Increasing evidence suggests that altered methionine/folate metabolism may contribute to the development of hepatic injury. We addressed the question of whether folic acid (FA) supplementation can affect serum alanine aminotransferase (ALT) level in hypertensive Chinese adults.
A total of 480 participants with mild or moderate essential hypertension and without known hepatic disease were randomly assigned to three treatment groups: (1) enalapril only (10 mg, control group); (2) enalapril–FA tablet (10 mg enalapril combined with 0.4 mg of FA, low FA group); and (3) enalapril–FA tablet (10 mg enalapril combined with 0.8 mg of FA, high FA group), once daily for 8 weeks.
This report included 455 participants in the final analysis according to the principle of intention to treat. We found a significant reduction in ALT level in the high FA group (median (25th percentile, 75th percentile), −0.6 (−6.9, 2.0)IU/l, P=0.0008). Compared with the control group, the high FA group showed a significantly greater ALT-lowering response in men (median ALT ratio (ALT at week 8 to ALT at baseline; 25th percentile, 75th percentile): 0.93 (0.67, 1.06) vs 1.00 (0.91, 1.21), P=0.032), and in participants with elevated ALT (ALT>40 IU/l) at baseline. There was no difference in ALT lowering between the control and the low FA group.
Compared with treatment using 10 mg of enalapril alone, a daily dose of 10 mg enalapril combined with 0.8 mg of FA showed a beneficial effect on serum ALT level, particularly in men and in participants with elevated (>40 IU/l) ALT.
Aminotransferase levels are sensitive indicators of liver cell injury. Aspartate aminotransferase (AST) is found primarily in the heart, liver, skeletal muscle and kidney, whereas the highest level of alanine aminotransferase (ALT) is found in the liver, and levels of this enzyme are accordingly more specific indicators of a variety of hepatic disorders (Pratt and Kaplan, 2000). Most importantly, ALT offers a valuable screening test to detect otherwise unapparent liver disease, such as non-alcoholic fatty liver disease, which represents an epidemic that remains largely undiagnosed (Kim et al., 2008). Furthermore, emerging data also suggest that ALT (both within the normal range and elevated) may serve as a good predictor of a range of health outcomes including the long-term development of multiple metabolic disorders, cardiovascular disease and mortality (Kim et al., 2004; Schindhelm et al., 2007; Ford et al., 2008; Goessling et al., 2008; Sato et al., 2008; Yun et al., 2009).
Although it is generally recognized that folate deficiency accompanied by elevated homocysteine concentration is causally related to cardiovascular diseases (Wald et al., 2002), its role in hepatic injury is uncertain. Increasing evidence suggests that altered methionine/folate metabolism may contribute to the development of hepatic injury. Folate deficiency may accelerate or promote hepatic injury by increasing hepatic homocysteine and S-adenosylhomocysteine concentrations decreasing hepatic S-adenosylmethionine and glutathione concentrations increasing lipid peroxidation and decreasing global DNA methylation (Purohit et al., 2007). Hyperhomocysteine also may promote insulin resistance (Björck et al., 2006; Li et al., 2008) and alter lipid metabolism (Liao et al., 2006). It was observed that low folate concentration and hyperhomocysteinemia were significantly associated with higher ALT concentration (Welzel et al., 2007) or liver damage (Gulsen et al., 2005), and that folic acid (FA) supplementation offered a hepatoprotective effect in animal models (Woo et al., 2006). Despite biological plausibility and evidence from animal models, a recent report based on the US populations raised some concern (Ioannou et al., 2006). Folic acid fortification of enriched grain products had been fully implemented in the U.S. and Canada by 1998 (Yang et al., 2006). However, this recent report (Ioannou et al., 2006) found that the prevalence of elevated ALT levels (>43 IU/l) in the United States from 1999–2002 was more than double what was previously reported based on data from the National Health and Nutrition Examination Survey, 1988–1994.
In light of the uncertainty regarding the effect of FA intervention or fortification on serum ALT concentration, we analyzed the data from a randomized controlled trial that compared three intervention groups: (1) enalapril only (10 mg, control group); (2) enalapril–FA tablet (10 mg enalapril combined with 0.4 mg of FA, low FA group); and (3) enalapril–FA tablet (10 mg enalapril combined with 0.8 mg of FA, high FA group), once daily for 8 weeks. We sought to test the hypothesis of whether FA intervention affects serum ALT level post 8weeks treatment.
We also were interested in exploring whether there were any differences in treatment effect between men and women, and/or between participants with elevated baseline ALT levels (>40 IU/l) and those without.
Materials and methods
We attempted to conform to the Consolidated Standards of Reporting Trials statement (Schulz et al., 2010) in the report of this randomized controlled trial.
Participants and Ethics
This was a multicenter, randomized, double-blind controlled trial in hypertensive Chinese adults (clinicaltrials.gov;. identifier: NCT00520247; Li et al., 2007; Mao et al., 2008). In all, 480 patients with mild or moderate hypertension were recruited from six hospitals in different Chinese regions (Ha’erbin, Shanghai, Shenyang, Beijing, Xi’an and Nanjing) from September to December 2005. All the six hospitals were certified as clinical pharmacology centers by the State Food and Drug Administration in China. The inclusion criteria were as follows: (1) aged 18–75 years; (2) seated systolic blood pressure (SBP) between 140 mm Hg and 180 mm Hg and/or seated diastolic blood pressure between 90 mm Hg and 110 mm Hg; (3) women of reproductive age agreed to use a reliable contraception method during the study; and (4) participants agreed to not take any other drugs that might affect blood pressure or any vitamin B supplements in the week before the study. Participants were excluded if they were pregnant or planned to be pregnant during the study period; had serious diseases (cardiovascular diseases, tumors, hepatic disease, and so on); or expected to take other drugs that might affect blood pressure or any vitamin B supplements during the study period.
This study was approved by the Ethics Committee of Peking University First Hospital, Beijing, China. The purpose and procedures of the study were carefully explained to all participants, and written informed consent was obtained from each participant.
Intervention and data collection
Eligible participants were randomly and double-blindly assigned to one of the three treatment groups: (1) enalapril tablet only (10 mg, control group); (2) enalapril–FA tablet (10 mg enalapril combined with 0.4 mg. of FA, low FA group); or (3) enalapril–FA tablet (10 mg enalapril combined with 0.8 mg of FA, high FA group), once daily for 8 weeks.
Demographic and clinical information were both obtained at baseline. Blood pressure was examined at baseline and every 2 weeks for a total period of 8 weeks. Serum ALT and AST concentrations were measured at baseline and post 8-week treatment.
The ALT ratio, which was defined as the ratio of ALT concentration at week 8 to ALT at baseline, was applied as the primary outcome. Secondary outcome: the ALT difference, which was defined as ALT concentration at week 8 minus ALT at baseline, was applied as the secondary outcome.
The sample of ∼150 participants in each treatment group gave the study 85% power to detect a 15% reduction in ALT ratio at a 0.05. significance level for a two-sided test. This calculation assumes that the ALT ratio in the control group was about 1.0 and that the coefficient of variation was around 0.5.
Randomization and blinding
To achieve a balance among the three treatment groups, permuted-fixed block randomization with a block length of 6 was used. The allocation of participants was programmed by the independent statistical coordinating center, encrypted and sent to each study center. Tablet containers were labeled only with the name of the trial and the allocated concealment number. Participants, care partners and all staff directly involved in the trial were blinded to interventions during the period of the trial. No request was ever made to break the blind.
Blood sample collection and laboratory methods
After 10–12 h of fasting, a venous blood sample was obtained from each participant. Serum or plasma samples were separated within 15 min of collection, and were analyzed within 30 min or stored at −80 °C for later analysis. Blood samples collected at baseline and at week 8 of the trial were used for the measurement of glucose, lipids (including total cholesterol, high-density lipoprotein cholesterol and triglycerides), ALT, AST, homocysteine and folate concentrations. Homocysteine in plasma was determined in duplicate by high-performance liquid chromatography. The intra- and inter-assay coefficients of variation were 3.5% and 4.2%, respectively. Folate in serum was determined by chemiluminescent immunoassay using a Beckman Coulter ACCESS Immunoassay System (Beckman-Coulter Canada, Mississauga, Canada). The intra- and inter-assay coefficients of variation were 2.3% and 3.7%, respectively. All sample collection and tests were performed in an identical manner following the same standard protocol.
Means (s.d.) or medians (25th percentile, 75th percentile) and proportions were calculated for baseline characteristics by treatment groups. Homocysteine, folate, ALT and AST concentrations in the natural logarithms (due to their positively skewed distributions) were analyzed as continuous variables. The difference in population characteristics among the three groups was compared via one-way analysis of variance for continuous variables or via χ2-test for categorical variables.
All analyses were performed according to the principle of intention to treat. The Wilcoxon signed rank test was applied for the paired ALT difference within groups, and the Mann–Whitney U-test was applied to ALT difference between the groups. Multivariable regression models were applied to compare the ln-transformed ALT ratio among different groups with or without adjustment of the major baseline characteristics. We also performed subgroup analysis, stratified by sex and baseline ALT levels (⩽40 IU/l versus >40 IU/l). All tests were two-sided and P<0.05 was set as the significance level. Statistical analysis was performed using SAS software version 6.12 (SAS Institute, Cary, NC, USA).
The flow of participants through the study is shown in Figure 1. A total of 480 participants were recruited for this study. This analysis excluded 25 participants who were ineligible to participate in the trial (seven in the control group, nine in the low FA group, and eight in the high FA group) or with known hepatic disease at baseline (one in the low FA group). Accordingly, a total of 455 participants were included in our final analysis.
Table 1 shows the baseline characteristics of the participants. The three groups were well balanced at baseline with regard to relevant demographic and clinical characteristics. In this study, 62 (13.6%) of the total participants, including 17 (11.1%) in the control group, 24 (16.0%) in the low FA group and 21 (13.8%) in the high FA group, had an elevated ALT concentration (ALT>40 IU/l). The proportions of participants with elevated ALT concentrations also were well balanced among the three treatment groups (P=0.475). Only three participants had an ALT concentration >80 IU/l (98.0, 98.0 IU/l and 81.2 IU/l, respectively). At the same time, only 24 (6.0%) of the total participants, including 9 (6.8%) in the control group, 9 (6.9%) in the low FA group and 6 (4.5%) in the high FA group, had an elevated (>40 IU/l) AST concentration.
Outcomes and estimation
Compared with the baseline, after the 8-week treatment we observed a significant reduction in ALT concentration; (median (25th percentile, 75th percentile): −0.6 (−6.9, 2.0), P=0.0008; Table 2) and AST (−1.0 (−6.0, 2.0), P=0.0094) only in the high FA group. Furthermore, compared with the control group, the high FA group showed a significantly greater ALT-lowering response in men (median ALT ratio (ALT at week 8 to ALT at baseline; 25th percentile, 75th percentile): 0.93 (0.67, 1.06) vs 1.00 (0.91, 1.21), P=0.032), and in participants with elevated ALT (ALT>40 IU/l) at baseline (median ALT ratio: 0.67 (0.38, 1.00) vs 0.94 (0.77,1.11), P=0.008; Table 3, Figure 2) or in participants with higher ALT (ALT>23 IU/l(median)) at baseline (median ALT ratio: 0.83 (0.62,1.00) vs 1.00 (0.76,1.07), P=0.025). There was no difference in ALT lowering between the control and the low FA group (Tables 2 and 3). The adjustments of baseline ALT, study center and other relevant demographic and clinical characteristics did not alter the results, and there appeared to be a dose–response relationship between treatment groups and ALT change (Table 3).
In women or participants without elevated ALT concentrations (ALT⩽40) or without higher ALT concentrations (ALT⩽23) at baseline (data not shown), there was no significant difference between groups (Tables 2 and 3 and Figure 2).
We obtained similar results when we restricted analyses to participants who fully complied with the protocol (consuming at least 80% of all of the prescribed drugs) during the treatment period (per protocol set, n=388, 130 in control group, 125 in low FA group and 132 in high FA group) or participants without any other disease and related drug treatment (n=358) at baseline (data not shown).
Finally, there were no severe adverse events during the study period. All adverse events were mild and reversible, and the incidence of adverse events was comparable among groups.
There is a relative paucity of clinical data regarding the effects of FA intervention on the liver damage or associated diseases. Our study was the first randomized, double-blind, controlled trial to investigate the effect of physiological doses (0.4 or 0.8 mg/day) of FA intervention on serum ALT concentration in the Chinese hypertensive patients without known hepatic disease. As all study participants were mild or moderate essential hypertensives, and enalapril had no effect on the change in ALT concentration, it is reasonable to select the enalapril tablet group as the control group and the enalapril–FA tablet group as the treatment group to evaluate the effect of FA intervention.
Among the listed physiological doses of FA (<1 mg) for preventive purposes recommended by the US National Academy of Sciences, a daily dose of 0.8 mg FA may achieve the maximum reduction in plasma homocysteine concentrations (Homocysteine Lowering Trialists’ Collaboration, 2005). In our study, after the 8-week treatment, we observed a significant reduction in ALT concentration only in the high FA group (0.8 mg/day), but the reduction was insignificant compared with the control group. However, compared with the control group, men in the high FA treatment group produced a significantly greater ALT-lowering response (median ALT ratio: 0.93 (0.67, 1.06) vs 1.00 (0.91, 1.21), P=0.032), as did participants with elevated ALT concentration (ALT>40 IU/l) at baseline (median ALT ratio: 0.67 (0.38, 1.00) vs 0.94 (0.77, 1.11), P=0.008). Adjustment for baseline ALT, study center, and other relevant demographic and clinical characteristics did not alter the results. Consistent with a previous study (Welzel et al., 2007), folate concentration was one of the major determinants of ALT concentration at baseline in our study (data not shown).
Our findings have important clinical and public health implications. First, our results showed that FA supplementation did not have any adverse effect on ALT change in our study population. Kim HC et al., 2005 reported that serum aminotransferase level was independently associated with the incidence of stroke in men, even after adjustment for age and other traditional risk factors. In a study by Yang et al., (2006) after FA fortification, the resulting population increase in serum folate concentration produced a significant decrease in stroke mortality. These results further indicate that low folate concentration may be one of the explanations for increased ALT concentration and the increased risk of mortality, stroke and related diseases. Most interestingly, we found a greater beneficial effect of FA intervention on ALT in men. The reason for this gender difference remains to be understood, and further study may shed more light on the sex difference observed in the relationship between ALT concentration and mortality or the incidence of stroke (Kim et al., 2004, 2005). In a recent open-label pilot study (Charatcharoenwitthaya et al., 2007), 6 months of therapy with FA at a dose of 1 mg/day did not lead to a significant reduction in ALT concentration in patients with nonalcoholic steatohepatitis. We speculate that FA fortification in this population may have attenuated the ALT-lowering effect of FA intervention. Additionally, in our study population without FA fortification, the prevalence of elevated ALT (>40 IU/l) was quite high (about 13.6%). We suspect that among participants with ‘unexplained’ elevated ALT concentrations, a considerable fraction of them may have elevated ALT due to folate deficiency and may benefit from FA intervention. Our trial, if further confirmed by future trials, may offer a simple, safe and effective means of lowering ALT, particularly in populations without FA fortification (Wang et al., 2007).
Our study has the following strengths. This was a randomized, multicenter and double-blind trial that minimized systematic bias and error. We evaluated two physiological doses of FA in the trial, which should help to inform future clinical and public health interventions. Caution is needed in generalizing our findings from this hypertensive Chinese population to other populations. Our sample size is relatively small and we only observed treatment effect for 8 weeks. Furthermore, we are not sure whether 0.6 IU differences in ALT levels due to FA supplementation in our study population (that included only hypertensive adults without known hepatic disease) are of clinical significance. Obviously, additional large randomized studies in diverse populations, particularly in participants with hepatic injury, are needed to further evaluate the ALT-lowering effect of FA intervention. Furthermore, we did not obtain detailed information on dietary intake and alcohol consumption in this study, but asked all participants not to take any nutritional supplements and to maintain their regular dietary habits during the study period. However, there were no significant changes in ALT concentration in the control group or in any control subgroups after treatment, which implies that these factors may not have had a significant impact on our results.
In summary, this short-term, randomized controlled trial demonstrated that a daily dose of 0.8 mg FA treatment may be beneficial in lowering serum ALT concentration, particularly in men and/or in participants with elevated (>40 IU/l) ALT concentration.
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We gratefully acknowledge the assistance and cooperation of the faculty and staff of the Anhui Medical University and thank all of the participants in our study. This study was conducted in accordance with the current regulations of People‘s Republic of China. The study was supported by Beijing Huaanfo Biomedical Research Center Inc. Beijing, China and in part by a grant from Anhui Provincial Ministry of Education (No. 2002kj174ZC), Anhui Provincial Ministry of Science and Technology, Anhui Medical University Biomedical Institute.
The sponsors did not participate in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, and approval of the manuscript.
The authors declare no conflict of interest.
Contributors: Study concept and design: XQ, JL, YC, ZL, ZZ, JG, DG, JH, YW, FZ, XX, XW, XX, YH. Acquisition of data: JL, YC, ZL, JG, DG, JH, YW, FZ, YH. Analysis of data: XQ, JL, XX, XW, XX, HY. Interpretation of Data: XQ, L, YC, ZL, ZZ, JG, DG, JH, YW, FZ, XX, XW, XX, YH. Drafting and critical review of manuscript for important intellectual content: XQ, JL, YC, ZL, ZZ, JG, DG, JH, YW, FZ, XX, XW, XX, YH.
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Qin, X., Li, J., Cui, Y. et al. Effect of folic acid intervention on ALT concentration in hypertensives without known hepatic disease: a randomized, double-blind, controlled trial. Eur J Clin Nutr 66, 541–548 (2012). https://doi.org/10.1038/ejcn.2011.192
- folic acid supplementation
- ALT concentration
- controlled trial
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