Plasma Homocysteine Concentrations in a Healthy Population Living in Burkina Faso

CURRENT THERAPEUTIC RESEARCH@ VOL 61, No 9, SEPTEMBER 2000 Plasma Homocysteine Concentrations in a Healthy Population Living in Burkina Faso Jacques Simpork, Salvatore Pignatelli, Sergio Barlati,* Mariano Malaguarnera,3 and Salvatore Musumeci4 Centre Medical St Camille, Ouugadougou, Burkina Faso, 2Department of Genetics, University of Bresciu, Brescia, 3Department of Internal Medicine, University of Catania, Catania, and 4Paediatric Department, University of Sassari, Sassari, Italy ABSTRACT Background: The low circulating levels of homocysteine observed in a South African black population have been associated with a low occurrence of coronary heart disease Objective: The aim of this study was to determine whether the observation of reduced homocysteine levels in South African black subjects is valid in a black population living in West Africa Methods: Plasma homocysteine and cysteine levels were determined by high-performance liquid chromatography in 171 healthy black adults of different tribal groups and 28 healthy black Mossi children, all living in Ouagadougou, Burkina Faso The same determinations were made in 67 healthy white adults and 25 healthy white children living in Ouagadougou The black subjects habitually consumed a diet of millet flour with vegetable sauce, whereas the white subjects consumed a Mediterranean-type diet consisting of pasta and tomato sauce, providing adequate amounts of calories and VihUIIiIlS Results: Plasma homocysteine levels were lower in the black adults and children, particularly the females, than in the white subjects However, no difference between males and females was observed in either white adults or children Conclusions: These results showed that the previously observed difference in homocysteine metabolism in black South Africans also exists in the black population of Burkina Faso, particularly in females of all ages This difference between black and white people living in the same area could be due to genetic and environmental factors, even if the role of diet cannot be excluded The lower plasma homocysteine levels in black people may play a protective role against coronary artery disease by reducing endothelial damage Key words: homocysteine, black people, Burkina Faso (Curr Ther Res CZin Exp 2000;61:659-668) Address correspondence to: S&&ore Musumeci, Paediatric Department, University of Sassari, Viale San Pietro no 12, Sassari, Italy Accepted for publication July 12, 2000 Printed in the USA Reproduction in whole or part is not permitted OOll-393x@o/$1900

CURRENT THERAPEUTIC RESEARCH@ INTRODUCTION Plasma levels of homocysteine are the result of an interplay between congenital and environmental factors Homocysteine is the demethylated derivative of methionine, an essential amino acid derived from dietary and recycled endogenous proteins Fifty percent of intracellular homocysteine is remethylated to methionine, and the remainder may be transsulfurated to cystathionine through a reaction catalyzed by the vitamin Bsdependent cystathionine p-synthase Cystathionine then forms cysteine, which is required for the synthesis of many compounds, including glutathione, the most important thiol Several studies carried out in the past 2 decades have demonstrated that mild to moderate hyperhomocystinemia may be associated with a higher risk of coronary and other vascular diseases2-5 The effect on thrombotic risk of reducing plasma levels of homocysteine through supplementation of vitamins Bs, Bi2, folic acid, and others has not been clearly demonstratedg8 A study of plasma homocysteine levels in a South African black population has demonstrated that the lower circulating levels of homocysteine observed in this population were associated with a low occurrence of coronary heart diseaseg Burkina Faso, formerly known as Upper Volta, is a country in West Africa near the Niger River with a population of 11 million living in an area of 274,200 km2 The inhabitants of Burkina Faso are distributed among 8000 villages, although 27% of the population currently lives in urban centers, the result of a strong movement from the country over recent years The landlocked nature of this region and its difficult environmental conditions have served to keep the genetic composition of the population largely unchanged The southern savanna in which Ouagadougou is located is inhabited by the Mossi people, whereas the arid Sahel contains a variety of ethnic groups, including the Songhrai s, Mellebe, Peuhl, and Tuaregs The economic system is based on agriculture, which involves >95% of the working population The rural living conditions of this population differ from those of the largely urban South African population The subjects in the present study consumed a traditional diet and lived in a depressed area with a high incidence of malaria and intestinal parasites The reduced levels of plasma homocysteine seen in black people living in South Africa suggest the existence of an imbalance in homocysteine metabolism in black compared with white people that is confirmed by the low increase in plasma homocysteine observed after oral methionine loading l1 If racial differences can explain these lower levels of homocysteine, then the higher levels of this amino acid reported in black people living in the United States, with their Western lifestyle and greater incidence of obesity, support a role for environmental factors12 660

J SIMPO& ET AL The aim of the present study was to measure circulating plasma homocysteine levels in healthy black and white subjects living in Burkina Faso, a depressed area of Africa with a high incidence of malaria, traditional nutritional habits, and a low incidence of coronary heart disease SUBJECTS AND METHODS Selection and Screening of Subjects Subjects were recruited at random from a population of 1200 patients visiting the Centre Medical St Camille in Ouagadougou for a basic health screening Exclusions included refusal to undergo more laboratory tests; weight >97th percentile for the subject s age; systolic blood pressure >2 SDS above normal for the subject s age; clinical evidence of atherothrombotic cardiovascular disease, diabetes mellitus, renal or hepatic disease, thyroid disease, or cardiomyopathy; use of anticonvulsant medication; chronic alcohol abuse; and the presence of other pathologies associated with increased plasma homocysteine levels (eg, low levels of folate or vitamin B& Written informed consent was obtained from all study participants The study protocol was reviewed and approved by the ethics committee of the Centre Medical St Camille Measures of subjects health status and organ function were recorded at the beginning of the study, including body weight, body mass index (BMI) (weight in kg/height in m2), and an electrocardiogram Routine laboratory tests were also performed Using a standard protocol, blood pressure was measured in triplicate in both arms with the subject in a seated position Other measures included heart rate (measured at the radial artery) and ventilation rate Clinical chemistry tests were performed in the medical center laboratory using standard methods These included hemoglobin electrophoresis, osmotic fragility of erythrocytes, and serum folate and vitamin Bi2 determinations, as required Blood Sampling Blood samples were collected at the end of the screening interview Ten milliliters of peripheral blood was collected, 5 ml in plain tubes and 5 ml in EDTA The tubes containing blood in EDTA were centrifuged at 15009 for 10 minutes at 4 C, whereas the tubes containing blood without additive were left to stand at room temperature for 30 minutes Plasma and serum were then separated and stored at -80 C (in 25O+L aliquots) Measurement of Plasma Homocysteine High-performance liquid chromatography was used to determine levels of circulating plasma homocysteine and cysteine13 661

CURRENT THERAPEUTIC RESEARCH@ Statistical Analysis Results are expressed as medians and ranges, or means * SD The SDS of the data from all groups were analyzed using the Student t test Statistical significance was set at P c 005 RESULTS The study enrolled 291 subjects with normal results on the health screening Subjects were separated into 2 groups by race The first group included 171 healthy black adults (41 males, 130 females) aged from 17 to 60 years (median, 26 years) from different tribal groups (151 Mossi, 7 Gaurussi, 5 Bisse, 4 Dagara, 4 Peuhl) and 28 healthy black Mossi children (14 males, 14 females) aged from 4 to 24 months (median, 11 months) The second group was composed of 67 healthy white adults (30 males, 37 females) aged from 10 to 60 years (median, 29 years) and 25 healthy white children (12 males, 13 females) aged from 6 to 24 months (median, 12 months) All subjects were from Ouagadougou and had conventional dietary habits The black subjects habitually ate cereals, millet flour with vegetable sauce, seasonal fruits, and, once a week, chicken, pork, mutton, or beef The white subjects ate a Mediterranean-type diet that included pasta and tomato sauce, beef or pork every day, cooked vegetables, and seasonal fruits, with adequate intake of calories and vitamins Results of the clinical and laboratory investigations showed all subjects to be in good health, with a BMI between 23 and 28 mg/dl and blood pressure, heart rate, and ventilation rate all in the normal range for their age (c95th percentile) Serum total cholesterol was 1782 * 666 mg/dl in black subjects and 1965 f 682 mg/dl in white subjects, a nonsignificant difference Triglyceride levels were also higher in white subjects (1037 * 389 mg/dl) compared with black subjects (979 * 316 mg/dl) The hemoglobin (Hb) level was significantly lower in black women compared with white women, a difference not observed between black and white males Hemoglobin electrophoresis demonstrated that 70 of 199 subjects were carriers of hemoglobinopathy (Hb S or Hb 0, but none were homozygous or heterozygous for both Levels of folic acid and vitamin B,, were within the normal range (folic acid, 152 f 67 ng/ml in black subjects and 136 f 58 ng/ml in white subjects; vitamin Bis, 4252 f 464 pg/ml in black subjects and 4189 + 441 pg/ml in white subjects) Mean homocysteine values were lower in black adults than in white adults (Table I> Black males had higher levels of homocysteine compared with black females No such difference between the sexes was observed in the white subjects Plasma homocysteine levels were also lower in black children than in white children (Table II) Furthermore, a statistically significant difference in homocysteine levels was observed between male 662

J SIMPOd ETAL Table I Homocysteine and cysteine levels in black and white adults living in Burkina Faso, by sex Values were measured as mean * SD Homocysteine (limol/l) Cysteine (pmol/l) Black adults (n = 171) Male (n = 41) Female (n = 130 6) White adults (n = 7) Male (n = 30 4 Female (n = 7) 412 f 230 8524 A 2628* 615 f 327*+ 9253 f 3524 359 f 173* 8321 * 2252 861 * 320 11354 f 2000 898 * 361 11823i1714 826 f 277+ 10924 i 2432 * P < 0001, black versus white ad&s (Student t test) + P < 0001, black males versus black females (Student t test) * P < 0001, black females versus white females (Student t test) and female black children (P = 002) Levels of cysteine were significantly lower in black adults than in white adults (Table I), with no significant difference between males and females in either group As with homocysteine levels, cysteine levels were lower in black children than in white children, with no significant difference between males and females As expected, a positive correlation was found between homocysteine and cysteine levels in both black adults (Figure 1) and black children (Figure 2) Homocysteine levels in black adults and children are compared with those in white adults and children in Figures 3 and 4 Considering the black subjects of Mossi origin, who were more numerous, no significant difference in mean homocysteine (427 f 250 kmol/l) or cysteine levels (8613 f 2632 pmol/l) was observed between them and black subjects of ethnic groups that were less well represented No significant correlation was found between subjects age and plasma homocysteine or cysteine levels DISCUSSION In the past 10 years, elevation in plasma homocysteine levels has been widely studied as an independent risk factor for atherosclerosis Homocys- Table II Homocysteine and cysteine levels in black and white children living in Burkina Faso, by sex Values were measured as mean * SD Black children (n = 28) Male (n = 14) Female (n = 13) Homocysteine (vmolr) 411 i 206* 661 A 423*+ 369 * 123 855 f 326 877 f 354 782 f 255 Cysteine (smol/l) 5572 * 3196* 5343 * 2126* 5772 zt 2381 9362 f 1905 10045 * 1853 9021 * 2124 * P < 0001, black versus white children (Student t test) + P = 002, black males versus black females (Student t test) 663

CURRENT THERAPEUTIC RESEARCH@ I:, 5 * 2 c _ : f 6- s E 4 I 2-01 ** -- - : _* w* * *, 0 20 40 60 60 100 120 140 160 160 Cysteine (pmol/l) Figure 1 Correlation between homocysteine and cysteine levels in 171 black adults tinemia, an inborn error of metabolism, was first described in 196214,15 This rare disorder, for which a homozygous defect in the enzyme cystathionine P-synthase is the most frequent etiology, produces multiple abnormalities, including virulent atherosclerotic plaque formation and widespread arterial and venous thrombosis, usually resulting in death as early as the first decade16 Similar vascular pathology observed in patients with elevated plasma homocysteine levels secondary to disparate enzymatic =6 E _ 5-3 $4 $3 E 0 2- + I l- - O,, 0 20 40 60 60 100 120 140 160 Cysteine (pmol/l) Figure 2 Correlation between homocysteine and cysteine levels in 28 black children 664

J SIMPOR~ ETAL Ir SS Figure 3 Homocysteine and cysteine concentrations in 171 black adults, by sex, as a proportion of those in white adults Homocys = homocysteine; T = total; M = male; F = female Whiie Chikiren Figure 4 Homocysteine and cysteine concentrations in 28 black children, by sex, as a proportion of those in white children Homocys = homocysteine; T = total; M = male; F = female 665

CURRENT THERAPEUTIC RESEARCH@ defects suggests that homocysteine is the causative agent Moreover, the findings in fraternal and identical twins suggest that homocysteine levels may be genetically controlled17 A study in a large population of American adolescents and adults has demonstrated that homocysteine concentrations are lower in females than in males in various racial and ethnic groups; and significantly lower in Mexican American females than in non-hispanic white and Hispanic black females A significant interaction between homocysteine level, age, and sex was seen in this study, with homocysteine levels in females diverging from those in males at a younger age and converging with those in males at an older age These observations confirm previous observations in black traditional people from South Africa9 and support our findings of a difference in homocysteine concentrations between males and females and between black and white subjects In another study, 6 weeks of supplementation with 10 mg of folic acid, 400 kg of vitamin Bis, and 10 p,g of vitamin Bs reduced mean fasting homocysteine concentrations in white subjects, whereas this effect was less evident in black subjects, suggesting a genetic factor Few studies to date have examined the risk associated with elevated plasma homocysteine levels in women1g920 However, like premenopausal white women with the same metabolic status, premenopausal black women living in the United States and having a high BMI, hypertension, greater consumption of saturated fat and cholesterol, and high levels of lipoprotein(a) have been shown to have high plasma homocysteine levels and an increased risk of coronary heart disease21 This suggests that environmental conditions may have a major or minor impact on the genetic factors that regulate plasma homocysteine levels The plasma homocysteine concentration in white subjects is positively skewed with age Furthermore, children with a positive parental history of coronary artery disease have significantly greater age-adjusted mean homocysteine levels than do those without such parental history22 Individuals with high plasma homocysteine concentrations probably acquire this characteristic during or after young adulthood, because the plasma homocysteine frequency distribution is normal in white children Diet may have been an important factor in our study, since there were several differences in the composition of the meals consumed by black and white subjects The black diet consisted primarily of millet flour and vegetables (beans and potatoes), whereas the white diet consisted of pasta, rice, and meats Both consumed sufficient amounts of local seasonal fruits to ensure similar intake of calories and vitamins Moreover, when we consider that black people living in the United States, where the Western lifestyle is conducive to obesity, have elevated levels of plasma homocysteine,12 it is probable that diet, as well as genetic and racial factors, may influence circulating homocysteine concentrations 666

J SIMPORk ET AL CONCLUSIONS The results of this study confirm previous observations that black people are biochemically less responsive to atherogenic factors than are white people and that these differences are present since infancy23 The lower incidence of coronary artery disease observed in black people living in Africa9 may be a consequence of the protective role of modified homocysteine metabolism on endothelial damage Acknowledgments The authors thank Centre Medical St Camille for its participation throughout the investigation They also thank RADIM, Pomezia, Italy, and remember Dr Franc0 Sparano with gratitude for economic support They acknowledge the economic assistance of the Conference Episcopale Italienne References: 1 Striver CR, Beaudet AL, Sly WS, Valle D The Metabolic and Molecular Bases of Znherited Disease 7th ed New York McGraw-Hill; 199512791327 2 Mayer EL, Jacobsen DW, Robinson K Homocysteine and coronary atherosclerosis JAm Co11 Cardiol 1996;27:517-527 3 Refsum H, Ueland PM, Nygard 0, Vollset SE Homocysteine and cardiovascular disease Annu Rev Med 1998;49:31-62 4 Neufeld EJ Update on genetic risk factors for thrombosis and atherosclerotic vascular disease Hematol Oncol Clin North Am 1998;12:1193-1209 5 Prasad K Homocysteine, a risk factor for cardiovascular disease Znt J Angiol 1999;8: 7686 6 van den Berg M, Franken DG, Beers GH, et al Combined vitamin B, plus folic acid therapy in young patients with arteriosclerosis and hyperhomocysteinemia J Vast Surg 1994;20:933-940 7 den Heijer M, Brouwer IA, Bos GM, et al Vitamin supplementation reduces blood homocysteine levels: A controlled trial in patients with venous thrombosis and healthy volunteers Arterioscler Thromb Vast Biol 1998;18:356-361 8 Ubbink JB, Vermaak WJ, van der Merwe A, et al Vitamin requirements for the treatment of hyperhomocysteinemia in humans J Nutr 1994;124:1927-1933 9 Ubbink JB, Delport R, Vermaak WJ Plasma homocysteine concentrations in a population with a low coronary heart disease prevalence J Nutr 1996;126(Suppl 4):1254S- 12578 10 Ubbink JB, Vermaak WJ, Delport R, et al Effective homocysteine metabolism may protect South African blacks against coronary heart disease Am J Clin Nutr 1995;62:802-808 11 van der Griend R, Haas FJ, Duran M, et al Methionine loading test is necessary for detection of hyperhomocysteinemia J Lab Clin Med 1998;132:67-72 667

CURREN THERAPEUTIC RESEARCH@ 12 Giles WH, Croft JB, Greenlund KJ, et al Total homocysteine concentration and the likelihood of nonfatal stroke: Results from the Third National Health and Nutrition Examination Survey, 1988-1994 Stroke 1998;29:2473-2477 13 Vester B, Rasmussen K High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum Eur J Clin Chem Clin Biochem 1991;29:549-554 14 Carson NA, Neil1 DW Metabolic abnormalities detected in survey of mentally backward individuals in Northern Ireland Arch Dis Child 1962;31:653-676 15 Gerritsent T, Vaugh JC, Waisman HA The identification of homocysteine in the urine Biochem Biophys Res Commun 1962;9493-496 16 Mudd SH, Skovby F, Levy HL, et al The natural history of homocystinuria due to cystathionine beta-synthase deficiency Am J Hum Genet 1985;37:1-31 17 Reed T, Malinow MR, Christian JC, Upson B Estimates of heritability of plasma homocysteine levels in aging adult male twins Clin Genet 1991;39:425-428 18 Greenlund KJ, Srinivasan SR, Xu JH, et al Plasma homocysteine distribution and its association with parental history of coronary artery disease in black and white children: The Bogalusa Heart Study Circulation 1999;99:2144-2149 19 Sheahan R, Graham I, Refsum H, et al Women with coronary artery disease: Lower folate and higher homocysteine Eur Heart J 1994;15:530-532 20 Cooper BA, Rosenblatt DS Inherited defects of vitamin B,, metabolism Annu Rev N&r 1987;7:291320 21 Gerhard GT, Sexton G, Malinow MR, et al Premenopausal black women have more risk factors for coronary heart disease than white women Am J Curdiol 1998;82:1040-1045 22 Jacques PF, Rosenberg IH, Rogers G, et al Serum total homocysteine concentrations in adolescent and adult Americans: Results from the third National Health and Nutrition Examination Survey Am J CZin Nutr 199969482-489 23 Vermaak WJ, Ubbink JB, Delport R, et al Ethnic immunity to coronary heart disease? Atherosclerosis 1991;89:155-162 668