Effect of Vitamin D Supplementation on Endothelial Function in Obese Adolescents

Vitamin D deficiency has been linked to endothelial dysfunction in adults. Obese adolescents have a high prevalence of Vitamin D deficiency as well as evidence of endothelial dysfunction. Our hypothesis is that supplementation of Vitamin D deficient adolescents with Vitamin D would lead to improvement in endothelial dysfunction.

Subjects had a brief screening visit with one of the study team members. Past medical history, current medications and a brief dietary history was taken. The dietary history was taken to assess calcium intake in a day and was obtained via the validated Short Calcium questionnaire. Blood pressure, heart rate as well as height,weight, waist and hip circumference measurement was obtained by study staff. In addition, a brief physical examination to determine Tanner stage was also completed. This involved examination of both breasts and genitalia for adolescent female and only genitalia in males. A physical activity questionnaire (IPAQ) was also administered by the study staff at this visit. A blood draw of 5 ml to measure plasma 25(OH) D, calcium, phosphorus was obtained as part of the screening visit. For girls who had started menstruating, a urine pregnancy test was also obtained. After the screening visit eligible subjects had baseline biochemical tests and an endothelial function assessment. Study participants received a pill container with six total pills of vitamin cholecalciferol (D3) (1 pill = 50,000 IU), with directions to take two pills once a month (100,000 IU) at the same time for a period of 3 months. Compliance was assessed at the 3 month visit by counting the number of pills remaining in the container. At 1 month, the subjects had tests for serum calcium and 25 hydroxy vitamin D levels. A urine test for random calcium to creatinine ratio was also obtained. At 2 months, the serum calcium, 25 hydroxy vitamin D levels, and calcium to creatinine ratio tests were repeated. At the end of 3 months, biochemical tests and an endothelial function assessment were repeated. Medical history, questionnaires, and the physical exam were also repeated.

Endothelial function was assessed by FMD, via a high-resolution Doppler ultrasonography examination of the right brachial artery. FMD was calculated as the maximal percentage increase in brachial artery diameter (BAD) from baseline after the release of cuff occlusion.

25(OH)D was measured using liquid chromatography-tandem mass spectrometry. Total 25(OH)D concentrations of each sample was calculated using internal standard, 25(OH)D_2 and 25(OH)D_3.

Body Mass Index (BMI) is a health index for comparing weight to height. BMI is a person's weight in kilograms (kg) divided by his or her height in meters squared. The body mass index is an indication if a person is at a suitable weight for his height on an approximation of body fat. A body mass index of under 20 is considered to be underweight, while a body mass index between 20 to 25 is considered healthy. A body mass index in the range of 25 to 30 is regarded as overweight. A body mass index over 30 is regarded as obese.

The IPAQ short form used asked 7 questions about activities in the last 7 days, covering vigorous physical activities, moderate activities, walking, and sitting, asking for days per week, hours per day or minutes per day. The score is reported in metabolic equivalent (MET)-minutes per week. Possible scores could range from 0 (inactive) to greater than 3000 MET-minutes/week (highly active). The definition of high activity was vigorous intensity activity on at least 3 days achieving a minimum total activity of at least 1500 MET-minutes/week OR 7 days of any combination of walking, moderate-intensity or vigorous-intensity activities achieving a minimum total physical activity of at least 3000 MET-minutes/week. Therefore a score of > 3000 MET-minutes/week was possible.

Calcium intake was measured using the validated Short Calcium Questionnaire (SCQ). This questionnaire is in the form of an spreadsheet, and asks the participant to enter the number of servings per week of various food items and vitamin or mineral supplements. The spreadsheet calculates the daily calcium intake (mg/day) from the data entered.

A parathyroid hormone (PTH) blood test measures the level of parathyroid hormone in the blood. This test is used to help identify hyperparathyroidism, to find the cause of abnormal calcium levels, or to check the status of chronic kidney disease. PTH controls calcium and phosphorus levels in the blood. PTH was measured by a two-site chemiluminescent immunometric assay.

This calculation measures insulin resistance, and requires U.S. standard units. The healthy range is 0.5 to 1.4. Less than 1.0 means the subject is insulin-sensitive, which is optimal. Above 1.9 indicates early insulin resistance. Above 2.9 indicates significant insulin resistance. The HOMA-IR was calculated as: HOMA-IR = fasting serum glucose (mmol/L) x fasting insulin (mU/mL)/22.5.

A high-sensitivity C-reactive protein (hs-CRP) test may be used to help evaluate an individual for risk of cardiovascular disease (CVD). C-reactive protein (CRP) is a protein that increases in the blood with inflammation. Studies have suggested that a persistent low level of inflammation plays a major role in atherosclerosis, the narrowing of blood vessels due to build-up of cholesterol and other lipids, which is often associated with CVD. The hs-CRP test accurately measures low levels of C-reactive protein to identify low but persistent levels of inflammation and thus helps predict a person's risk of developing CVD. hs-CRP was measured using particle-enhanced immunonephelometry.

The test for low-density lipoprotein cholesterol is used as part of a lipid profile to predict an individual's risk of developing heart disease. A desirable level is <3.36 mmol/L; borderline high is 3.36 - 4.11 mmol/L; high is >/= 4.14 mmol/L. LDL cholesterol was calculated as: LDL = Total cholesterol - HDL cholesterol - Triglycerides/5.

Total HDL cholesterol levels were measured by an enzymatic colorimetric assay. The test for high-density lipoprotein cholesterol (HDL-C) is used along with other lipid tests to screen for unhealthy levels of lipids and to determine the risk of developing heart disease. If a subject has a negative risk factor, a desirable HDL level would be >/= 1.55 mmol/L.

Urine calcium/creatinine ratio (unit mg/g) on random urine sample was calculated by dividing calcium in mg by creatinine in g.

The cuff of a sphygmomanometer was placed on the forearm and inflated to 50 mm Hg above the participant's systolic blood pressure for a period of 5 min. The increase in resting brachial blood flow was calculated as the maximum flow recorded in the first 15 seconds after cuff deflation and expressed as a percentage increase from baseline reactive. Higher values are considered normal or improved endothelial function.

Inclusion Criteria: Age 12-18 years BMI >95% for age and gender 25 (OH) D levels less than 30 ng/ml Exclusion Criteria: 25 (OH) D levels >30 ng/mL Serum calcium >10.4 mg/dL Serum phosphorus > 4.7 mg/dl Pregnancy or nursing Current cancer Patients on vitamin D3 supplementation exceeding 400 IU/day Hypertension defined as Blood Pressure over the 95th percentile for age, gender and height Dietary calcium intake exceeding 1500 mg/day Hepatic or renal disorders Type 1 or type 2 diabetes mellitus Subjects receiving insulin, metformin, or oral hypoglycemic medications Subjects with malabsorption disorders (celiac disease, cystic fibrosis, inflammatory bowel disease)

Javed A, Kullo IJ, Balagopal PB, Kumar S. Effect of vitamin D3 treatment on endothelial function in obese adolescents. Pediatr Obes. 2016 Aug;11(4):279-84. doi: 10.1111/ijpo.12059. Epub 2015 Aug 14.