Sample size determination was done at the household level. This involved enrollment of equal number of men and woman pairs in each group. A total of 30 (15 were men and 15 women) in the placebo group and 30 (15 were men and 15 women) in the lysine group was arrived at. However, the sample size was increased to 45 males and 45 females to give 90 participants in each group making a total of 180 from 90 households containing one man and one woman from each household [4].
Exactly 428 households made up of 856 individuals were screened for eligibility. To be eligible, participants were supposed to be between the ages 18–45 years and should not be on any anti-hypertensive medications. Of these (428), 90 households totaling 180 individuals of 90 men and 90 women were recruited. Each household was randomly placed in the lysine or placebo group. Randomization was done at the household level to prevent supplements (lysine or placebo) from mixing between household members. The study was a double-blind and the supplements were identical in appearance.
The study protocol was approved by Ethical Review Board of Noguchi Memorial Institute for Medical Research (FWA 00001824-IRB000176). Informed consent form was signed by each recruited household head. The intervention (lysine supplemented) group consumed 1000 mg of lysine per day in two divided doses for a period of 112 continuous days.
Based on the calculations of Pellett [2], it is noted that at 41.1 mg lysine /g protein, lysine availability in Ghanaian diets is low. Table 1 shows the supplementary lysine required to complete the protein quality. An extra 15.2 mg lysine/g protein or 900 mg of lysine per day was needed to supplement the diets of Ghanaians. The lysine was provided in tablet form. Each subject was supplied two tablets of lysine-HCl (each tablet equivalent to 500 mg lysine per tablet or 500 mg of di-calcium phosphate as a placebo) per day for 112 days. Supplements were formulated by Disto Pharmaceuticals, in Hyderabad.
Table 1 Lysine requirement and addition calculation for Ghana
Lysine mg/g Protein from the formula: Lysine values in milligrams per day were calculated from the equation: Lysine = (86.3 × APg/day) + (19.8 × CPg/day) + (63.6×PSg/day) + 599 where AP, CP and PS were animal, cereal and pulse-soy protein respectively. Lysine values in milligrams per gram of protein were derived by further dividing by the amount of total protein [5] FAO/FAOSTAT (2004) (Adapted from Pellett 2004).
Information collected at starting point included: dietary, anthropometry and blood pressure (BP) measurements. A repeated 24-h dietary recall was done for 3 non-consecutive days for each subject including one weekend to obtain a representative data on dietary intake. For the 24 h recall, the participants were asked to mention all the foods they had consumed in the past 24 h prior to the interview. With the help of food models they were made to estimate the quantities or portions they consumed and with the help of food composition tables, their nutrient intake was estimated Blood plasma lysine is known to be poor indicator of dietary lysine status thus only dietary information was used to determine lysine levels [8].
Dietary data were converted into nutrient content using Ghanaian Food Composition Tables and [9] supplemented by US Department of Agriculture Table [10], owing to absence of amino acid data in the ‘Ghanaian Food Composition Tables’. Nutrient examination included type of protein (animal, legume, vegetable and roots and tubers), utilizable protein levels based on the PDCAAS (Protein Digestibility Corrected Amino Acid Score) method and expressed as total utilizable protein per day and utilizable protein g/kg body weight, lysine, sulphur amino acids, threonine and tryptophan (mg/day and mg/g protein), total energy, carbohydrate and fat.
Adequacy of nutrients intake was assessed using FAO/WHO Recommended Dietary Allowance (RDA) and Estimated Average Requirements (EAR) [5]. Adequacy of nutrient intake were determined employing the EAR cut-point method [11] to evaluate the nutrients adequacy of subjects. The population prevalence of inadequacy of a given nutrient is the proportion of the population with intakes below the EAR [12]. Nutrients intakes were compared to the RDAs for each subject. Protein intakes were categorized by sources namely cereal protein, legume protein, animal protein, protein from fruits and vegetables, protein from roots and tubers, and protein from other plant sources. Energy intakes were expressed as the percentage contributions from the various sources namely carbohydrate, protein and fats.
All the anthropometric measurements including weight and height were collected using standard procedures. Weight and height were taken using the weighing scale and Stadiometer respectively. Body Mass Index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Clinical assessments involved the determination of BP before and after supplementation/intervention. Blood pressure was recorded using a digital sphygmomanometer. Here, the participants were made to relax and sit comfortably. It was made sure that participants had recently emptied their bladder prior to the measurement. Tight sleeved clothing were removed and lose ones were rolled up. They were made to rest in a chair next to a table for about 5–10 min. Their left arm was gently positioned at heart level with palm facing up. Seated up straight with legs uncrossed and forearm on the table, the arm cuff was wrapped around it. The start button of the sphygmomanometer was pressed and the reading were recorded with alongside with the date and time after the automated process. All measurements were done in triplicates and their averages were used. A subject was classified as hypertensive if SBP ≥140 mmHg (systolic BP). The design of the monitoring was such that the tablets were provided on a weekly basis during the intervention period.
Epi-Info 2000, MS Excel 2007 and SPSS version 16 were used for data analyses. Statistical analysis included comparisons between groups for differences in diets, anthropometry and blood pressure. Data analysis was carried out using paired and independent t-test for comparison of measured parameters followed by analysis of variance (ANOVA). Changes in BP (Delta) values were calculated for both men and women groups (lysine and placebo). The chi-square test was used for comparison of categorical variables between the two treatment groups. Significance level was set at 0.05. An unpaired t-test was employed to compare the lysine-supplemented and the placebo-supplemented subjects. The paired t-test was used to compare the delta of baseline and posttest data of all categories (normotensive, and hypertensive).