with average normal endothelial RHI values ranging between 1.7
and 2.0.

Vitamin D measurements:

Blood will be drawn by Houston Methodist Hospital laboratory for measurement of serum
25(OH)D concentrations by chemiluminescence immunoassay( Abbott Architect). Vitamin D
deficiency will be defined as serum 25(OH)D levels less than 20 ng/mL (50 nmol/L) and
vitamin D insufficiency as 21 to 29 ng/mL (50 to 80 nmol/L).

Statistical analyses:

Descriptive statistics, including means and standard deviations for continuous variables and
frequencies and percentages for categorical variables, will be calculated for study
variables. Age, BMI, blood pressure, serum 25(OH)D concentration, and RHI will be treated as
continuous variables. Prior to conducting primary analyses, exploratory and preliminary
analyses will be conducted to test the statistical assumptions of planned analyses.
Specifically, these assumptions include normality, homogeneity of variance, equivalent group
size, and data that is missing by random. In the event that any of these assumptions have
been violated, the data will either be transformed to meet the parametric assumptions of
planned analyses or non-parametric equivalencies will be conducted as an alternative. In
addition, correlation analyses, including Pearson's product moment correlations for normally
distributed variables and Spearman's rho for non-normally distributed variables, will be
conducted to examine the variables for potentially collinear relationships, which may impact
primary analyses.

To answer the primary research questions, within subjects repeated measures analysis of
variance (ANOVA) tests will be conducted to test if differences in blood pressure, serum
25(OH)D concentrations, and RHI are statistically different after 10 weeks of
supplementation with 2,000 IU/d of vitamin D. Furthermore, exploratory multivariate linear
regression models will be constructed to determine relationships between vitamin D status
and vascular function parameters (blood pressure, RHI) before and after adjustment for age
and BMI. All statistical analyses will be performed using Statistical analyses SPSS (version
19; SPSS IBM) and p < 0.05 will be used to determine statistical significance.

A priori power analyses was conducted using G*Power (v. 3.0.10) in order to determine an
adequate sample size to detect statistical significance if significance in fact exists. In
order to obtain a desired level of power at .80, estimating a moderate effect size (.25) and
an alpha level of .05, a total of 34 participants will be needed to test the within subjects
effects in a repeated measures ANOVA. Accounting for attrition of approximately 15%, a total
of 40 participants will be recruited. Repeated measures-ANOVAs will be utilized to detect
significant changes across the following outcome variables: serum 25(OH)D, blood pressure,
and RHI. As such, a total of 40 participants should be sufficient to detect significance if
significance in fact exists across all planned analyses.

Inclusion Criteria:

- Healthy pre-menomausal African American women

Exclusion Criteria:

- History of elevated blood pressure >140/90 mm Hg

- Pregnant or planning to become pregnant in the next three months

- Lactating

- Known Crohn's

- Inflammatory bowel disease

- Previous bowel resections

- Bariatric surgeries, or psoriasis

- Artificial or long fingernails they do not want to cut for testing

- Regularly taking medications that might affect endothelial function such as
anti-inflammatory medications, statins, anti-hypertensive medication, daily aspirin,
non-SSRI psychotropic medication, and dietary supplements including vitamin D
supplements