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  1. 1. Journal of Pediatric Gastroenterology and Nutrition 43:234Y239 August 2006 Lippincott Williams Wilkins, Philadelphia Alanine Amino Transferase Concentrations Are Linked to Folate Intakes and Methylenetetrahydrofolate Reductase Polymorphism in Obese Adolescent Girls *†Marie-Laure Frelut, ‡Nathalie Emery-Fillon, §Jean-Claude Guilland, kHunh Han Dao, and ¶Genevie`ve Potier de Courcy *Pediatric Therapy Center, 95580 Margency, France; ÞPediatric Endocrinology, Saint Vincent de Paul Hospital, University of Paris 5, Paris, France; þBiochemistry Department, University Hospital, Vandoeuvre-le`s-Nancy, France; §Physiology Department, University Hospital, Dijon, France; kRheumatology Department, Pitie´ Salpeˆtrie`re University Hospital, AP-HP, Paris, France; and ¶INSERM U557, Scientific and Technical Institute for Food and Nutrition, ISTNA-CNAM, Paris, France ABSTRACT Objective: The objective of this study was to investigate the consequences of low dietary folate intake and the impact of the 677 CYT methylenetetrahydrofolate reductase (MTHFR) common mutation on liver function in obese adolescents. Methods: Fifty-seven obese girls (BMI = 36.1 T 6.0 kg/m2) aged 14.1 T 1.5 years were included before starting a weight reduction program. Dietary intakes for folate were assessed by means of an adapted food frequency questionnaire (n = 50). Liver enzymes, plasma lipids, glucose metabolism parameters, ferritin, homocysteine and erythrocyte folate content were measured in plasma or blood obtained under fasting con-ditions. The MTHFR 677 CYT polymorphism, which is associated with decreased enzyme activity, was determined using PCR. Body composition was assessed using dual x-ray absorptiometry. Results: Twenty-three subjects were heterozygote (CT) for the mutation and 5 were homozygote (TT). An increase in alanine amino transferase (ALT) and ALT/aspartate aminotransferase ratio was associated with the mutation (F = 4.46, P = 0.016 and F = 5.92, P = 0.0049, respectively). Alanine amino transferase was correlated negatively to folate intake (r = j0.32, P = 0.024) (n = 50) and positively to homocysteine concentrations (r = 0.30, P = 0.025). Body composition was similar among the 3 genotypic groups. Ferritin was also correlated to ALT concentrations of the entire group (P = 0.009). Conclusion: Our data suggest that folate intake and the MTHFR polymorphism represent a part of the link between antioxidant status and liver disease in obese adolescent girls. JPGN 43:234Y239, 2006. Key Words: ObesityVAdolescentsV LiverVFolateVHomocysteineVMethylenetetrahydrofolate reductase. 2006 Lippincott Williams Wilkins INTRODUCTION The risk of nonalcoholic fatty liver disease (NAFLD) is 4.6-fold higher in obese adults with a BMI more than 30 kg/m2 (1). In children and adolescents, 6% of overweight and 10% of obese adolescents have been reported to have elevated serum alanine aminotransfer-ase (ALT) levels (2), whereas up to 35% of the whole group have steatosis when assessed by ultrasound (3). The fatty liver condition is thus acknowledged as an increasing clinical problem in childhood obesity (4). Magnetic resonance imaging revealed that abnormalities in serum ALT occur exclusively in the most severe cases of fatty liver (5), which could lead to severe fibrosis and end-stage liver disease, even in children (5Y13). Thus far, however, weight reduction is the only clearly identified factor that leads to liver function improvement in children (14,15) and adults (16), although supplementing vitamin E (17) may also play a role. The molecular events that result in hepatic steatosis and liver injury are thought to result from a Btwo hit^ phenomenon. The first hit consists of deposition of triglycerides (TG) in the hepatocytes. For the disease to progress, a Bsecond hit^ is required that promotes inflammation, cell death and fibrosis that are the hallmark of nonalcoholic steatohepatitis (NASH). Antioxidants are therefore putative protective factors against the progression from simple fat accumulation to more severe conditions (18). However, conflicting results have been published regarding a link with glucose and lipid metabolism: Elevated TG and gly-cated haemoglobin (2) and hyperinsulinemia (12,13) have been reported by some authors, whereas responses to oral glucose tolerance tests were found similar among obese children with or without increased ALT concen-trations (3). Ferritin has also been reported to be a major Received March 9, 2005; accepted April 5, 2006. Address correspondence and reprint requests to Marie-Laure Frelut, MD, Pediatric Therapy Center, Obesity Unit, 18 rue Roger Salengro, PO Box 6 95580, Margency, France (e-mail: 234 Copyr ight © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  2. 2. ALT ARE LINKED TO FOLATE INTAKES AND MTHFR POLYMORPHISM 235 determinant of NAFLD in association with insulin resistance in apparently healthy obese adults (19). Nutritional factors that could contribute to increasing the risk level in children are decreased plasma concen-trations of vitamins E, A-carotene, vitamin C with antioxidant properties and even modest alcohol consump-tion (2). In animal models, folate depletion and elevated plasma homocysteine (Hcy) promote oxidative stress in the liver (20), whereas in adults suffering liver cirrhosis, Hcy has been shown to induce cellular toxicity and to play a role in the development of liver fibrosis (21). Food consumption patterns in the general population of French children and adolescents are currently shifting toward higher fat and sugar intakes (22). Obese adolescents often report a dislike of fruits and vegeta-bles and a preference for high-carbohydrateYhigh fat diets (23). Fruit and vegetables bring significant amounts of not only antioxidants and fibers but also folic acid (24). Tetrahydrofolate and its derivatives are the biolog-ically active forms of folic acid. They function as cosubstrates for a variety of enzymes associated with one-carbon metabolism. 5,10-Methylenetetrahydrofolate (MTHF) is a hydroxyl methyl group donor for thymi-dylate synthase and hence plays an important role in cell division through nucleic acid (purines and pyrimidine) synthesis. The 677 CYT missense mutation of the MTHF reductase (MTHFR) decreases its activity by approximately 20% and enhances the accumulation of Hcy (25), an independent cardiovascular risk factor (26). We previously reported that in France, approxi-mately 16% of the population living in the suburbs of Paris is homozygote (TT) for this mutation (27). We hypothesized that liver damage in obese adoles-cents might be enhanced by poor folate status or altered folate metabolism. The objective of this study was to examine whether folate consumption and biological status may be determinants of altered liver function in severely obese adolescents. MATERIALS AND METHODS Fifty-seven girls who were admitted in our unit to inves-tigate their health status before entering a weight reduction program were included. Investigations were performed accord-ing to the Helsinki 2 declaration. All participants were provided a detailed information on the purpose of the study. The parents and the adolescent signed an informed consent form approved by the Medical Ethics Committee of Robert Debre´ University Hospital. Folate intakes were evaluated in 50 out of 57 subjects using an adapted food questionnaire developed by our team as previously detailed. In 5 subjects, for whom no parental control was obtained, data were not used. Data were also missing in 2 other subjects. In the survey, the folate content of edible portions of 7 categories of food (vegetables, fruit, starchy foods, fish and meat, eggs, cheese, other dairy products) was analyzed and an adjustment made according to the average food patterns of the French population. This allowed us to estimate folate intakes and to detect high-risk subjects, that is, those who eat food with high folate content less than twice per week (28). Blood samples were taken by venous puncture in fasting conditions after an overnight fast. Erythrocyte folate (EF) content, which reflects folate body stores, was measured by a microbiological method, as previously detailed (27,28). Methylenetetrahydro-folate reductase polymorphism was determined by PCR as previously reported (27,29). Plasma Hcy was measured by HPLC (Biorad laboratories, Marnes-la-Coquette, France). Liver damage was evaluated by serum levels of aspartate aminotransferase (AST), ALT, gamma glutamyl transferase (FGT), alkaline phosphatase (AP) and 5¶ nucleotidase (30), and lipids were measured using a Hitachi 911 automat. Alanine amino transferase and AST were measured using Randoxi, FGT and AP using Roche Diagnostici and 5¶ nucleotidase was measured using BioMe´rieuxi respective methods. Total cholesterol and TG were measured by enzymatic methods (CHOD PAP and GPO PAP, respectively, Rochei). High-density lipoprotein cholesterol (HDL-C) was measured using a corresponding nonprecipitating method. Low-density lipoprotein cholesterol (LDL-C) concentrations were calculated using the Friedewald formula (LDL-C = Total cholesterol - HDL-C - TG/5) (31). Plasma glucose concen-trations were measured using the glucose oxidase method and fasting insulin concentration by radioimmunoassay using a double antibody method. Insulin resistance was estimated using the homeostasis model assessment of insulin sensitivity (HOMA IR = insulin (U/mL) glycemia (mmol/L)/22.5), which has been shown to mirror the glucose clamp technique in the assessment of insulin sensitivity. A higher HOMA IR score reflects a greater degree of insulin resistance (32). Glycated haemoglobin (HbA1c) was measured by chromato-graphic method. Ferritin was measured by immunological assay (TINA QUANT Ferritin, Roche). Body composition was assessed using dual x-ray absorptiometry by means of a Hologic QRD 1000/Wi device (version 5.11, Hologic, Inc., Waltham, Mass). Statistics were performed using Statview 5 (Abacus Concept, Inc., Berkeley, Calif). The effect of MTHFR was analyzed using ANOVA. P values for trends were calculated using the post hoc Bonferroni/Dunn test. Correla-tions were calculated using Fisher r coefficient, and unpaired comparisons were performed using Student t test. Significance level was set at 0.05. RESULTS Age, anthropometrical characteristics and body com-position data are shown in Table 1 as a function of the genotype of the MTHFR polymorphism. Five subjects were homozygote (TT) and 23 were heterozygote (CT) for the mutation. Mean ages, anthropometrical and body composition measures did not differ between the 3 genotypes. Biological characteristics are shown in Table 2. Parameters of the lipid metabolism and of glycore-gulation did not differ among the 3 groups. As expected, mean insulin values were slightly above the reference value of our laboratory (15 IU/mL), whereas all other mean values remained within normal range. J Pediatr Gastroenterol Nutr, Vol. 43, No. 2, August 2006 Copyr ight © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  3. 3. 236 FRELUT ET AL. Mean folate intake, which ranged from 104 to 412Kg/d, did not differ (P = 0.12) among the 3 groups and are below national recommended allowances (300 Kg/d) in 43 (80%) subjects (33). Erythrocyte folate was corre-lated neither to BMI (P = 0.18) nor to ferritin (P = 0.23). Alanine amino transferase levels were above the upper limit of 50 IU/mL in 3 subjects (1 CC, 2 CT subjects). Mean ALT concentrations were increased significantly in subjects displaying the mutation TT (F = 4.46, P = 0.016). 5¶ Nucleotidase was also marginally increased (F = 3.11, P = 0.054), whereas AP plasma concentrations did not differ. Alanine amino transferase concentrations were not correlated to abdominal fat mass (FM) (P = 0.50). Ferritin concentrations were G200 ng/mL in all subjects and did not differ among the 3 groups. However, calculation of P for trend showed that mean insulin levels were higher in the TT than in the CC group (P = 0.012), and ferritin levels were higher in the CT than in the CC subjects (P = 0.011). Antibodies to hepatitis A or C and cytomegalovirus were checked only in patients with elevated ALT and found to be negative in all cases. We also verified individual records on the absence of any past vacci-nations against hepatitis B before entering the program. No patient had a history of liver dysfunction. Erythrocyte folate (P = 0.12) and Hcy (P = 0.15) were similar in the 3 groups, with the exception that EF was significantly lower in TT subjects when compared with CC subjects (t = 2.28, P = 0.027). Alanine amino transferase concentrations were negatively correlated to folate intake (r = j0.32, P = 0.024) as shown in Figure 1, positively correlated to both Hcy (Fig. 2; r = 0.30, P = 0.029) and ferritin concentrations (data not shown; r = 0.57, P = 0.0007). When multiple regression is performed between ALT on one hand and ferritin, Hcy and EF on the other, as shown in Table 3, ALT is linked with ferritin (P = 0.009) and Hcy (P = 0.039), whereas a nonsignificant trend is found with EF (P = 0.16). Simple regression shows that ferritin and insulin are marginally correlated (r = 0.33, P = 0.052). DISCUSSION This is the first report in obese girls of high levels of ALT associated not only with a low intake of folate, a major methyl donor nutrient with antioxidant properties, but also with a common mutation in a key enzyme of folate metabolism, the 677 CYT mutation of MTHFR. The frequency (9%) of the TT genotype, slightly below that reported for a large sample (16%) of adults in the same area (27), is likely to be due to our sample size. Anthropometrical measurements and folate intake did not differ between the 3 genotypes. Obese adolescents have been shown to often underreport low-quality food (34). Our food frequency questionnaire did not focus on energy intake, but rather on categories of foods classified according to their folate content, and were completed with the help of a dietician and of the mother of the adolescent, all of whom were advised of the purpose of the study. Underreporting is thus unlikely to provide an explanation for these findings. Careful attention had also been paid to alcohol consumption, which could be ruled out in this population of girls. Gamma GT was normal in all and did not differ among the genotypes (data not shown). Alanine amino transferase concentrations reveal liver injury in a more consistent way than do AST, which is also present in striated muscle. 5¶ Nucleotidase reveals TABLE 2. Biological characteristics of obese girls as a function of the MTHFR polymorphism for the 677 CYT mutation CC (n = 29) CT (n = 23) TT (n = 5) ALT, IU/mL 20 T5 23 T8 34 T 11* AST, IU/mL 22 T5 23 T8 26 T 11 AP, IU/mL 131 T 78 146 T 77 127 T 56 5¶ Nucleotidase, IU/mL 3.0 T 1.7 3.6 T 1.6 5.2 T 2.3† Insulin, IU/mL 17.0 T 12.4‡ 19.6 T 9.3 24.2 T 17.0 Glycemia, mmol/L 4.42 T 0.53 4.53 T 0.48 4.62 T 0.81 HOMA IR 3.53 T 2.87§ 4.01 T 2.13 4.73 T 3.17 HbA1c, % 5.3 T 0.5 5.2 T 0.6 5.2 T 0.5 Triglycerides, mmol/L 0.93 T 0.37 0.96 T 0.30 1.14 T 0.50 Cholesterol, mmol/L 4.60 T 0.34 4.42 T 0.83 5.27 T 1.60 HDL-C, mmol/L 1.60 T 0.78 1.37 T 0.36 1.09 T 0.13 LDL-C, mmol/L 2.61 T 0.57‡ 2.73 T 0.620 3.46 T 1.73 EF, Kg/L 264 T 65 263 T 104 177 T 80 Hcy, Kmol/L 6.3 T 1.8 5.7 T 2.0 7.9 T 4.6 Ferritin, ng/mL 38 T 20 60 T 38 46 T 14 Results are expressed as mean T SD. *ANOVA for ALT: F = 4.46, P = 0.016. ***ANOVA for ALT/AST: F = 5.92, P = 0.0049. †ANOVA for 5¶ nucleotidase: F = 3.11, P = 0.054. ‡P for trend: P = 0.012 between CC and TT subjects for insulin and LDL-C. §P for trend: P = 0.067 for HOMA IR. P for trend: P = 0.011 between CC an CT subjects for ferritine. ALT, alanine amino transferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; EF, eythrocytic folate; HOMA IR, homeostasis model assessment of insulin sensitivity; Hcy, homocysteine. TABLE 1. Characteristics and body composition data in obese girls as a function of the polymorphism of the MTHFR for the 667 CYT mutation CC (n = 29) CT (n = 23) TT (n = 5) Age, y 14.5 T 1.2 14.2 T 1.7 14.7 T 1.3 Weight, kg 100.9 T 20.2 95.8 T 23.6 96.3 T 18.1 Height, m 1.65 T 0.08 1.62 T 0.12 1.64 T 0.05 BMI, kg/m2 37.8 T 6.7 36.4 T 7.1 35.9 T 6.2 Total FM, kg 40.1 T 15.3 39.9 T 17.2 36.1 T 8.7 Total LM, kg 51.5 T 8.3 47.8 T 8.4 51.2 T 13.7 Abdominal FM, kg 17.5 T 8.0 17.4 T 7.3 15.7 T 4.4 Folate intakes, Kg/d 242 T 78 240 T 76 218 T 75 Results are expressed as mean T SD. All comparisons (ANOVA) were not significant. FM, fat mass; LM, lean mass. J Pediatr Gastroenterol Nutr, Vol. 43, No. 2, August 2006 Copyr ight © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  4. 4. ALT ARE LINKED TO FOLATE INTAKES AND MTHFR POLYMORPHISM 237 cholestasis or biliary obstruction in a highly specific way (30). The increase in ALT beyond the upper references of our laboratory in 3 subjects is consistent with the slight increase in the 5¶ nucleotidase (P = 0.054) in CT and TT genotypes, although the concen-trations of the latter enzyme remained within normal range. Increased ALT concentrations in obese children (3) and in adults with ALT/AST ratio 91 (1) have been reported as a criteria of risk of severe fibrosis in NASH. However, ALT is the key liver enzyme that allows the reversible transamination of alanine to pyruvate, which can be further degraded to acetyl CoA. Alanine acts, therefore, as a source of carbon and energy within the glucose-alanine cycle that allows gluconeogenesis. Intracellular concentrations of ALT may thus be modulated by the metabolic conditions. Obesity is characterized by an insulin resistant state. Patients suffering NAFLD have been shown to have specific metabolic abnormalities, namely, a more marked hyper-insulinemia and resistance to the insulin-mediated suppression of lipolysis (12). An enhanced production of glucose and a conversion rate of glucose to pyruvate under the action of increased concentrations of insulin may, in turn, result in an increased ALT activity. Cysteine, which derives from Hcy, can also be con-verted to pyruvate. Intracellular concentrations of ALT may be further modulated by the accumulation of Hcy that results from the MTHFR 677 CYT mutation. The link between a hypothesized alteration of intracellular and plasma ALT concentrations would still have to be fully demonstrated (35). Therefore, additional studies that would include histopathological assessment, follow-up and assessment of the impact of weight loss are required to determine the predictive value and sensitivity of the early elevation of each criteria (1) and to improve the biological assess-ment of liver disease severity in this population as has been recently stated (36). Similar levels of obesity and similar total and segmental body compositions strongly suggest that neither increased total FM nor trunk FM is sufficient to individually explain the degree of abnormal liver function, although dual x-ray absorptiometry has shown some limitations in analyzing body composition in this population of severely obese adolescents, as we have shown previously (37). Although the increase in insulin levels and HOMA IR values were similar among the 3 genotypes, basal insulin concentrations tended to be higher in TT than in CC subjects (P = 0.01). Insulin resistance has been, thus far, identified in obese adults as a major risk factor of NASH (1,19). The reduction of the ability of insulin to suppress lipolysis and the release of VLDL by the liver(1,18) would lead to higher free fatty acid and TG content in hepatocytes. However, additional factors are required to explain the heterogeneity of the disease such as oxidative stress and its modulation.(1,2,4,17Y19,21) We also found that ferritin was highly correlated to ALT (P = 0.009), but not to BMI (P = 0.70) nor to EF (P = 0.16), whereas its correlation with insulin was only marginal (P = 0.053). Plasma ferritin concentrations are not only directly proportional to intracellular ferritin concentrations and iron stores (38) but also a marker of inflammation (39). Indeed, obesity can be considered an inflammatory condition (40). In rats fed diets varying in folate concentrations, the Fe2+-induced lipid peroxida-tion of liver homogenates had the strongest correlation with folate-depletion variables (20). Whether increased ferritin concentrations would enhance liver damage because of increased oxidant stress of iron is still a matter of debate (36,39). Recent data suggest that in a group of adults with a low prevalence of obesity, who are suffering NASH, increased ferritin levels are markers of severe histological damage but not of iron overload (39). In obese but otherwise healthy adults, FIG. 1. Correlation between folate intakes and plasma ALT concentrations (r = j0.32, P = 0.024). ALT, alanine amino transferase. FIG. 2. Correlation between plasma ALT and Hcy concentrations (r = 0.30, P = 0.025). ALT, alanine amino transferase; Hcy, homocysteine. J Pediatr Gastroenterol Nutr, Vol. 43, No. 2, August 2006 Copyr ight © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  5. 5. 238 FRELUT ET AL. both insulin resistance and ferritin are independent major contributors to NAFLD (19). In this group of obese but otherwise healthy population of adolescents, we did not measure other markers of inflammation. Blood counts were within normal range and the food frequency questionnaire was indicative of standard meat consumption for all subjects. Therefore, focusing on antioxidant consumption and status has a major potential interest in the young. Data in children are scarce and still conflicting but underline the protective effect of vitamins E and C, which have strong antioxidant properties (2,17). Nevertheless, folate metabolism in humans remains poorly understood (41). In animals, folate depletion and elevated plasma Hcy have been shown to promote oxidative stress in the liver by decreasing Zn-Cu super-oxide dismutase and gluthatione peroxidase activities. This results in an increased lipid peroxidation in the liver (20). The links between folate and glucose metabolism are derived, so far, from 2 sets of data obtained in animals that need to be confirmed: as stated before, intracellular methylation seems to be important to allow the neoglucogenesis derived from the reutilization of Hcy (40). Low folate intakes markedly alter exocrine pancre-atic function (41), but no data are available on pancreatic endocrine function. The conversion of Hcy into methio-nine requires the methyl group to be transferred from MTHF after its reduction by MTHFR (42). Higher levels of serum Hcy had been reported in the general population in children with low serum levels of folic acid and among nonusers of multivitamin supplements (43). In adults, mean Hcy levels were 18% lower among persons who used vitamin B supplements (44). In obese girls, insulin seems to also be an independent risk factor of hyperhomocysteinemia (45). Data from animal studies also suggest that Hcy could play an additional role in oxidative stress instead of being a simple indicator of decreased MTHF production (20). Children suffering strokes have also been found to have lower folate intakes. This group was shown to have a higher frequency of TT genotype for the 677 CYT mutation of the MTHFR. A negative correlation between Hcy and folate intake was also reported in this study (46). In conclusion, our data suggest that in this population of young obese girls, low folate intakes and MTHFR polymorphism are additional factors that may increase the relative contribution of oxidant stress on liver alteration. Further studies are required to confirm this hypothesis both from a histological point of view and also on a long-term basis. So far, only weight reduction (14Y16) and vitamin E (17) have been demonstrated to reduce ALT levels in obese children, whereas ursodeox-ycholic acid therapy proved to be unsuccessful (47). These data also provide arguments for a potential additional link that should be further explored between cardiovascular risk, eating pattern and genetic back-ground (26). Folate intakes and the impact of the common 677 C Y T MTHFR mutation require further investigation for the understanding and management of obesity and its short- and long-term complications in children and adolescents. REFERENCES 1. Angulo P. Non alcoholic fatty liver disease. N Engl J Med 2002; 346:1221Y31. 2. Strauss RS, Barlow SE, Dietz WH. Prevalence of abnormal serum aminotransferase values in overweight and obese adolescents. J Pediatr 2000;136:727Y33. 3. Guzzaloni G, Grugni G, Minocci A, et al. Liver steatosis in juvenile obesity: correlations with lipid profile, hepatic biochem-ical parameters and glycemic and insulinemic responses to an oral glucose tolerance test. Int J Obes 2000;24:772Y6. 4. Marion AW, Baker AJ, Dhawan A. Fatty liver disease in children. Arch Dis Child 2004;89:648Y52. 5. 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Multiple regression analysis of the influence of ferritin, Hcy and EF on ALT in obese girls N = 57 A (slope) SE P Adjusted R2 Dependent variable ALT 0.27 Independent variable Ferritin 0.40 0.062 0.008 Hcy 0.29 0.78 0.040 EF 0.20 0.020 0.16 ALT, alanine amino transferase; EF, eythrocytic folate; Hcy, homocysteine. J Pediatr Gastroenterol Nutr, Vol. 43, No. 2, August 2006 Copyr ight © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
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