disease regression in non-alcoholic fatty liver

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Author's Personal Copy Clinical Nutrition 36 (2017) 1490e1497

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Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu

Randomized Control Trials

Effect of daily calcitriol supplementation with and without calcium on disease regression in non-alcoholic fatty liver patients following an energy-restricted diet: Randomized, controlled, double-blind trial Hamid Lorvand Amiri a, Shahram Agah b, Javad Tolouei Azar c, Sharieh Hosseini d, Farzad Shidfar a, b, *, Seyedeh Neda Mousavi e, ** a

Department of Nutrition, School of Public Health, Colorectal Research Center, Rasoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran Colorectal Research Center, Rasoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran Department of Exercise Physiology, Faculty of Physical Education and Sport Sciences, Urmia University, Urmia, Iran d Department of Applied Chemistry, Faculty of Pharmaceutical Chemistry, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran e Department of Biochemistry and Nutrition, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran b c

a r t i c l e i n f o

s u m m a r y

Article history: Received 23 June 2016 Accepted 22 September 2016

Background & aims: Despite evidence for beneficial effects of vitamin D, to our knowledge, no study has compared the effects of calcium supplementation with vitamin D on non-alcoholic fatty liver disease (NAFLD) regression during a hypo-energetic program. We compared the effect of the vitamin D supplementation with and without calcium on anthropometric measures and biochemical parameters in NAFLD patients during a weight-loss program. Methods: A 12-week, randomized, controlled, double-blind trial was conducted in 120 NAFLD patients randomly assigned to receive 25 mg calcitriol (n ¼ 37), 500 mg calcium carbonate þ 25 mg calcitriol (n ¼ 37), or placebo (n ¼ 36) every day with their lunch meals while following a weight-loss program. Results: Weight, BMI and fat mass reduction were significant in each group after 12 wk of intervention (p < 0.001), but differences among the groups was not significant after 12 wk of the study, adjusted to the baseline measurements. Significant reduction in fasting plasma glucose (FPG), insulin, insulin resistance (by HOMA-IR) and TG concentrations and an increase in HDL.C was seen over the 12 wk of study in each group (p < 0.001). Adjusting to the baseline measurements, there was significant difference in FPG (p < 0.001), HOMA-IR (p < 0.001), serum insulin (p ¼ 0.01), TG (p ¼ 0.01) and HDL.C (p < 0.001) among the groups after 12 wk of the study. The calcium plus calcitriol group showed a significant decrease in ALT and FPG and increase in HDL.C level compared with the calcitriol group, adjusted to the baseline measures (p < 0.001). Conclusions: Our results suggest that calcium plus calcitriol supplementation for 12 weeks may be potentially effective for biochemical parameters in NAFLD patients. Further additional larger controlled trials are needed to confirm these findings. Registration: Registered under ClinicalTrials.gov Identifier no. IRCT201408312709N29. © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords: Non-alcoholic fatty liver disease Supplements Anthropometry Calcium Calcitriol

Abbreviations: NAFLD, non-alcoholic fatty liver disease; HOMA-IR, homeostasis model assessment for insulin resistance; FPG, fasting plasma glucose; VDR, vitamin D receptor; IR, insulin resistance; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TC, total cholesterol; P þ P, placebo þ placebo; D þ P, vitamin D þ placebo; Ca þ D, calcium carbonate þ vitamin D. * Corresponding author. Hemmat Highway, Iran University of Medical Sciences, Tehran, Iran. Fax: þ98 2188622533. ** Corresponding author. Mahdavi Blv, Zanjan University of Medical Sciences, Zanjan, Iran. Fax: þ982433449553. E-mail addresses: [email protected] (F. Shidfar), [email protected] (S.N. Mousavi). http://dx.doi.org/10.1016/j.clnu.2016.09.020 0261-5614/© 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Author's Personal Copy H. Lorvand Amiri et al. / Clinical Nutrition 36 (2017) 1490e1497

1. Introduction Non-alcoholic fatty liver disease (NAFLD) progression, as the one of the common metabolic problems, has a direct link with central obesity, glucose intolerance, inflammatory signaling pathways and dyslipidemia [1]. Central obesity leads to insulin resistance and inflammation which are the main causes of NAFLD [2]. Due to inconclusive results about drugs, weight loss remains the standard of care for the treatment of this condition. Drugs have been variable success on NAFLD patients [3e5]. Because dietary, lifestyle modifications and drugs often fail or cannot be implemented effectively, new pharmacologic agents are needed to be designed and studied for reducing NAFLD progression. Increasing evidence suggest that serum levels of 25(OH)D is associated with the chronic metabolic diseases including insulin resistance (IR), type 2 diabetes, cardiovascular disease, metabolic syndrome and fatty liver [6]. Vitamin D receptor (VDR) regulates lipid and glucose metabolism in the liver [7]. Vitamin D plays as an anti-inflammatory, antifibrotic and insulin sensitizing agent. Then, its deficiency may be involved in the pathogenesis of NAFLD [8]. In addition, emerging evidence indicates that serum calcium is associated with dyslipidemia, glucose disturbances and insulin resistance [9], suggesting physiological link between calcium levels and NAFLD progression. Studies in transgenic mice have shown that calcium supplementation can increase lipolysis and body temperature, as well as reduce fatty acid synthase activity and body weight [10]. It has been claimed that the consumption of calcium supplements may facilitate weight loss. However, a systematic review and RCT failed to find any beneficial effect of calcium supplements on weight loss [11,12]. One study showed the close relationship between NAFLD and both renal dysfunction and bone mineral dysregulation. Subjects at the highest quartile of serum calcium and phosphorus levels had higher dyslipidemia, fatty liver and serum ALT level [13]. Moreover, in some studies there was a direct link between serum calcium levels and the key pathogenic causes of NAFLD (IR) [14,15]. Despite some evidence, although inconclusive, of the beneficial effects of calcium supplements on weight loss as the best approach to control NAFLD, no study, to our knowledge, has evaluated whether calcitriol supplementation with calcium, compared with calcitriol alone, results in a greater beneficial effect on disease progression in NAFLD patients when they are following an energy-restricted diet with controlled physical activity. The purpose of this study was to compare the effects of calcitriol supplementation with and without calcium with respect to body weight (and body fat mass) as a primary outcome, as well as carbohydrate and lipid metabolism as secondary outcomes, in NAFLD patients after a 12-wk weight-loss program. 2. Subjects and methods 2.1. Study participants and recruitment In total,120 NAFLD patients were selected between June 2015 and March 2016 from patients with NAFLD diagnosis who were attending the Rasool-Akram Hospital, Tehran, Iran. Inclusion criteria were as follows: patients aged 18e65 y, BMI (in kg/m2) of 25e35, serum 25 (OH) D level< 15 ng/ml, reporting a daily calcium intake 700e800 mg/day, and willingness to introduce a dietary change to lose weight. The exclusion criteria were as follows: reporting a daily calcium intake800 mg/day (in diet or as a supplement), drugs for blood glucose or lipid control, pregnancy or having given birth in the past year or planning a pregnancy in the next 6 mo, lactation, weight loss of 10% of body weight within the 6 mo before enrollment in the study, participation in a competitive sport, abnormal thyroid hormone concentration, intake of medications that could affect body weight and/or energy expenditure,

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allergy, smoking, diagnosis of chronic diseases including inflammatory diseases, heart, liver and renal failure, cancer, acute myocardial infarction, diabetes, stroke, or serious injuries and any other conditions that were not suitable for the trial as evaluated by the physician. The study was approved by the Ethical Committee of Iran University of Medical Sciences, Tehran, Iran. All subjects provided their signed consent before study enrollment. 2.2. Study design and interventions A randomized, double-blind, controlled trial was designed that aimed to the effects of calcitriol supplementation with and without calcium on weight loss, body fat, fasting plasma glucose (FPG), serum insulin concentrations, lipid profiles, and liver function tests in NAFLD patients. After measuring baseline variables (e.g. age, height, weight, …), eligible participants were randomly assigned by using a computer-generated random-numbers method by the project coordinator. One hundred-twenty subjects who were eligible for the study were randomly assigned to one of the 3 groups: Calcitriol and placebo (D þ P) group (n ¼ 37) received one tablet containing 1000 IU of vitamin D (25 mg/d as calcitriol; Jalinus Arya Co, Iran) plus exactly the same calcium carbonate placebo (25 mg/d as lactose; Jalinus Arya Co, Iran), calcium þ calcitriol (Ca þ D) group (n ¼ 37) received a 500 mg calcium tablet (500 mg/ d as calcium carbonate; Jalinus Arya Co, Iran) plus one tablet containing 1000 IU of vitamin D (25 mg/d as calcitriol; Jalinus Arya Co. Iran), placebo þ placebo (P þ P) group (n ¼ 36) received placebo of calcitriol and placebo of calcium (25 mg/d as lactose; Jalinus Arya Co. Iran) after lunch with a glass of water for 12 weeks. Shape, color and packaging of placebo were exactly similar to the supplements in the supplemented group. Products were administered by a blinded researcher assistant to blinded patients (Fig. 1). 2.3. Screening visit, diet and physical activity planning At the screening visits, new diagnosed NAFLD patients proven by ultra-sonography underwent an examination by an Internist. In this study, the dietary program was designed to enable weight loss of 5% of starting body weight. The dietary program was designed to introduce a 500-kcal energy deficit based on estimated energy requirements at the start of the study by an expert dietitian. Diets containing 55% of calorie as carbohydrate, 15% of calorie as protein and 30% of calorie as fat were designed for all the participants. A prescribed diet was designed for each participant and during a council meeting, details explained to the participants. Physical activity level was assessed by an expert sport specialty by International Physical Activity questionnaire (IPAQ) at the beginning of the study, which was not significantly different among the groups. Daily 30 min moderate walking advised to all the participants and compliance was assessed throughout the study in 3 days (two regular days and one holiday) in each month. Compliance with the supplement consumption instructions and dietary program were monitored once a week by telephone interviews and double checked by using a 3d dietary food recall questionnaire that was completed 3 times during the study period (baseline and at the end of the 6th weeks and 12th weeks of intervention). This dietary intake data was analyzed by the N4 software (Nutritionist 4, First Data Bank, San Bruno, CA, USA). Researchers advised to the participants which control the direct sun-light exposure to less than 1 h/day. Duration of sun-light exposure was monitored once a week in each telephone interview. 2.4. Measurements Anthropometric measurements of all subjects were taken at the baseline and after 12 wk (except height, which was taken only at

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Fig. 1. Screening, enrollment, random assignment, and follow-up of study participants.

the first visit) by the dietitian. Blood samples of all subjects were taken from the antecubital vein after 10e12 h fasting, between 0700 and 0800 AM, at baseline and at 12 wk for biochemical measurements. After centrifugation for 20 min (3000 g), the serum samples were frozen simultaneously and stored at 80  C until analyzed. In order to eliminate the probable effects of sex hormones on blood lipids, blood sampling was not performed between days 1 and 5 of the menstrual cycle in women. At the baseline and after 12 weeks of treatment, the liver fat accumulation, serum lipid profile, FPG and insulin levels were measured for the both groups. Fasting blood glucose, lipid profile and liver enzymes were measured by an enzymatic method (Pars Azmoon Co. kit, Tehran, Iran) using Liasys autoanalyzer while insulin was measured by IRMA method (Immunotech Co. kit); homeostasis model assessment for insulin resistance (HOMA-IR) [16] [fasting insulin (mU/ L)  FPG (mmol/L)]/405 was used as the major outcome measurement. 25-OH vitamin D was measured by ELISA kit (SE120139, Sigma Aldrich). Body weight was taken to the nearest 0.1 kg by using a calibrated Seca scale to the nearest 100 g with light clothing and no shoes. Body height was measured to the nearest 0.5 cm by using a wall-mounted stadiometer (Seca) in a barefoot and freestanding position. Waist circumference was measured with a rigid measuring tape and recorded to the nearest 0.5 cm. Waist circumference was measured at the halfway between the lower rib and the iliac crest [17]. BMI (Body Mass Index) was calculated according to the formula: BMI ¼ weight/height2 (kg/m2). The demographic data were collected during the initial anthropometric assessment.

2.5. Grades of fatty liver classification Liver ultrasound device Siemens brand Sonoline G50 series and 3.5e5 MHz probe made of Germany was used for liver sonography. Liver steatosis classified through sonographic echogenicity of liver as: 1) normal: echogenicity as the same as renal cortex 2) grade I: mild steatosis; increased hepatic echogenicity with visible periportal and diaphragmatic echogenicity 3) grade II: moderate steatosis; increased hepatic echogenicity with imperceptible periportal echogenicity, without obscuration of diaphragm 3) grade III: sever steatosis; increased hepatic echogenicity with imperceptible periportal echogenicity and obscuration of diaphragm [18]. 2.6. Sample size and statistical analysis In designing of the study, we considered a power of 80% with a two-sided test with a ¼ 0.05 (type I error) and standard deviation (SD) difference of 3.5 for waist circumference and 2 for BMI. On the basis of SDs, reported in similar studies [19], the number of subjects needed to be treated in order to detect this difference was 36/ group. Given an anticipated dropout rate of 10 percent, we set the enrollment target at 40 subjects. All data were expressed by means ± SD. The level of significance was set at P < 0.05. Statistical analyses were performed with IBM SPSS Statistics software (version 18; IBM Corp). Normal distribution of the variables was checked by Kolmogorov Smirnov Test. One-way ANOVA followed by post-hoc test was used to test whether the differences between the mean values of the items studied in the groups were significant. The comparison of mean values of variables

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Table 1 Daily dietary intake of energy and some nutrients of two groups of the study. Dietary intake Total Energy:(Kcal) Before intervention After intervention Total protein(g/day) Before intervention After intervention Total carbohydrate(g/day) Before intervention After intervention Total fat(g/day) Before intervention After intervention SFA1 (g/day) Before intervention After intervention PUFA2 (g/day) Before intervention After intervention MUFA3 (g/day) Before intervention After intervention Fiber (g/day) Before intervention After intervention Vitamin C (mg/day) Before intervention After intervention Vitamin E (mg/day) Before intervention After intervention calcium (mg/day) Before intervention After intervention Vitamin D (mg/day) Before intervention After intervention

P þ P (n ¼ 36)

D þ P (n ¼ 37)

Ca þ D (n ¼ 37)

p value*

2157.5 ± 437.1 1657.8 ± 391.3

2160.2 ± 385.2 1667.5 ± 345.9

2162.3 ± 426.4 1658.5 ± 364.7

0.4 0.5

75.8 ± 19.9 60.1 ± 17.1

79.8 ± 20 62.4 ± 22.7

76.6 ± 19.5 60.5 ± 19.3

0.1 0.1

298.42 ± 72.6 223.9 ± 68.52

296.72 ± 52.33 216.3 ± 63.21

295.9 ± 68.5 219.8 ± 62.3

0.31 0.8

73.3 ± 20 57.9 ± 19.5

72.8 ± 24.3 60.3 ± 24.7

73.9 ± 22.6 59.7 ± 23.3

0.8 0.51

13.6 ± 6.5 3.4 ± 6.1

13 ± 6.3 3.5 ± 6.6

12.8 ± 5.9 3.9 ± 6.5

0.42 0.52

43.4 ± 14.7 33.4 ± 19.2

37.7 ± 17.9 32.1 ± 21.4

39.2 ± 16.4 33.6 ± 20.8

0.9 0.4

6.1 ± 4.5 21.1 ± 2.3

12.5 ± 3.1 24.7 ± 2.7

11.9 ± 4.2 22.2 ± 3.1

0.54 0.6

16.7 ± 7.6 13.9 ± 9.8

14.8 ± 5.8 13.8 ± 5.5

15.4 ± 6.7 13.5 ± 7.6

0.3 0.7

127.8 ± 85.8 130.9 ± 83.3

107.3 ± 75.9 110.3 ± 74.8

1165 ± 81.8 124.6 ± 79.9

0.35 0.34

10.7 ± 8.1 10.6 ± 7.9

13.1 ± 7.5 12.7 ± 7.1

12.4 ± 7.9 11.5 ± 6.9

0.25 0.3

777 ± 386 725 ± 454

773 ± 586 729 ± 533

758 ± 432 792 ± 214

0.8 0.63

0.39 ± 0.37 0.37 ± 0.35

0.53 ± 0.6 0.4 ± 0.47

0.51 ± 0.4 0.39 ± 0.2

0.32 0.75

Data are expressed as means ± SD. *Differences between groups were evaluated by independent t-test. p þ p, placebo þ placebo group; D þ P, calcitriol þ placebo group; Ca þ D, calcium þ calcitriol group. 1 SFA: Saturated Fatty Acid. 2 PUFA: Polyunsaturated Fatty Acid. 3 MUFA: Monounsaturated Fatty Acid.

before and after the intervention in each group was examined by paired t-tests. ANCOVA test was used to compare mean difference of outcomes, adjusted for the baseline measures as covariate. Chisquare test was used to compare the qualitative variables before and at the end of the study. 3. Results 3.1. Baseline characteristics From 122 NAFLD-proven patients with serum 25(OH) D levels lower than15 ng/ml, 2 patients had not inclusion criteria. The 120 remaining patients gave written consent and participated in the study. There was no significant difference in energy and nutrient intake at the beginning of the study among groups and also no significant change was seen at the end of the study (Table 1). The level of physical activity was not different among groups at the baseline, as well as at the end of the study. At baseline, there were no statistically significant differences in physical characteristics and biochemical measurements among the intervention groups (Table 2). 3.2. Anthropometric measurements Time of sun-light exposure (hr/day) was not significantly different among the groups (p > 0.05). As shown in Table 4, there

Table 2 Patient characteristics before the intervention.a Variables

P þ P (n ¼ 36)

D þ P (n ¼ 37)

Ca þ D (n ¼ 37)

Age, y Sex

44±10.8b 35.3% 64.7% 7% 32.5% 60.5% 25% 75% 91.2 ± 2.3 31.3 ± 0.58 0.98 ± 0.01 34.9 ± 1.3 86.7% 90.6 ± 1.1 16.1 ± 0.94 3.6 ± 0.22 189.7 ± 5.5 113.1 ± 4.2 38.7 ± 1.6 199.4 ± 14.9 46.5 ± 3.01 31.1 ± 1.6 10 ± 0.63

39.8 ± 11 40.5% 59.5% 5% 30% 65% 32.5% 67.5% 90.1 ± 2.3 30.3 ± 0.64 0.97 ± 0.01 35.3 ± 1.2 87.5% 92.2 ± 0.84 19.04 ± 1.1 4.3 ± 0.25 185.1 ± 5.3 111.5 ± 4.9 36.3 ± 1.1 197.6 ± 13.02 45.9 ± 2.4 30.4 ± 1.3 9.9 ± 0.64

38.3 ± 10.1 37.8% 62.3% 7% 35.1% 57.9% 30% 70% 89.5 ± 3.1 30.5 ± 0.93 0.99 ± 0.01 38.4 ± 0.98 82.2% 89.1 ± 1.4 18.1 ± 0.97 3.9 ± 0.21 198.3 ± 6.1 122.1 ± 4.2 35.1 ± 0.8 211.8 ± 10.4 50.2 ± 2.6 32.5 ± 1.2 9.9 ± 0.64

Female, % Male, % Education Illiterate, % Diploma, % College, % Job Employee Self-employed Body weight, Kg BMI, Kg/m2 WHR Body fat, % Married, % FPG, mg/dl Insulin (mU/L) HOMA-IR TC, mg/dl LDL cholesterol, mg/dl HDL cholesterol, mg/dl TG, mg/dl ALT, mmol/L AST, mmol/L 25 (OH) D, ng/ml

a Group difference, p > 0.05. WHR, waist to hip ratio; FPG, fasting plasma glucose; TC, total cholesterol; TG, triglyceride; ALT, alanine aminotransferase; AST, aspartate aminotransferase; P, placebo. b Means ± SE (all such values).

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Table 3 Grade of fatty liver in the groups before and after the 12-wk intervention. Group

Grade of fatty liver

Before N (%)

After N (%)

Changesa, N

*

PþP

Normal Stage 1 Stage 2 Stage 3 Normal Stage 1 Stage 2 Stage 3 Normal Stage 1 Stage 2 Stage 3

0 22 (61.1) 11 (30.6) 3 (8.3) 0 19 (51.3) 16 (43.2) 2 (5.4) 0 15 (40.5) 19 (51.4) 3 (8.1)

3 (8.3) 19 (52.8) 12 (33.3) 2 (5.6) 15 (40.5) 20 (54) 2 (5.4) 0 10 (27) 25 (67.6) 2 (5.4) 0

Without change: 32 1 reduction: 4 2 reduction: 0