Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia and atherogenesis

aln Universi b Pathology and Laboratory Medicine Service, VA Boston cDepartment of Pathology, Harvard Medical School, Bos a r t i c l e i n f o Article history: Received 30 December 2010 Accepted 27 April 2011 Keywords: Protein malnutrition Lean body mass constituting an international pandemic [2]. More than half of these deaths occur in developing countries where vegetarian diets consisting of several varieties of legumes and starchy foods have prevailed for millennia. The beneficial health effects of such regimens in the prevention of chronic disorders such as CVD, Plant-based regimens, however, do not optimally fulfill the nutritional requirements of population groups living in devel- oping countries, as illustrated by the increasing incidence of hyperhomocysteinemia (HHcy) states [7,8], now recognized as a critical factor in occurrence of CVD and stroke [9]. HHcy was originally associated with the pathogenesis of atherosclerosis by the study of the vascular pathology of children with inherited disorders of methionine (Met) metabolism [10]. Dietary insuffi- This work was funded by University Louis Pasteur, Strasbourg, France. Contents lists availab rit ww Nutrition 28 (2012) 148–153 * Corresponding author. Tel.: þ857-203-5990; fax: þ857-203-5623. Conclusion: The low dietary intake of protein and sulfur amino acids by a plant-eating population leads to subclinical protein malnutrition, explaining the origin of hyperhomocysteinemia and the increased vulnerability of these vegetarian subjects to cardiovascular diseases. Published by Elsevier Inc. Introduction According to World Health Organization estimates [1], 16.7 million deaths are attributable to cardiovascular disease (CVD) each year, accounting for 30% of all deaths worldwide and diabetes, and cancer have been documented by many investi- gators [3,4], explaining their growing popularity in Westernized countries during the past several decades. It is estimated that about 2% to 5% of individuals living in developed countries have adopted vegetarianism as a lifestyle choice [5,6]. Transthyretin Homocysteine Sulfur deficiency Cardiovascular disease E-mail address: [email protected] (K. S. McC 0899-9007/$ - see front matter Published by Elsevier doi:10.1016/j.nut.2011.04.009 Healthcare System, West Roxbury, Massachusetts, USA ton, Massachusetts, USA a b s t r a c t Objective: To explain why vegetarian subjects develop morbidity and mortality from cardiovascular diseases unrelated to vitamin B status and Framingham criteria. Methods: A study of 24 rural male subjects 18 to 30 y old and 15 urban male controls was con- ducted in the Sahel region of Chad. Food consumption was determined from a dietary question- naire, and overall health status was assessed by body weight, body mass index, serum albumin, plasma transthyretin, urinary nitrogen, and creatinine. Plasma lipids, vitamins B6, B9 and B12, homocysteine, and related sulfur amino acids were measured as selected cardiovascular disease risk factors. Results: Body weight, body mass index, blood, and urinary markers of protein status were signif- icantly lower, with an estimated 10% decrease of lean body mass in the study group compared with urban controls. Neither lipid fractions nor plasma levels of vitamins B6, B9, and B12 were significantly different between the two groups. Although the mean consumption of sulfur amino acids (10.4 mg$kg�1$d�1) by rural subjects was significantly below the recommended dietary allowances (13 mg$kg�1$d�1), plasma methionine values were similar in the two groups. In contrast, homocysteine concentration was significantly increased (18.6 mmol/L, P < 0.001), and the levels of cysteine and glutathione were significantly decreased in the study group, demonstrating inhibition of the trans-sulfuration pathway. The strong negative correlation (r ¼ �0.71) between transthyretin and homocysteine implicated lean body mass as a critical determinant of hyperhomocysteinemia. Yves Ingenbleek M.D. , Kilm a Laboratory of Nutrition, Faculty of Pharmacy, . McCully M.D. ty Louis Pasteur, Strasbourg, France a er S b,c,* Applied nutritional investigation Vegetarianism produces subclinical m and atherogenesis Nut journal homepage: ully). Inc. utrition, hyperhomocysteinemia le at ScienceDirect ion w.nutr i t ionjrnl .com ciency or malabsorption of any of three water-soluble B vitamins / Nu Materials and methods Study participants and controls The study was conducted in 2009 in the Sahel region of southern Chad, about 400 km from the capital city of N’Djamena. The Sahel belt is a shrubby countryside, which extends across Africa from Senegal to Somalia, and is locally populated by the Sara ethnic group. The men are physically active farmers engaged in sowing, irrigation from well water, and harvesting. In this semiarid area, the staple foods are cassava, sweet potatoes, beans, millets, and ground nuts. Cassava leaves, cabbages, and carrots provide adequate carotenes, folates, and pyridoxine, but contain little protein or SAAs. The lack of satis- factory grazing land explains why livestock are undernourished and provide few dairy foods. Meat is rarely eaten, but poultry and eggs are occasionally consumed. The study was approved by the Ministry of Public Health and by regional health authorities. From 47 candidates, 24 men 18 to 30 y old in satisfactory health were recruited from three remote rural hamlets and examined for health status, dietary habits, and anthropomorphic parameters. Women were excluded because of the difficulty in collecting valid urine specimens in an animist pop- ulation. Alcoholism (1 of 47), smoking (3 of 47), medications (4 of 47), micro- albuminuria (2 of 47), and cytokine-induced overproduction of acute-phase reactants (13 of 47) were exclusion criteria. In our clinical experience, the combined measurement of C-reactive protein (CRP; >10 mg/L) and of a1-acid glycoprotein (>800 mg/L) allows the identification of nearly all viral, bacterial, fungal, and parasitic infestations [20], notably hepatitis B, human immunodefi- ciency virus, tuberculosis, and malaria, which have a significant prevalence in this area. CRP and a1-acid glycoprotein inflammatory markers were determined using immunoassays with, in the case of CRP, high-sensitivity Denka reagents. The minimally detectable CRP threshold was 0.03 mg/L and the interassay coefficient of variation (CV) was 4%. Our 24 rural participants and 15 control subjects had a1-acid glycoprotein values below 700 mg/L. Their high-sensitivity CRP values were 0.34 � 0.41 and 0.23 � 0.32 mg/L, respectively, with no statis- tically significant difference between groups (P > 0.05). Identification of micro- albuminuria was accomplished by testing a morning urine specimen. The control participants consisted of 15 healthy men 18 to 29 y old, belonging to the same (B6, pyridoxal-50-phosphate; B9, folates; or B12, cobalamins) regulating the Met–homocysteine (Hcy) cycle are regarded as key determinants of acquired HHcy [11]. In particular, cobalamin deficiency constitutes a worldwide problem [12] and is currently regarded as the principal causal factor of HHcy in vegetarian populations [7,8] because vitamin B12 is almost totally absent from plant foods. In contrast, folates are abundant in plant foods, and pyridoxal-50-phosphate is ubiquitous in unprocessed foods from plant and animal sources. Nevertheless, some studies undertaken in developing areas have shown that HHcy status may develop despite normal B12 status [13], prompting the search for alternative explanations. A possible causal factor for HHcy in vegetarian populations is related to the observation that most plant foods contain lower concentrations of protein and essential amino acids, especially lysine and Met, than animal foods [14]. White beans, for example, contain about half the sulfur amino acids (SAAs; 422 mg/100 g of edible portion) contained in bovine meat (930 mg/ 100 g) [15]. As a result, the Met provided by vegetarian diets does not meet the tissue requirements of mammalian species [15]. Clinical investigations conducted in subclinically malnourished patients [16,17] and recent experimental studies of the mouse [18] and rat [19] models have revealed that restriction of dietary Met intake inhibits the trans-sulfuration cascade and leads to HHcy, irrespective of cobalamin status. The present field study was undertaken to investigate the importance of dietary vitamin B12 and Met deficiency in the development of HHcy. The results explain how the altered body composition of vegetarian subjects affects SAA metabolism and produces HHcy, a risk factor for CVD in vegetarian populations. Y. Ingenbleek, K. S. McCully Sara ethnic group but residing in an upper-class urban environment. The diet of the control group was similar to that of the study group except for occasional consumption of bovine meat, canned or powdered milk, and smoked fish. Dietary and health assessments A questionnaire assessing detailed information regarding family status, occupation, and dietary habits was recorded from the 24 subjects who success- fully completed the screening protocol. During the week before collection of blood and urine samples, the typical daily portion sizewas provided for each food item and expressed in grams. The results were compared with values reported in food composition tables [21,22]. The data allowed the calculation of daily consumption of energy, fiber, carbohydrate, lipid, protein, and SAAs after adjustment for body weight (BW). Body mass index (BMI) was calculated as BW (kilograms) divided by height (meters) squared. Venous blood samples obtained after overnight fast were centrifuged to yield plasma and serum that were frozen at �20�C and shipped on dry ice. A 24-h urine sample was collected into sterile polyvinylchloride flasks under night and day supervision of field staff, and, after volume measuring, an aliquot was stored at �20�C for determination of urinary nitrogen by the Kjeldhal method and creatinine by the Jaff�e method, allowing estimates of total body nitrogen turn- over rate and muscle mass, respectively. Specimen analysis Serum cholesterol was determined by an enzymatic photometric assay (CHOD-PAP method) using a Hitachi 917 analyzer (Roche, Basel, Switzerland). Serum high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were determined by homogeneous enzymatic colorimetric assays (Roche). The between-run CVs were 1.4% for cholesterol, 1.9% for LDL, and 2.3% for HDL. Serum triacylglycerols were assayed by the GPO-PAP method (Advia Trig, Bayer, Leverkusen, Germany), and the CV was 1.4%. Plasma Hcy was determined using a reverse-phase high-performance liquid chromatographic method [23]. The detection limit was �0.5 mmol/L, and the between-run CV was 5.5%. Plasma cysteine (Cys) was determined after depro- teinization of plasma by sulfosalicylic acid and chromatography on a Beckman 121MB Amino Acid Analyzer (Beckman-Coulter, Villepinte, France) using a single column and a sequence of lithium citrate buffers of increasing pH. Glutathione (GSH) was determined by a high-performance liquid chromatographic method [24]. Plasma vitamin B12 and folates were determined by chemoluminescence (ACS 180, Bayer Vital). The between-run CVs for vitamin B12 and folates were 6.9% and 5.8%, respectively. Plasma pyridoxine was determined by high-perfor- mance liquid chromatography by fluorometric detection, with a CV of 5.1%. Statistical analysis All values are presented as mean � standard deviation. For normal data, differences between study and control participants were compared using the Student t test. For non-normal data such as values for plasma Hcy and vitamins B6, B9, and B12, median values were calculated. The Mann-Whitney test, a non- parametric procedure, was used to compare the median values between the study and control groups. Linear regression models were applied to determine the relation between variables. Spearman rank correlation coefficients (r) eval- uated the relations and degree of significance (P) between the different variables. Differences with P � 0.05 were considered statistically significant. Results Food consumption differed between the study and control groups, as assessed from the food questionnaire (Table 1). Rural vegetarian participants consumed a comparable energy intake, but they consumed less protein and fat and more carbohydrate and fiber than the urban omnivorous partici- pants. The control group consumed a well-balanced dietary regimen with 54.1% of total energy as carbohydrate, 32.6% as fat, and 13.3% as protein, close to the recommended dietary allowances for each nutrient class, in contrast with the significantly (P < 0.05) larger proportion of carbohydrate (63.1%) but less fat (25.7%) and less protein (11.2%) consumed by the study group. The consumption of SAAs (10.4 mg$kg�1$d�1) by the study group was significantly (P < 0.001) lower than the control group (14.6 mg$kg�1$d�1). Measurements of anthropometric and biochemical markers of health status of the study group were significantly lower than those of the control group (Table 2). The study group was char- trition 28 (2012) 148–153 149 acterized by normal height but significantly (P< 0.05) lower BW, BMI, and serum albumin (SA) and much lower plasma transthyretin (TTR; P < 0.001) compared with the control group. Despite an apparently satisfactory state of health, plasma TTR values of all rural subjects were �240 mg/L (Fig. 1), indicating that an element of protein malnutrition was present in all members of the study group. The lower excretion of urinary nitrogen and creatinine by the study group reflected lower protein consumption and decreased lean body mass (LBM) and muscle mass, respectively, compared with the control group. There was no difference in serum levels of triacylglycerols, The Spearman correlation coefficients among plasma Hcy, nutritional indicators, and dietary variables (Table 3) indicated Fig. 1. Relation of plasma homocysteine and transthyretin. Regression line analysis plots total plasma homocysteine and plasma transthyretin values in participants with protein malnutrition (diamonds) and in controls (circles). Decreased plasma transthyretin values are negatively correlated (r ¼ �0.71) with increased plasma homocysteine values in the study group, indicating that a decrease of lean body Table 3 Table 1 Characteristics of food consumption Nutrient classes Study group Control group P (n ¼ 24) (n ¼ 15) Energy (MJ/d) 7.33 (6.1–8.4) 7.22 (6.4–8.9) NS Fiber (g/d) 73.4 (62–97) 49.4 (38–73) a significant association of decreased plasma TTR, urinary nitrogen, and creatinine with Hcy in the study group but no association, except for plasma TTR, in the control group. Corre- lations between BMI and SA with Hcy were not significant for either group. There was no correlation of serum triacylglycerols, cholesterol, HDL, or LDL with Hcy in either group. The data in Figure 1 show a strongly negative correlation (r ¼ �0.71) between plasma TTR and Hcy in the study group. The data in Figure 2 show a strongly negative correlation between plasma Cys and Hcy (r ¼ �0.67) in the study group but not in the control group. Discussion The blood lipid profiles of rural subjects were within normal ranges (Table 2), the likely result of low fat and high fiber intakes and strenuous physical activity, ruling out these parameters as causal risk factors for CVD. The normal concentrations measured for vitamins B6, B9, and B12 (Table 2) excluded deficiencies of these micronutrients as determinants of HHcy. In contrast, the Y. Ingenbleek, K. S. McCully / Nu Fig. 2. Relation of plasma homocysteine and cysteine. Regression line analysis plots total plasma homocysteine and plasma cysteine values in participants with protein malnutrition (diamonds) and in controls (circles). Decreased plasma cysteine values consumption of a protein-deficient diet caused significant alterations in body composition (Table 2). All members of the study group had significantly lower BW, BMI, SA, and plasma TTR levels (Table 2, Fig. 1) than the control group, despite an appar- ently good state of health. Recent observations have demon- strated that LBM constitutes a critical determinant of Hcy status in healthy subjects [25] and in diseased patients [26]. There exist close relations between the evolutionary patterns of TTR and LBM values in health and disease [26]. The Foundation for Blood Research (Scarborough, ME, USA) recently published TTR results in 68 720 healthy US citizens 0 to 100 y, constituting reference data for evaluating the relation of LBM fluctuations with TTR in relation to sex and age [26]. Decreased SA was a less sensitive index of protein malnutrition than TTR [27]. Nevertheless, because LBM represents about two-thirds of BW [26], the concomitant lowering of BW, BMI, SA, and TTR concentrations and of urinary nitrogen and creatinine excretions (Table 2) were characteristic biomarkers of subclinical protein malnutrition, reflecting an average decrease of LBM by 10% in the study group. Calculation of the height–creatinine index was established by are negatively correlated (r ¼ �0.67) with increased plasma homocysteine values in the study group, indicating that downregulation of cystathionine-b-synthase activity is dependent on the bioavailability of methionine. comparison of 24-h creatinine excretion of the rural participants versus 24-h excretion of control subjects of the same height. The normal height–creatinine index is 1.0, and this ratio progres- sively decreases as the muscle mass decreases toward sarcope- nia. In our study, the rural group had a height–creatinine index of 0.83 (17% lower than controls). A decrease of LBM by about 10% suggested that muscle mass was more severely protein depleted than the visceral organs in the study group. The consumption of 10.4 mg$kg �1$d�1 of the two SAAs (approximately two-thirds Met and one-third Cys [15]) by the study group was below the recognized recommended dietary allowance of 13 mg$kg�1$d�1 [21] for adequate nutrition. Despite the low consumption of SAAs, plasma Met levels were the same in the two groups, contrasting with increased Hcy (18.6 mmol/L) and decreased Cys and GSH concentrations in the study group (Table 2). These data indicate that maintenance of Met plasma concentration is mandatory for the synthesis and func- tioning of many Met-dependent metabolic processes described in detail elsewhere [15,28]. These changes in SAA metabolism demonstrate downregulation of the trans-sulfuration pathway at the cystathionine-b-synthase (CbS) level, producing increased Hcy in biological fluids and increased methylation of Hcy to Met. This remarkable adaptive response was first observed during a field study conducted in 1986 in Senegal [16] and was later confirmed in clinical studies [17] and animal experiments [18,19]. These observations show that dietary Met deprivation causes post-transcriptional downregulation of CbS activity without a decrease in hepatic S-adenosylmethionine [18], the classic allosteric regulator of this enzyme [29]. The strong negative correlation (r ¼ �0.71) found between TTR and Hcy, also repor- ted in previous studies [15,26,28], indicates that decreased LBM is the primary determinant of HHcy in malnourished individuals [26]. Because the concentrations of nitrogen and sulfur are closely correlated in body tissues [15,26], a decrease in LBM causes concomitant shrinking in total body nitrogen and in total body sulfur that is proportional to the severity of protein malnutrition. The data are consistent with mouse [18] and rat [19] experiments in displaying a BW loss of 33% and 44%, respectively, at the end of the Met-deprivation period. We postulate that depletion of total body sulfur and of total body Met, its principal constituent, activates an adaptive feedback loop downregulating CbS activity [15]. The significantly low plasma Cys levels in the study group (33.4% of control value; Table 2, Fig. 2) may be explained by lower dietary SAAs (Table 1), a decreased release of Cys from protein catabolism in malnutrition [30], and a decreased conversion of Met to Cys because of decreased CbS activity. The significantly low plasma GSH level in the study group (62.8% of control value; Table 2) is in part explained by the almost complete absence of GSH from cereals, roots, and dairy products [31] and by the restriction of Met and Cys intake, reflecting primary substrate limitations [32]. Cys undergoes reversible GSH conversion and is catabolized to hydrogen sulfide (H2S) using alternate reactions catalyzed by two pyridoxal-50-phosphate–dependent enzymes, CbS and cystathionine-g-lyase [33]. Our data show that the most decreased Cys and GSH values were found in subjects with the lowest TTR concentrations and decreased LBM, reflecting the most marked protein-depleted states. Cys is a limiting substrate for the production of GSH and H2S, suggesting that decreased availability of Cys, GSH, and H2S causes a loss of their reducing properties in proportion to the decrease in LBM. Besides nitrogen oxide and carbonmonoxide, H S has been recently recognized as trition 28 (2012) 148–153 151 2 the third gaseous signaling molecule that mediates vasodilation effects [34]. The reducing property of H2S functions as a major [18] Tang B, Mustafa A, Gupta S, Melnyk S, James SJ, Kruger WD. Methionine- deficient diet induces post-transcriptional down-regulation of cys- [24] Toyo’oka T, Uchiyama S, Saito Y, Imai K. Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with / Nu scavenging agent against HHcy-induced accumulation of reac- tive oxygen species [35]. Of additional importance is the discovery that Cys and GSH participate in the non-enzymatic conversion of elemental sulfur (S8) into H2S [36]. This converting process may be impeded in the case of insufficient dietary S8 intake and underlying malnutri- tion, causing decreased Cys and GSH bioavailability. Pedologists and agronomists have demonstrated for many decades that S8 is an essential element in plant nutrition, because deficiency of S8 produces growth retardation, decreased chlorophyll synthesis, and decreases in fertility and crop yields [37]. Because plant foods supply inadequate SAAs for mammalian tissue require- ments [15,28], consumption of foods with imbalanced nitro- gen:sulfur ratios grown in S8-deficient agricultural soils will exacerbate protein malnutrition [38]. S8 deficiency is a world- wide soil problem affecting more than half the countries producing mainly foodstuffs of plant origin, including rice, soybeans, wheat, maize, and oilseeds [37]. In contrast to the status of plant agronomy, the mammalian requirements for elemental S8 are presently unknown. Only very recently has the essentiality of sulfur has been recognized in human nutrition, causing Hcy elevation in deficient subjects [39]. The Indian subcontinent, notably the northern region extending from Pakistan to Bangladesh, is one of the areas with soil that is most severely deficient in S8 [40], contributing to the public health problem of CVD. More than 40% of Indian patients with HHcy-induced CVD are refractory to combined administration of folates and cobalamins [41], supporting the concept of S8 defi- ciency as the key etiologic factor. The concept of sulfur deficiency may explain why many clinical trials using B vitamin interven- tions have proved ineffective in the prevention of recurrent vascular disease in patients with advanced CVD, cerebrovascular disease, renal failure, and inflammatory disorders [42]. Many other countries in southeastern Asia with vegetarian lifestyles, such as China, Taiwan, Indonesia, Philippines, Pakistan, and Sri Lanka, are also affected by increasing CVD morbidity and mortality [7–9,37]. These developments call attention to the need to redefine CVD risk factors in South Asia [43] and explain why cardiologists from five of these countries, Bangladesh, India, Pakistan, Nepal, and Sri Lanka, have exhorted their governments to focus more attention on CVD epidemiology [44]. We assume that our Chadian data are representative of the health status of most vegetarian African populations [13,16,17]. The first African report showing that HHcy is significantly asso- ciated with ischemic stroke came from a study carried out in the Sahel region of Nigeria, close to the Chadian border [45]. In that study, 50 adult patients had a clinical diagnosis of cerebral infarction confirmed by computerized tomographic scanning. At the onset of thrombovascular injury, HHcy (20.8 mmol/L) was found in all patients, revealing a slightly higher concentration than that found in our apparently healthy Sara farmers (18.6 mmol/L). The impact of our observational study is limited because of the modest number of participants in the study and control groups, resulting from the difficulty in selecting valid candidates who fulfill the requirements of field study conditions. Additional larger investigations in vegetarian populations with protein malnutrition and dietary S8 deficiency in other regions of the world are needed to confirm the present findings. In conclusion, our investigation shows that a vegetarian population consuming a diet deficient in SAAs is characterized by normal B vitamin status, normal serum lipids, subclinical Y. Ingenbleek, K. S. McCully152 protein malnutrition, and decreased LBM, leading to an adaptive downregulation of the trans-sulfuration pathway, fluorescence detection. Anal Chim Acta 1988;205:29–41. [25] Battezzatti A, Bertoli S, San Romerio A, Testolin G. Body composition: an important determinant of homocysteine and methionine concentrations in tathionine b-synthase. Nutrition 2010;26:1170–5. [19] Elshorbagy AK, Valdivia-Garcia M, Refsum H, Smith AD, Mattocks DA, Perrone CE. Sulfur amino acids in methionine-restricted rats: hyper- homocysteinemia. Nutrition 2010;26:1201–4. [20] Ingenbleek Y, Carpentier YA. 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Association of plasma homo- cysteine and ischaemic stroke in a Nigerian population. Pak J Med Sci 2006;22:405–8. Y. Ingenbleek, K. S. McCully / Nutrition 28 (2012) 148–153 153 Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia and atherogenesis Introduction Materials and methods Study participants and controls Dietary and health assessments Specimen analysis Statistical analysis Results Discussion Acknowledgments References