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Association between serum folate levels and fatty liver disease
Clinical Nutrition Experimental xxx (xxxx) xxx
Contents lists available at ScienceDirect
Clinical Nutrition Experimental journal homepage: http:// www.clinicalnutritionexperimental.com
Original Article
Association between serum folate levels and fatty liver disease Hamid Vahedi a, Niloufar Bavafaetousi b, Pouneh Zolfaghari c, Maryam Yarmohammadi d, Mohammad Bagher Sohrabi e, * a
Department of Gastroenterology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran Student Research Committee, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran Vice-chancellery of Health, Shahroud University of Medical Sciences, Shahroud, Iran d Department of Pathology, Imam Hossain Center for Education, Research and Treatment, Shahroud University of Medical Sciences, Shahroud, Iran e School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 30 May 2019 Accepted 25 November 2019 Available online xxx
Background: Non-alcoholic fatty liver disease is one of the most common chronic liver diseases. Due to the lack of definitive documentation for the effect of folic acid on fatty liver, this study was conducted to determine the relationship between serum folate levels and fatty liver in patients referred to Imam Hossain Hospital in Shahroud, in 2016. Methods: This caseecontrol study was performed on 150 patients. The case group was fatty liver patients whose disease was diagnosed based on laboratory and ultrasound findings and a control group of patients without fatty liver. After liver ultrasonography, blood samples were taken from all patients and folic acid levels were measured. Results: Of the 150 patients examined, mean BMI of patients was (25.3 ± 6.1 kg/m2 in case group and 24.8 ± 7.2 kg/m2 in control group), which was significantly higher in case group (p < 0.035). The mean fasting blood glucose level in patients was (137.5 ± 53.5 mg/dl in case group and 124.5 ± 62.5 mg/dl in control group), which was significantly higher in case group (p < 0.01). The mean LDL of the patients was (175.3 ± 65.5 mg/dl in case group and 125.5 ± 42.5 mg/dl in control group), which was significantly higher (P < 0.003) in patients with fatty liver. The mean of folic acid in patients was (3.7 ± 5.3 ng/ml in case group and 5.3 ± 4.1 ng/
Keywords: Non-alcoholic fatty liver Folic acid Risk factor
* Corresponding Author. School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran. Fax: þ982332394800. E-mail address: [email protected] (M.B. Sohrabi). https://doi.org/10.1016/j.yclnex.2019.11.004 2352-9393/© 2019 The Authors. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
2
H. Vahedi et al. / Clinical Nutrition Experimental xxx (xxxx) xxx
ml in control group), which was significantly lower in patients with fatty liver (p < 0.01). Conclusion: The results of this study showed that folic acid deficiency was significantly higher in patients with fatty liver than in other patients. But in order to fully confirm this, more research is needed with a larger sample size. © 2019 The Authors. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Fatty liver disease (FLD) is a spectrum of clinical and pathological conditions that is due to the deposition of fat droplets in the liver of patients who have no history of alcohol consumption, it is determined and includes a range of diseases simple steatos to liver disease such as non-alcoholic steatohepatitis, fibrosis, cirrhosis and eventually hepatocellular cancers [1,2]. The natural liver contains 5 g of fat per 100 g of his weight, and if the fat content is more than 5 to 10 percent of the liver's weight, the person has a FLD (3). Fatty liver is one of the most common liver diseases in the world and its prevalence varies, different in different societies. The prevalence of this disease is estimated to be between 7% and 40% in Asian regions. Outbreak of FLD in western developed countries 2 to 3 times more hepatitis B and C and alcohol-related liver disease and the most common cause of liver tests disorders [1e3]. The symptoms are nonspecific and includes fatigue, lethargy, and pain in the upper right abdomen and so patients may not be aware of their illness or accidently discovered in abdominal ultrasound or other causes [4]. Risk factors associated with this disease include: increased age, high weight, abdominal obesity, abnormal blood lipids profiles, high blood pressure, high blood sugar, insulin resistance and metabolic syndrome, cigarette and non-standard diet [5]. Also, taking medications such as Corticosteroids, Aspirin, Methotrexate, Tamoxifen, Tetracycline, Valproic acid, Amiodarone, viruses can also secondary to this complication, but the main risk factor is diabetes and resistance to insulin [6,7]. Diet plays an important role in non-alcoholic fatty liver disease. In some studies, there has been a correlation between the incidence of this disease and the high intake of saturated fat or carbohydrates. Vitamins can play a largely protective role in the development of fatty liver. One of these compounds is folic acid and folate [8]. Folate or vitamin B9 or folic acid is found in a range of foods such as leafy green vegetables, cereals, meat and fruits. This vitamin for many body exercises, including the health of the nervous system, blood and cells are vital and essential. Folic acid also has a supportive role in reducing the incidence of type 2 diabetes and heart disease, but its mechanism is still not well defined [2]. Given that these cases (diabetes and high blood lipids) are risk factors for fatty liver, therefore, it is conceivable that serum folate levels will also affect reduce the incidence of fatty liver [1,2]. The main mechanism of folate in reducing the incidence of fatty liver is still not fully understood but several hypotheses have been raised in this regard. Possibly, it has a positive effect on glucose metabolism and insulin resistance, or as a catalyzer for the metabolism of fats. Because of brief studies in Iran regarding folic acid on the development of fatty liver and the association of this vitamin with other risk factors has been done, it is necessary to study in patients with fatty liver. This study was conducted to determine the relationship between serum folate levels and fatty liver in patients referred to Imam Hossain Hospital of Shahroud, in 2016. 2. Materials & methods This research is a caseecontrol study among patients referring to Imam Hossain Hospital of Shahroud (Northeastern of Iran) following is done between January and December 2017. The case group was selected from patients with non-alcoholic liver disease diagnosed on the basis of liver tests and sonography of their disease was selected and categorized. Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
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Mild fatty liver: a brief increase in liver echogenicity so that the diaphragm and vascular wall are well visible in ultrasound (grid 1). Medium fatty liver: moderate increase in liver echogenicity so that the diaphragmatic view and the intra-liver vessel wall are slightly disturbed in ultrasound (grid 2). Sever fatty liver: increased liver echogenicity so that the posterior lobe of the liver disappears in ultrasound and the observation of the diaphragm and the wall of the intra-liver vessel is difficult or unobservable (grid 3) [9]. The best diagnostic test for non-alcoholic liver fatty liver is biopsy [9,10] but ethical and medical considerations have limited the use of it in patients. Increased liver enzymes are commonly found in these patients, but the characteristics of these tests are low. Therefore, clinical evaluation of nonalcoholic fatty liver was performed based on a combination of ultrasound findings and laboratory tests. The control group had no symptoms in ultrasound and liver tests in favor of the fatty liver and referred to the ultrasound section for other reasons, was chosen. None of the patients and controls was not infected with liver disease, malignancy, chemotherapy, diabetes, pregnancy and had no history of taking corticosteroids, alcohol and vitamin supplements and folic acid over the past year. In these two groups, age, sex, BMI, fasting blood glucose, triglyceride, total cholesterol, HDL-LDL, liver enzymes, history of diabetes and folate intake were investigated and the two groups were compared. Folic acid can be measured in plasma. Folic acid levels can be measured in red blood cells. But plasma folic acid is a better way to measure the amount of folic acid stored in the body. The preferred method of measuring folic acid is RIACPB method. The natural values in this method for adults are 2e10 ng/ml, less than 2 ng/ml was considered deficient and more than 2 ng/ml was considered normal. The following sampling conditions were also followed: Preferably the patient was fasting for 8 h, no alcohol at least 24 h before the test, and no blood transfusion at least 2 weeks before the test [1]. Descriptive statistics including mean and standard deviation, as well as relative frequency were used to describe the data. To examine the relationships and comparisons between the two groups, was used the chi-square test and multivariate logistic regression was used to evaluate the odds of each of the variables. All analyzes were performed using SPSS software version 16 and significant level (p < 0.05). Sample size using Epi info 7.2 at a significant level of 5% and a power of 80%, equal to 75 people in each group and a total of 150 people. This study has an ethics code number (IR.SHMU.REC. 1395.90) from research deputy of Shahroud University of Medical Sciences. The essential information and the objectives of the study were explained to the patients, and written consent was obtained for participation in the plan. 3. Results In this study, 64 (42.7%) of the participants were male and the rest were female. The mean age of the all patients was 49.52 ± 23.29 years that were no significant difference between the two groups. The mean BMI of the case group was 25.3 ± 6.1 kg/m2 and the control group was 24.8 ± 7.2 kg/m2, which was significantly higher in the case group (p ¼ 0.035). The mean of folic acid in the case group was 3.7 ± 5.3 ng/ml and in the control group was 5.3 ± 4.1 ng/ml, which was significantly lower in the case group (p ¼ 0.01). The results of biochemical and liver tests of patients in the two groups are shown in Table 1 Results of biochemical and liver tests in two groups. Lab Test
Case group Mean ± SD
Control group Mean ± SD
Total Mean ± SD
p-value
Fasting blood sugar (mg/dl) Triglyceride (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) AST (IU/L) ALT (IU/L) ALK-P (IU/L)
137.5 ± 53.5 223.5 ± 130.5 318.3 ± 155.5 175.3 ± 65.5 39.5 ± 6.8 95.3 ± 32.5 108.5 ± 28.5 380.5 ± 75.5
124.5 ± 62.5 218.5 ± 125.5 265.5 ± 144.5 125.5 ± 42.5 44.5 ± 5.5 29.5 ± 6.5 37.5 ± 7.4 295.5 ± 45.5
128.5 ± 59.5 220.5 ± 128.5 284.5 ± 151.5 155.5 ± 53.5 42.8 ± 5.3 63.5 ± 15.8 75.4 ± 20.8 345.4 ± 58.2
0.01 0.077 0.051 0.003 0.109 0.001 0.001 0.003
Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
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Table 1. In this study independent variables with FLD were investigated in multivariate regression model. As shown in Table 2, fatty liver variables were significantly associated with LDL (p < 0.001) and FBS (p < 0.001) and there was no significant relationship with other variables. The results of the multivariate logistic regression model are presented in Table 2. 4. Discussion The results of this study showed that placeholders similar to BMI, LDL and fasting blood glucose there was a significant relationship with fatty liver and other variables were not significantly correlated with logistic regression model. Therefore, it is possible to increase BMI, LDL and fasting blood glucose as important factors in the development of fatty liver disease. According to logistic regression model findings, folic acid does not increase the risk of fatty liver and there was no significant relationship between the deficiency of this vitamin and the incidence of fatty liver disease. This finding is based on Ganji et al. [10],. study of the relationship between folic acid serum levels and fatty liver disease, do not find significant relationship, it is perfectly consistent [10]. In the Hu et al. study that was done on 239 patients, BMI, lipid profiles and diabetes were identical in both fatty and non-fatty liver group, and examined folic acid levels in these two groups. In this study, a significant difference was found between the two groups in the level of folic acid but, in the study of logistic regression, it was found that folic acid deficiency does not correlate with the appearance of fatty liver. This finding is consistent with the result of our study [7]. However, in some studies, including Fabbrini et al. which performed on 211 samples, reductions in serum levels of vitamins, including folic acid, in fatty liver patients compared to controls, a significant relationship was found which contrasts with the present research to a great extent, perhaps this Table 2 Relationship between independent variables with fatty liver in multivariate logistic regression model. Independent variables Age category Less than 30 years 30e50 years More than 50 years Sex Male Female History of diabetes mellitus Negative Positive Body mass index (kg/m2) 18e25 <18 >25 Triglyceride (mg/dl) <150 >150 Total cholesterol (mg/dl) <180 >180 LDL cholesterol (mg/dl) <130 >130 HDL cholesterol (mg/dl) >40 <40 Fasting blood sugar (mg/dl) <126 >126 Folic acid (ng/ml) >2 <2
Odds Ratio
95% Confidence
P- Value
1.000 1.076 1.091
1.342e0.0652 1.451e0.0712
0.065 0.055
1.000 0.791
1.013e0.615
0.069
1.000 1.105
1.248e0.811
0.063
1.000 0.723 1.375
0.985e0.556 1.735e0.912
0.059 0.033
1.000 1.151
1.323e0.871
0.83
1.000 1.326
1.542e1.109
0.052
1.000 1.489
1.763e1.216
0.001
1.000 1.028
1.282e0.857
0.079
1.000 1.551
1.854e1.256
0.001
1.000 1.081
1.218e0.855
0.068
Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
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difference is due to the sample size being measured, the food and cultural habits of those patients or general levels of folic acid at the community level [11]. In a study by Heemskerk et al., Who conducted research on risk factors affecting fatty liver, variables such as age, waist circumference, BMI, FBS, triglyceride and cholesterol showed a significant correlation with fatty liver that among these variables, a significant correlation was found between BMI and this study and other variables contradict our study results, that perhaps the reason for this inconsistency is the existence of demographic and epidemiological differences between the subjects [8]. In the study of Wong et al., which was performed on 185 patients with metabolic syndrome, parameters such as weight gain and obesity, lipid profiles, and the amount of folic acid and fatty liver were evaluated and was found that all of these factors can be associated with the development of a fatty liver as an independent agent, but there was no statistically significant difference between the groups that this finding was consistent with the result of the present study [12]. As it was observed, the present study showed a strong relationship between obesity and overweight with the incidence of fatty liver disease [13,14]. The role of fat tissue storage and release of energy in the form of triglycerides in excessive consumption of food and hunger can be one of the most important of these factors [15]. Also, fat tissue produces hormones such as leptin, adiponectin, and proinflammatory cytokines such as interleukin-6 and alpha-tumor necrosis factor. Increased levels of obesity and obesity lead to imbalance of hormones and cytokines, as a result, the inflammatory mechanisms and insulin resistance increase, especially in the liver and increases the risk of hepatic steatosis and nonalcoholic fatty liver [16e19]. Since the efficacy and safety of drug therapy in patients with non-alcoholic fatty liver is not definitively known, lifestyle changes, diet modifications, and weight loss are commonly used treatments for non-alcoholic liver fatty acids, so that 5 to 10 percent weight loss can dramatically improve the metabolic function of the liver [20e22]. 5. Conclusions The results of this study showed that folic acid deficiency was significantly higher in patients with fatty liver than in other patients, however, this factor was not confirmed as a contributing factor in the incidence, exacerbation and persistence of fatty liver. But in order to fully confirm this, more research is needed with a larger sample size. Therefore, all patients with fatty liver needing attention to dietary deficits and lifestyle changes, should be carefully evaluated for their vitamin deficiencies and, if such deficiencies are detected, be corrected as soon as possible [23,24]. 6. Limitation The limitations of this research which is mentioned, include the impossibility of a biopsy to confirm the ultimate fatty liver (due to ethical barriers) and self-declaration of patients about chronic diseases, by performing accurate and complete liver ultrasonography, along with relevant tests and full justification of patients for research purposes, it has tried to reduce this limitation. Conflict of Interest The authors declared that they have no conflict of interest. Acknowledgements The present study was supported by Shahroud University of Medical Sciences as a Medical Doctor (MD) Thesis. We hereby acknowledge the research deputy. Also we would like to thank all participated patients. Funding This study received funding from the research deputy of Shahroud University of Medical Sciences. Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
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Ethical standards This study has an ethics code number (IR.SHMU.REC. 1395.90) from research deputy of Shahroud University of Medical Sciences. Informed consent Informed consent was obtained from all individual participants included in the study. References [1] Polyzos SA, Kountouras J, Patsiaoura K, Katsiki E, Zafeiriadou E, Zavos C, et al. Serum vitamin B12 and folate levels in patients with non-alcoholic fatty liver disease. Int J Food Sci Nutr 2012;63(6):659e66. [2] Koplay M, Gulcan E, Ozkan F. Association between serum vitamin B12 levels and the degree of steatosis in patients with nonalcoholic fatty liver disease. J Investig Med 2011;59(7):1137e40. ~ o M, Csendes A, Burdiles P, Smok G, et al. Serum folate and homocysteine levels in obese [3] Hirsch S, Poniachick J, Avendan females with non-alcoholic fatty liver. Nutrition 2005;21(2):137e41. [4] Tilg H, Moschen AR. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 2010;52:1836e46. [5] Ganji SH, Kamanna VS, Kashyap ML. Folic acid and cholesterol: role in cardiovascular disease (review). J Nutr Biochem 2003;14(6):298e305. [6] Ganji SH, Kashyap ML, Kamanna VS. Niacin inhibits fat accumulation, oxidative stress, and inflammatory cytokine IL-8 in cultured hepatocytes: impact on non-alcoholic fatty liver disease. Metabolism 2015;64(9):982e90. [7] Hu M, Chu WC, Yamashita S, Yeung DK, Shi L, Wang D, et al. Liver fat reduction with niacin is influenced by DGAT-2 polymorphisms in hypertriglyceridemic patients. J Lipid Res 2012;53(4):802e9. [8] Heemskerk MM, van den Berg SA, Pronk AC, van Klinken JB, Boon MR, Havekes LM, et al. Long-term niacin treatment induces insulin resistance and adrenergic responsiveness in adipocytes by adaptive down regulation of phosphodiesterase 3B. Am J Physiol Endocrinol Metab 2014;306(7):E808e13. [9] Leaf A, Lansdowne Z. Vitamin's conventional uses and new insights. World Rev Nutr Diet 2014;110:152e66. [10] Ganji SH, Kukes GD, Lambrecht N, Kashyap ML, Kamanna VS. Therapeutic role of niacin in the prevention and regression of hepatic steatosis in rat model of nonalcoholic fatty liver disease. Am J Physiol Gastrointest Liver Physiol 2014;306(4): G320e7. [11] Fabbrini E, Mohammed BS, Korenblat KM, Magkos F, McCrea J, Patterson BW, et al. Effect of fenofibrate and niacin on intrahepatic triglyceride content, very low-density lipoprotein kinetics, and insulin action in obese subjects with nonalcoholic fatty liver disease. J Clin Endocrinol Metab 2010;95(6):2727e35. [12] Wong RJ, Aguilar M, Cheung R, Perumpail RB, Harrison SA, Younossi ZM, et al. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States. Gastroenterology 2015; 148(3):547e55. [13] Gusdon AM, Song KX, Qu S. Nonalcoholic fatty liver disease: pathogenesis and therapeutics from a mitochondria-centric perspective. Oxid Med Cell Longev 2014;2014:637027. [14] Deshmukh U, Katre P, Yajnik CS. Influence of maternal vitamin B12 and folate on growth and insulin resistance in the offspring. Nestle Nutr Inst Workshop Ser 2013;74:145e54. discussion 154-6. [15] Clouston A, Powell EE. Nonalcoholic fatty liver disease: is all the fat bad? Intern Med J 2004;34(4):187e91. [16] Da Silva HE, Arendt BM, Noureldin SA, Therapondos G, Guindi M, Allard JP. A cross-sectional study assessing dietary intake and physical activity in Canadian patients with nonalcoholic fatty liver disease vs healthy controls. J Acad Nutr Diet 2014; 114(8):1181e94. [17] Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology 2012;142(7):1592e609. [18] Aykut UE, Akyuz U, Yesil A, Eren F, Gerin F, Ergelen R, et al. A comparison of FibroMeter NAFLD Score, NAFLD fibrosis score, and transient elastography as noninvasive diagnostic tools for hepatic fibrosis in patients with biopsy-proven non-alcoholic fatty liver disease. Scand J Gastroenterol 2014;49(11):1343e8. [19] Zhu JZ, Dai YN, Wang YM, Zhou QY, Yu CH, Li YM. Prevalence of nonalcoholic fatty liver disease and economy. Dig Dis Sci 2015;60(11):3194e202. [20] Chitturi S, Farrell GC, George J. Non- alcoholic steatohepatitis in the Asiapacific region: Future shock? J Gastroenterol Hepatol 2004;19(4):368e74. [21] Argo CK, Caldwell SH. Epidemiology and natural history of non-alcoholic steatohepatitis. Clin Liver Dis 2009;13:511e31. [22] Bahrami H, Daryani NE, Mirmomen S, Kamangar F, Haghpanah B, Djalili M. Clinical and histological features of nonalcoholic steatohepatitis in Iranian patients. BMC Gastroenterol 2003;3:27. [23] Vos MB, Colvin R, Belt P, Molleston JP, Murray KF, Rosenthal P, et al. Correlation of vitamin E, uric acid, and diet composition with histologic features of pediatric NAFLD. J Pediatr Gastroenterol Nutr 2012;54(1):90e6. [24] Miele L, Valenza V, La Torre G, Montalto M, Cammarota G, Ricci R, et al. Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology 2009;49(6):1877e87.
Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
Contents lists available at ScienceDirect
Clinical Nutrition Experimental journal homepage: http:// www.clinicalnutritionexperimental.com
Original Article
Association between serum folate levels and fatty liver disease Hamid Vahedi a, Niloufar Bavafaetousi b, Pouneh Zolfaghari c, Maryam Yarmohammadi d, Mohammad Bagher Sohrabi e, * a
Department of Gastroenterology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran Student Research Committee, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran Vice-chancellery of Health, Shahroud University of Medical Sciences, Shahroud, Iran d Department of Pathology, Imam Hossain Center for Education, Research and Treatment, Shahroud University of Medical Sciences, Shahroud, Iran e School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 30 May 2019 Accepted 25 November 2019 Available online xxx
Background: Non-alcoholic fatty liver disease is one of the most common chronic liver diseases. Due to the lack of definitive documentation for the effect of folic acid on fatty liver, this study was conducted to determine the relationship between serum folate levels and fatty liver in patients referred to Imam Hossain Hospital in Shahroud, in 2016. Methods: This caseecontrol study was performed on 150 patients. The case group was fatty liver patients whose disease was diagnosed based on laboratory and ultrasound findings and a control group of patients without fatty liver. After liver ultrasonography, blood samples were taken from all patients and folic acid levels were measured. Results: Of the 150 patients examined, mean BMI of patients was (25.3 ± 6.1 kg/m2 in case group and 24.8 ± 7.2 kg/m2 in control group), which was significantly higher in case group (p < 0.035). The mean fasting blood glucose level in patients was (137.5 ± 53.5 mg/dl in case group and 124.5 ± 62.5 mg/dl in control group), which was significantly higher in case group (p < 0.01). The mean LDL of the patients was (175.3 ± 65.5 mg/dl in case group and 125.5 ± 42.5 mg/dl in control group), which was significantly higher (P < 0.003) in patients with fatty liver. The mean of folic acid in patients was (3.7 ± 5.3 ng/ml in case group and 5.3 ± 4.1 ng/
Keywords: Non-alcoholic fatty liver Folic acid Risk factor
* Corresponding Author. School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran. Fax: þ982332394800. E-mail address: [email protected] (M.B. Sohrabi). https://doi.org/10.1016/j.yclnex.2019.11.004 2352-9393/© 2019 The Authors. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
2
H. Vahedi et al. / Clinical Nutrition Experimental xxx (xxxx) xxx
ml in control group), which was significantly lower in patients with fatty liver (p < 0.01). Conclusion: The results of this study showed that folic acid deficiency was significantly higher in patients with fatty liver than in other patients. But in order to fully confirm this, more research is needed with a larger sample size. © 2019 The Authors. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Fatty liver disease (FLD) is a spectrum of clinical and pathological conditions that is due to the deposition of fat droplets in the liver of patients who have no history of alcohol consumption, it is determined and includes a range of diseases simple steatos to liver disease such as non-alcoholic steatohepatitis, fibrosis, cirrhosis and eventually hepatocellular cancers [1,2]. The natural liver contains 5 g of fat per 100 g of his weight, and if the fat content is more than 5 to 10 percent of the liver's weight, the person has a FLD (3). Fatty liver is one of the most common liver diseases in the world and its prevalence varies, different in different societies. The prevalence of this disease is estimated to be between 7% and 40% in Asian regions. Outbreak of FLD in western developed countries 2 to 3 times more hepatitis B and C and alcohol-related liver disease and the most common cause of liver tests disorders [1e3]. The symptoms are nonspecific and includes fatigue, lethargy, and pain in the upper right abdomen and so patients may not be aware of their illness or accidently discovered in abdominal ultrasound or other causes [4]. Risk factors associated with this disease include: increased age, high weight, abdominal obesity, abnormal blood lipids profiles, high blood pressure, high blood sugar, insulin resistance and metabolic syndrome, cigarette and non-standard diet [5]. Also, taking medications such as Corticosteroids, Aspirin, Methotrexate, Tamoxifen, Tetracycline, Valproic acid, Amiodarone, viruses can also secondary to this complication, but the main risk factor is diabetes and resistance to insulin [6,7]. Diet plays an important role in non-alcoholic fatty liver disease. In some studies, there has been a correlation between the incidence of this disease and the high intake of saturated fat or carbohydrates. Vitamins can play a largely protective role in the development of fatty liver. One of these compounds is folic acid and folate [8]. Folate or vitamin B9 or folic acid is found in a range of foods such as leafy green vegetables, cereals, meat and fruits. This vitamin for many body exercises, including the health of the nervous system, blood and cells are vital and essential. Folic acid also has a supportive role in reducing the incidence of type 2 diabetes and heart disease, but its mechanism is still not well defined [2]. Given that these cases (diabetes and high blood lipids) are risk factors for fatty liver, therefore, it is conceivable that serum folate levels will also affect reduce the incidence of fatty liver [1,2]. The main mechanism of folate in reducing the incidence of fatty liver is still not fully understood but several hypotheses have been raised in this regard. Possibly, it has a positive effect on glucose metabolism and insulin resistance, or as a catalyzer for the metabolism of fats. Because of brief studies in Iran regarding folic acid on the development of fatty liver and the association of this vitamin with other risk factors has been done, it is necessary to study in patients with fatty liver. This study was conducted to determine the relationship between serum folate levels and fatty liver in patients referred to Imam Hossain Hospital of Shahroud, in 2016. 2. Materials & methods This research is a caseecontrol study among patients referring to Imam Hossain Hospital of Shahroud (Northeastern of Iran) following is done between January and December 2017. The case group was selected from patients with non-alcoholic liver disease diagnosed on the basis of liver tests and sonography of their disease was selected and categorized. Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
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Mild fatty liver: a brief increase in liver echogenicity so that the diaphragm and vascular wall are well visible in ultrasound (grid 1). Medium fatty liver: moderate increase in liver echogenicity so that the diaphragmatic view and the intra-liver vessel wall are slightly disturbed in ultrasound (grid 2). Sever fatty liver: increased liver echogenicity so that the posterior lobe of the liver disappears in ultrasound and the observation of the diaphragm and the wall of the intra-liver vessel is difficult or unobservable (grid 3) [9]. The best diagnostic test for non-alcoholic liver fatty liver is biopsy [9,10] but ethical and medical considerations have limited the use of it in patients. Increased liver enzymes are commonly found in these patients, but the characteristics of these tests are low. Therefore, clinical evaluation of nonalcoholic fatty liver was performed based on a combination of ultrasound findings and laboratory tests. The control group had no symptoms in ultrasound and liver tests in favor of the fatty liver and referred to the ultrasound section for other reasons, was chosen. None of the patients and controls was not infected with liver disease, malignancy, chemotherapy, diabetes, pregnancy and had no history of taking corticosteroids, alcohol and vitamin supplements and folic acid over the past year. In these two groups, age, sex, BMI, fasting blood glucose, triglyceride, total cholesterol, HDL-LDL, liver enzymes, history of diabetes and folate intake were investigated and the two groups were compared. Folic acid can be measured in plasma. Folic acid levels can be measured in red blood cells. But plasma folic acid is a better way to measure the amount of folic acid stored in the body. The preferred method of measuring folic acid is RIACPB method. The natural values in this method for adults are 2e10 ng/ml, less than 2 ng/ml was considered deficient and more than 2 ng/ml was considered normal. The following sampling conditions were also followed: Preferably the patient was fasting for 8 h, no alcohol at least 24 h before the test, and no blood transfusion at least 2 weeks before the test [1]. Descriptive statistics including mean and standard deviation, as well as relative frequency were used to describe the data. To examine the relationships and comparisons between the two groups, was used the chi-square test and multivariate logistic regression was used to evaluate the odds of each of the variables. All analyzes were performed using SPSS software version 16 and significant level (p < 0.05). Sample size using Epi info 7.2 at a significant level of 5% and a power of 80%, equal to 75 people in each group and a total of 150 people. This study has an ethics code number (IR.SHMU.REC. 1395.90) from research deputy of Shahroud University of Medical Sciences. The essential information and the objectives of the study were explained to the patients, and written consent was obtained for participation in the plan. 3. Results In this study, 64 (42.7%) of the participants were male and the rest were female. The mean age of the all patients was 49.52 ± 23.29 years that were no significant difference between the two groups. The mean BMI of the case group was 25.3 ± 6.1 kg/m2 and the control group was 24.8 ± 7.2 kg/m2, which was significantly higher in the case group (p ¼ 0.035). The mean of folic acid in the case group was 3.7 ± 5.3 ng/ml and in the control group was 5.3 ± 4.1 ng/ml, which was significantly lower in the case group (p ¼ 0.01). The results of biochemical and liver tests of patients in the two groups are shown in Table 1 Results of biochemical and liver tests in two groups. Lab Test
Case group Mean ± SD
Control group Mean ± SD
Total Mean ± SD
p-value
Fasting blood sugar (mg/dl) Triglyceride (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) AST (IU/L) ALT (IU/L) ALK-P (IU/L)
137.5 ± 53.5 223.5 ± 130.5 318.3 ± 155.5 175.3 ± 65.5 39.5 ± 6.8 95.3 ± 32.5 108.5 ± 28.5 380.5 ± 75.5
124.5 ± 62.5 218.5 ± 125.5 265.5 ± 144.5 125.5 ± 42.5 44.5 ± 5.5 29.5 ± 6.5 37.5 ± 7.4 295.5 ± 45.5
128.5 ± 59.5 220.5 ± 128.5 284.5 ± 151.5 155.5 ± 53.5 42.8 ± 5.3 63.5 ± 15.8 75.4 ± 20.8 345.4 ± 58.2
0.01 0.077 0.051 0.003 0.109 0.001 0.001 0.003
Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
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Table 1. In this study independent variables with FLD were investigated in multivariate regression model. As shown in Table 2, fatty liver variables were significantly associated with LDL (p < 0.001) and FBS (p < 0.001) and there was no significant relationship with other variables. The results of the multivariate logistic regression model are presented in Table 2. 4. Discussion The results of this study showed that placeholders similar to BMI, LDL and fasting blood glucose there was a significant relationship with fatty liver and other variables were not significantly correlated with logistic regression model. Therefore, it is possible to increase BMI, LDL and fasting blood glucose as important factors in the development of fatty liver disease. According to logistic regression model findings, folic acid does not increase the risk of fatty liver and there was no significant relationship between the deficiency of this vitamin and the incidence of fatty liver disease. This finding is based on Ganji et al. [10],. study of the relationship between folic acid serum levels and fatty liver disease, do not find significant relationship, it is perfectly consistent [10]. In the Hu et al. study that was done on 239 patients, BMI, lipid profiles and diabetes were identical in both fatty and non-fatty liver group, and examined folic acid levels in these two groups. In this study, a significant difference was found between the two groups in the level of folic acid but, in the study of logistic regression, it was found that folic acid deficiency does not correlate with the appearance of fatty liver. This finding is consistent with the result of our study [7]. However, in some studies, including Fabbrini et al. which performed on 211 samples, reductions in serum levels of vitamins, including folic acid, in fatty liver patients compared to controls, a significant relationship was found which contrasts with the present research to a great extent, perhaps this Table 2 Relationship between independent variables with fatty liver in multivariate logistic regression model. Independent variables Age category Less than 30 years 30e50 years More than 50 years Sex Male Female History of diabetes mellitus Negative Positive Body mass index (kg/m2) 18e25 <18 >25 Triglyceride (mg/dl) <150 >150 Total cholesterol (mg/dl) <180 >180 LDL cholesterol (mg/dl) <130 >130 HDL cholesterol (mg/dl) >40 <40 Fasting blood sugar (mg/dl) <126 >126 Folic acid (ng/ml) >2 <2
Odds Ratio
95% Confidence
P- Value
1.000 1.076 1.091
1.342e0.0652 1.451e0.0712
0.065 0.055
1.000 0.791
1.013e0.615
0.069
1.000 1.105
1.248e0.811
0.063
1.000 0.723 1.375
0.985e0.556 1.735e0.912
0.059 0.033
1.000 1.151
1.323e0.871
0.83
1.000 1.326
1.542e1.109
0.052
1.000 1.489
1.763e1.216
0.001
1.000 1.028
1.282e0.857
0.079
1.000 1.551
1.854e1.256
0.001
1.000 1.081
1.218e0.855
0.068
Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
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difference is due to the sample size being measured, the food and cultural habits of those patients or general levels of folic acid at the community level [11]. In a study by Heemskerk et al., Who conducted research on risk factors affecting fatty liver, variables such as age, waist circumference, BMI, FBS, triglyceride and cholesterol showed a significant correlation with fatty liver that among these variables, a significant correlation was found between BMI and this study and other variables contradict our study results, that perhaps the reason for this inconsistency is the existence of demographic and epidemiological differences between the subjects [8]. In the study of Wong et al., which was performed on 185 patients with metabolic syndrome, parameters such as weight gain and obesity, lipid profiles, and the amount of folic acid and fatty liver were evaluated and was found that all of these factors can be associated with the development of a fatty liver as an independent agent, but there was no statistically significant difference between the groups that this finding was consistent with the result of the present study [12]. As it was observed, the present study showed a strong relationship between obesity and overweight with the incidence of fatty liver disease [13,14]. The role of fat tissue storage and release of energy in the form of triglycerides in excessive consumption of food and hunger can be one of the most important of these factors [15]. Also, fat tissue produces hormones such as leptin, adiponectin, and proinflammatory cytokines such as interleukin-6 and alpha-tumor necrosis factor. Increased levels of obesity and obesity lead to imbalance of hormones and cytokines, as a result, the inflammatory mechanisms and insulin resistance increase, especially in the liver and increases the risk of hepatic steatosis and nonalcoholic fatty liver [16e19]. Since the efficacy and safety of drug therapy in patients with non-alcoholic fatty liver is not definitively known, lifestyle changes, diet modifications, and weight loss are commonly used treatments for non-alcoholic liver fatty acids, so that 5 to 10 percent weight loss can dramatically improve the metabolic function of the liver [20e22]. 5. Conclusions The results of this study showed that folic acid deficiency was significantly higher in patients with fatty liver than in other patients, however, this factor was not confirmed as a contributing factor in the incidence, exacerbation and persistence of fatty liver. But in order to fully confirm this, more research is needed with a larger sample size. Therefore, all patients with fatty liver needing attention to dietary deficits and lifestyle changes, should be carefully evaluated for their vitamin deficiencies and, if such deficiencies are detected, be corrected as soon as possible [23,24]. 6. Limitation The limitations of this research which is mentioned, include the impossibility of a biopsy to confirm the ultimate fatty liver (due to ethical barriers) and self-declaration of patients about chronic diseases, by performing accurate and complete liver ultrasonography, along with relevant tests and full justification of patients for research purposes, it has tried to reduce this limitation. Conflict of Interest The authors declared that they have no conflict of interest. Acknowledgements The present study was supported by Shahroud University of Medical Sciences as a Medical Doctor (MD) Thesis. We hereby acknowledge the research deputy. Also we would like to thank all participated patients. Funding This study received funding from the research deputy of Shahroud University of Medical Sciences. Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004
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Please cite this article as: Vahedi H et al., Association between serum folate levels and fatty liver disease, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.11.004