Effectiveness of a Micronutrient Supplement to Lower Plasma Homocysteine MDEG2 Pilot Supplementation Trial

In rural Gambia women experience considerable variation in their diet by season. In the dry season women have particular micronutrient deficiencies that can disrupt one-carbon metabolism pathways. In the dry season women have lower levels of most of the B vitamins and higher levels of homocysteine compared to the rainy season. The goal of this trial is to test two nutritional interventions to see which one works best in providing micronutrients in the ratio and quantity necessary for optimal 1-carbon metabolism in the dry season. The primary end point is to assess which supplement is most effective in reducing plasma homocysteine.

This is a 3-arm randomized controlled trial, unblinded, with 125 women per arm. Non-pregnant, non-lactating healthy women of reproductive age in West Kiang will be randomized to 12 weeks of daily supplementation of either a) novel micronutrient drink powder supplement, b) existing available micronutrient supplement (UNIMMAP) or c) no intervention (control). The novel drink powder provides 800 µg folic acid, 5.2 µg cyanocobalamin (B12), 2.8 mg Riboflavin-5'-phosphate (B2), and 4g trimethylglycine (betaine). This dose is twice the Recommended Daily Allowance for folic acid, B12 and B2. The UNIMMAP tablet provides 15 micronutrients (vitamins A, D, E, B1, B2, B6, B12, C, Niacin, Folic Acid, Iron, Zinc, Copper, Iron, Selenium) at the Recommended Daily Allowance level.

Potentially eligible participants will be identified through the Keneba Health and Demographic Surveillance System in all 35 villages of West Kiang region of The Gambia. Field assistants will visit the homes of potentially eligible participants to provide full information about the purpose and methods of the study, potential risks and benefits, and participants' rights. Full inclusion and exclusion criteria will be assessed. Participants will then be asked to provide a signed or thumb-printed informed consent before being enrolled into the study.

The supplements will be supplied to participants on a daily basis by Community-based Birth Companions (CBCs). The novel drink powder will be provided in daily sachets and dissolved in 200ml water. UNIMMAP will be provided in capsule form to be taken with water. The CBCs will observe consumption of the supplement. Participants will be given 7 coloured cards at the start of each week. Every day they will give the CBC one card and receive their supplement. Each week a field assistant will collect data on compliance by recording card collection in liaison with the CBC.

Women will provide one 10mL fasted venous blood sample at baseline and another after 6 and 12 weeks of supplementation. At each time point they will also have their blood pressure and anthropometry (weight and height) assessed and provide a urine pregnancy test. Women will be brought to the Medical Research Council Gambia (MRCG) field station at Keneba for the baseline, 6 week (mid-line) and 12 week (end-line) data collection visits. Participants will be followed-up after the intervention period for a further 3 weeks to monitor any adverse effects.

The fasted 10mL baseline, 6 week and 12 week blood samples will be taken by venepuncture into EDTA monovettes and kept on ice. Within one hour of collection the samples will be processed by the laboratory in Keneba to centrifuge the samples, separate the plasma and store plasma and red blood cell aliquots at -70°C. One plasma aliquot will be analysed in Keneba to measure homocysteine using the Cobas Integra 400 Plus analyser.

The hypothesis is that the novel micronutrient supplement will reduce plasma by at least 1 µmol/L compared to the control group after 12 weeks of daily supplementation. It will be more effective in reducing plasma homocysteine than existing UNIMMAP tablets.

This trial is powered to study to detect a decrease of 1 µmol/L homocysteine with 80% power and 95% confidence. The trial statistician will use a linear regression model to determine the mean difference between intervention and control homocysteine at 12 weeks adjusted for baseline homocysteine, week 12 age and week 12 body mass index.

Inclusion criteria

• Pre-menopausal women aged 18-45 years

• Non-pregnant, confirmed by pregnancy urine test at eligibility screen
• Non-lactating (at least 9 months post-partum)
• No plan to conceive in the ensuing 3 months, asked verbally by field worker
• No plans to travel
• Healthy with no current illness and no chronic health problems, asked verbally by field worker

Exclusion criteria

• Known history of chronic illness (particularly cardiovascular disease, renal disease, thyroid disease, cancer)

• Taking B vitamin or multivitamin supplements.
• Taking medication for prevention of seizures (e.g. Carbamazepine).