efficacy of docosahexaenoic acid -choline -vitamin e (dha ... · draft 1 efficacy of...

Draft

Efficacy of docosahexaenoic acid-choline-vitamin E (DHA-

CHO-VE) in paediatric NASH:

a randomized controlled clinical trial

Journal: Applied Physiology, Nutrition, and Metabolism

Manuscript ID apnm-2016-0689.R2

Manuscript Type: Article

Date Submitted by the Author: 11-Apr-2017

Complete List of Authors: Zöhrer, Evelyn; Medizinische Universitat Graz Alisi, Anna; Bambino Gesù Children’s Hospital – IRCCS Jahnel, Jörg; Medizinische Universitat Graz Moscla, Antonella; Bambino Gesù Children’s Hospital – IRCCS Della Corte, Claudia; Bambino Gesù Children’s Hospital – IRCCS Crudele, Annalisa; Bambino Gesù Children’s Hospital – IRCCS Fauler, Günter; Medizinische Universitat Graz Nobili, Valerio; Bambino Gesù Children’s Hospital – IRCCS

Is the invited manuscript for consideration in a Special

Issue? :

Keyword: docosahexaenoic acid, choline, vitamin E, non-alcoholic fatty liver disease, children

https://mc06.manuscriptcentral.com/apnm-pubs

Applied Physiology, Nutrition, and Metabolism

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1

Efficacy of docosahexaenoic acid-choline-vitamin E (DHA-CHO-VE) in paediatric NASH:

a randomized controlled clinical trial

Evelyn Zöhrer1*

, PhD, and Anna Alisi2*

, PhD, Jörg Jahnel1, MD, Antonella Mosca

3, MD,

Claudia Della Corte3, Annalisa Crudele

2, BSc, Günter Fauler

4, PhD, Valerio Nobili

2, 3, MD

Affiliations:

1Department of Pediatrics and Adolescent Medicine, Medical University Graz, Graz, Austria;

2Liver Research Unit, Bambino Gesù Children’s Hospital – IRCCS, Rome, Italy;

3Hepato-Metabolic Disease Unit, Bambino Gesù Children’s Hospital – IRCCS, Rome, Italy

4Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz,

Graz, Austria.

* Evelyn Zöhrer and Anna Alisi contributed equally to this work.

Running title: Combination therapy in paediatric NASH

Address correspondence to: Valerio Nobili, MD, Bambino Gesù Children’s Hospital IRCCS

(Instituto di Ricovero e Cura a Carattere Scientifico), P.le S. Onofrio, 4 – 00165, Rome, Italy,

Phone/Fax: +39-06-68592192, e-mail: [email protected]

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Abstract

Non-alcoholic steatohepatitis (NASH), a progressive form of non-alcoholic fatty liver disease

(NAFLD), is one of the most common hepatic diseases in children. We conducted a randomized

controlled clinical trial on children with biopsy-proven NASH based on a combinatorial nutritional

approach compared to placebo.

Participants were assigned to lifestyle modification plus placebo, or lifestyle modification

plus a mix containing docosahexaenoic acid, choline and vitamin E (DHA-CHO-VE). Forty

children and adolescents concluded the trial. The primary outcome was the improvement of liver

hyperechogenicity. Secondary outcomes included alterations of ALT and other metabolic

parameters. Furthermore, changes of serum bile acids (BA) and plasma fibroblast growth factor 19

(FGF19) levels were evaluated as inverse biomarkers of disease severity.

At the end of the study, we observed a significant decrease in severe steatosis in the

treatment group (50% to 5%, p=0.001). Furthermore, although the anthropometric and biochemical

measurements in the placebo and DHA-CHO-VE groups were comparable at baseline, at the end of

the study ALT and fasting glucose levels improved only in the treatment group. Finally, we found

that BA levels were not influenced whereas FGF19 levels were significantly increased by DHA-

CHO-VE.

The results suggest that a combination of DHA, vitamin E and choline could improve

steatosis and reduce ALT and glucose levels in children with NASH. However, further studies are

needed to assess the impact of a DHA and vitamin E combination on repair of liver damage in

paediatric NASH.

Key words: bile acids, choline; docosahexaenoic acid; FGF19; non-alcoholic fatty liver disease;

steatosis; vitamin E.

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Introduction

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disease that is becoming more

prevalent due to a rapid rise in obesity. In fact, NAFLD is currently the most common chronic

hepatopathy in both adults and children in most of the western world (Loomba and Sanyal 2013;

Nobili et al. 2013). Besides obesity, other factors affect the prevalence of paediatric NAFLD,

including genetic susceptibility, social-environmental variables and dysmetabolism (Bedogni et al.

2005). Metabolic syndrome traits, including insulin resistance (IR), dyslipidaemia and type two

diabetes, are well known risk factors associated with NAFLD in children (Nobili et al. 2015; Nobili

et al. 2016).

Since in children with NAFLD clear guidelines on pharmacological interventions are still

lacking, lifestyle interventions designed to reduce body weight, including diet and exercise, remain

the first-line treatments (Africa et al. 2016). However, several lines of evidence demonstrate that in

children, as well as in adults, lifestyle interventions improve only metabolic parameters and simple

steatosis but not the histological features of non-alcoholic steatohepatitis (NASH), which is the

most severe form of NAFLD (Vilar-Gomez et al. 2015; Nobili et al. 2008). Therefore, during the

last several decade drugs acting upon mechanisms that are involved in the pathogenesis of NAFLD,

including insulin resistance (IR) and oxidative stress, also have been tested in paediatric settings

(Nobili et al. 2008; Lavine et al. 2011).

To discover the medication and dose that are the best for children with NASH, while

avoiding adverse pharmacological effects, recent studies have evaluated the effects of nutritional

supplementation, such as vitamins, probiotics and omega-3 fatty acids (Nobili et al. 2013; Nobili et

al. 2014a; Alisi et al. 2014; Janczyk et al. 2015). Lavine et al. (2011) have reported that vitamin E

favourably affects hepatocellular ballooning. We have demonstrated that treatment with

docosahexaenoic acid (DHA), an omega-3 fatty acid, may improve liver steatosis, insulin sensitivity

and hepatocellular necro-inflammation in children with NASH (Nobili et al. 2013; Nobili et al.

2014a). However, none of the tested therapeutic approaches has proved entirely satisfactory,

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suggesting that a mix of multiple drugs and/or dietary supplements targeting specific pathogenic

features could be more effective (Alisi et al. 2012).

The liver is centrally responsible for the metabolism of choline, an essential nutrient.

Choline is mainly phosphorylated and used to make phospholipids, or oxidized and used as a major

source of methyl groups (Corbin and Zeisel 2012). An important choline metabolite in liver is

phosphatidylcholine (PC), required for the generation of very low density lipoprotein (VLDL), the

lipoprotein involved in the export of fat from liver. Impaired biosynthesis of VLDL is a crucial

mechanism in hepatic steatosis development (Vance 2008). In addition, PC is a major component of

cellular membranes and decrease in PC synthesis can impair the integrity of the hepatocyte

membrane, leading to ALT increase and NASH (Li et al. 2006). However, choline is derived not

only from the diet. In fact, endogenous biosynthesis of PC may occur in liver, catalysed by

phosphatidylethanolamine N-methyltransferase (PEMT) (Corbin and Zeisel 2012). Moreover,

choline may be also oxidized to betaine in humans; both betaine and choline are methyl donors in

pathways involving the re-methylation of homocysteine to methionine, thereby reducing blood

levels of homocysteine, which are positively correlated with NAFLD severity in both adults and

children (de Carvalho et al. 2013; Pastore et al. 2014). These findings, together with experimental

studies demonstrating that a choline-deficient diet may predispose to NAFLD in rodent models,

strongly suggest that choline supplementation could ameliorate liver damage in NAFLD (Al Rajabi

et al. 2014).

Here, we report the results of a randomized clinical trial to investigate the effect on

paediatric NAFLD of these mixed compounds compared with placebo.

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Patients and Methods

Study population and design

Sixty Italian children or adolescents (age range, 4-16 years) referred to the Hepato-metabolic

Disease Unit of Bambino Gesù Children Hospital (Rome, Italy) with liver biopsy-proven NASH

and without other causes of liver disease were screened for the study. Excluded were patients with

Wilson disease, hepatitis B and C, acute systemic disease, autoimmune hepatitis, hypothyroidism,

cystic fibrosis, celiac disease, suspected muscular dystrophy, alpha-1-antitrypsin deficiency and

other metabolic inherited diseases. Patients were also excluded if body weight and carbohydrate

metabolism were altered by parenteral nutrition, protein malnutrition, previous gastrointestinal

surgery, structural abnormalities of the gastrointestinal tract or neurological impairment. Finally, the

use of nonsteroidal anti-inflammatory drugs, antibiotics, probiotics or antisecretory drugs capable of

causing achlorhydria within the two months before enrolment was also an exclusion criterion.

Forty-three of 60 screened children with NAFLD met these inclusion criteria: age 4-16 years [13

(30.2%) children and 30 (69.8%) adolescents], persistently elevated serum aminotransferase levels,

diffusely echogenic liver on imaging studies suggestive of fatty liver and biopsy findings consistent

with NASH. All these children were enrolled in this placebo-controlled clinical trial.

Children were randomized by computer to receive every day for six months either pearls

combining 250 mg of DHA, 39 UI of vitamin E and 201 mg of choline (DHA-CHO-VE; Pro DHA

Steatolip Plus®) or externally identical pearls as placebo. Both were provided by DMF Dietetic

Metabolic Food (Italy). Concomitantly, all patients were recommended to follow a hypocaloric diet

(25-30 kcal/kg/day) and to engage in twice weekly 1-hour physical activity during the treatment and

for further 6 months of follow-up. Compliance with treatment and diet was monitored on monthly

visits by counting the amount of investigational product left in the bottle.

Complete medical histories were recorded for all participants. Anthropometrical data,

biochemical data and liver ultrasound findings were collected at baseline and after 12 months from

trial start. Patients and investigators were blinded before and after intervention assignment.

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Symptoms and side effects were assessed at each visit by an investigator. Adverse events were

defined as injuries caused by the treatments under study. At each visit, parents were specifically

asked about adverse events, and the first author checked for any association between the adverse

events and morbidity.

Clinical and research investigators of the Bambino Gesù Children Hospital designed the

study and managed the DHA-CHO-VE trial. The Hospital Ethics Research Committee approved the

study, in accordance with the Declaration of Helsinki (as revised in Seoul, Korea, October 2008),

and monitored that all experimental procedures were carried out in accordance with the relevant

guidelines included in the protocol. Parents of the included patients gave their written informed

consent to therapies, to the tests performed and to publication of the results. The study was

registered at ClinicalTrials.gov as NCT01934777 on 30 August 2013. However, we underline that

the current primary endpoint differs from that in the protocol submitted to ClinicalTrials.gov

because liver biopsy at 12 months in the placebo group was not performed for ethical reasons. The

original trial, in fact, had as primary endpoint improvement in NAFLD Activity Score (NAS) and as

secondary endpoints all parameters of metabolic syndrome and bright liver at ultrasonography.

Physicians responsible for patients decided to change the primary endpoint after the trial had begun

but when data were still blinded. Thus, at the end of the trial (12 months), each participant/guardian

was informed of his or her group in the study and, if he or she belonged to the DHA-CHO-VE

group, we proposed a second liver biopsy. Consequently, we compared, as primary endpoint,

improvement in liver hyperechogenicity in the DHA-CHO-VE group with that in the placebo group

after 12 months. The secondary endpoints were amelioration in ALT and in other metabolic

parameters in both groups. The safety of the mixture was also evaluated, monitoring possible side

effects.

Anthropometrics

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Anthropometric data were collected at baseline and after 12 months. Weight was measured

using a conventional scale with a precision of 100 g and height was measured by a Harpenden

stadiometer with a precision of 1 mm. Body mass index (BMI) was expressed in kilograms per

square meters (kg/m2). Waist circumference (WC) was evaluated by using a tape to the nearest 0.5

cm measure, at the narrowest circumference between the lower costal margin and the iliac crest in

standing position.

Laboratory parameters

Blood collected after overnight fasting at baseline and at month 12 was immediately

analysed by standard laboratory methods for alanine aminotransferase (ALT), aspartate

aminotransferase (AST) and gamma-glutamyl-transferase (GGT) activity and for total triglycerides,

cholesterol, glucose and insulin. Insulin resistance was evaluated by the homeostatic model

assessment (HOMA) equation [fasting insulin (µU/mL) X fasting glucose (mg/dL)/405] (Matthews

et al. 1985).

Ultrasound

The same blinded physician evaluated ultrasound (US) liver brightness at baseline and after

the 12-month treatment. Liver US was performed by an experienced radiologist, using an Acuson

Sequoia C512 scanner equipped with a 15L8 transducer (Universal Diagnostic Solutions,

Oceanside, CA). Normal liver without steatosis (grade 0) was defined as having normal liver echo-

texture; mild steatosis (grade 1) as slight and diffuse increase in fine parenchymal echoes with

normal visualisation of diaphragm and portal vein borders; moderate steatosis (grade 2) as a

moderate and diffuse increase in fine echoes with slightly impaired visualisation of diaphragm and

portal vein borders; and severe steatosis (grade 3) as fine echoes with poor or no visualisation of

diaphragm, portal vein borders and posterior portion of the right lobe.

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Liver biopsy

As already stated, for ethical reasons set out in the position paper of the European Society of

Paediatric Gastroenterology, Hepatology and Nutrition (Vajro et al. 2012), liver biopsy was

performed at baseline (T0) in both groups and after 12 months only in the treatment arm.

US-guided liver biopsies were fixed in 10% buffered formalin and interpreted by an

experienced pathologist (RDV) employing criteria proposed by the NAFLD Clinical Research

Network (Kleiner et al. 2005). Steatosis was graded 0–3 (0 = <5% steatosis, 1 = 5–33%, 2 = 33–

66% and grade 3 = >66%); lobular inflammation was scored by number of inflammatory foci per

200 per field (0 = no inflammatory foci, 1 = <2 foci; 2 = 2–4 foci and 3 = >4 foci); ballooning was

graded 0–2 (0 = none; 1 = few balloon cells present and 2 = prominent ballooning). Portal

inflammation (PI; 0 = no PI, 1 = mild PI, 2 = more than mild) was scored as proposed by Brunt et

al. (2009). Fibrosis was staged 0–4 (0 = no fibrosis; 1 = periportal or perisinusoidal; 1A = mild,

Zone 3, perisinusoidal; 1B = moderate, Zone 3, perisinusoidal; 1C = portal/periportal; 2 =

perisinusoidal and portal/periportal; 3 = bridging fibrosis and 4 = cirrhosis). A NAS > 5 was used

for further comparisons with variables of interest.

Intestinal fibroblast growth factor 19 assay

Blood was centrifuged at 2000 g for 12 min and plasma was stored at -80°C pending further

analysis. A specific sandwich enzyme-linked immunosorbent assay was used to determine

fibroblast growth factor 19 (FGF19) levels (Biovendor; Brno; Czech Republic).

Bile acids quantification

For bile acids (BA) quantitation, blood samples were collected from patients after 12 hours

of fasting, with sera immediately separated and stored at -80 °C until assays. Total BA (tBA) and

individual BA levels, including unconjugated and taurine (T)- and glycine (G)- conjugated species,

were measured using high-performance liquid chromatography coupled with tandem mass

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spectrometry (HPLC-MS/MS). A full 15-BA profile was determined using 10 µl of serum; it

included cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic (DCA), lithocholic (LCA),

and ursodeoxycholic (UDCA) acid and their T-conjugates TCDCA, TLCA, TDCA, TCA and

TUDCA and G-conjugates GCA, GCDCA, GLCA, GDCA and GUDCA. Individual BA were

separated by HPLC using a reversed-phase C18 column with a methanol and water gradient.

Deuterium-labeled internal standards served for quantification. Mass spectrometer Q Exactive™

MS/MS (Thermo Fisher Scientific, Waltham, MA) and high-performance quadrupole precursor

selection with high-resolution and accurate-mass (HR/AM) Orbitrap™ detection were used for

identification of single BA.

Statistical analysis

The SPSS Statistics package 21 (IBM SPSS, Armonk, NY) was used for statistical analyses

of data. Patient characteristics and biochemical variables are expressed by mean ± standard

deviation (SD) or number (%) as appropriate. The p value used for checking significance level was

<0.05. To compare all studied variables between groups, independent sample t-testing for

quantitative data was used. Comparisons between groups were performed using analysis of

variance. Difference between proportions were evaluated by Chi-square testing. Pearson's

correlation test was used to evaluate a possible correlation between BA and FGF19 or biochemical

variables and histological pattern in NAFLD patients. Furthermore, multivariate regression analysis

was used to test the independence of associations between steatosis and anthropometrics and

laboratory parameters.

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Results

Effect of DHA-CHO-VE on liver histology

Forty children with biopsy-proven NASH (of 60 screened and 43 enrolled) completed the

trial (Supplementary Figure S1). No adverse events were reported. Table 1 shows all histological

traits and score assessed at baseline. Histological traits did not differ significantly between placebo

and treatment group at baseline. Comparing the treatment group over the time, we found some

interesting differences. In particular, we found a significant improvement in steatosis (p = 0.01),

ballooning (p = 0.001) and NAS (p = 0.0003) at 12 months compared to baseline data (Table 2).

Second liver biopsy was not offered to the placebo group.

Effect of treatments on liver echogenicity and metabolic parameters

Changes in hepatic US findings differed between the placebo and DHA-CHO-VE groups.

Patients in the DHA-CHO-VE group exhibited significant improvement in liver steatosis at 12

months (Table 3), with severe steatosis significantly decreased from 50% to 5% of patients

(p=0.001).

Anthropometric and biochemical parameters were similar in the placebo and DHA-CHO-VE

groups at entry (Table 4). However, after 12 months, statistically significant improvement in ALT

and fasting glucose levels was found in the treatment group only.

Effect of treatment on BA metabolism

Serum BA levels are low in non-fibrotic and fibrotic patients with NAFLD potentially

caused by metabolic changes, particularly by hyperinsulinemia (Jahnel et al. 2015). Al Rajabi et al.

(2014) demonstrated that choline supplementation normalized hepatic cholesterol levels and

circulating levels of BA, thus preventing progression to NASH and liver failure in Pemt−/−

/Ldlr−/−

mice on a high fat diet. Therefore, we evaluated if DHA-CHO-VE supplementation restored serum

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levels of BA. As shown in Figure 1, mean levels of serum BA after 12 months of DHA-CHO-VE

treatment were similar to those observed at baseline both in placebo (1.48±1.21 µM ; 1.91±1.85

µM) and in treatment group (1.97±1.91 µM ; 1.10±1.07 µM). No significant changes after 12

months were seen in G- and T-conjugates in the placebo group (G-conjugates: 1.02±1.01 µM;

0.80±0.79 µM and T-conjugates: 0.03±0.02 µM; 0.05±0.02 µM) or after DHA-CHO-VE treatment

(G-conjugates: 1.23±1.11 µM; 0.55±0.47µM and T-conjugates: 0.08±0.04 µM; 0.01±0.01 µM).

Effect on FGF19 circulating levels

Circulating FGF19 levels are inversely correlated with fibrosis grade and positively

correlated with UDCA levels in children with NAFLD (Alisi et al. 2013; Jahnel et al. 2015).

Moreover, FGF19 may lower serum glucose levels in diabetic mice (Fu et al. 2004). Therefore, we

analysed FGF19 circulating levels and investigated their possible correlations with fasting insulin,

fasting glucose and individual BA in placebo and DHA-CHO-VE groups. In the placebo group, we

found that FGF19 levels were similar at baseline and after 12 months: 41.3±18.9 pg/mL (p =0.14) ;

56.6±33.9 pg/mL (Figure 2). By contrast, in the DHA-CHO-VE group FGF19 levels were

significantly (p <0.0001) higher after 12 months (110.1±45.3 pg/mL) than at baseline (46.8±28.1

pg/mL) (Figure 2). No statistically significant correlation was found between serum total BA levels

and improvement in liver steatosis or in metabolic parameters. However, Pearson's correlation

analysis with individual BA showed that UDCA levels were positively associated with FGF19

levels (0.828; p <0.0001).

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Discussion

In this study, we evaluated the efficacy and safety of DHA-CHO-VE in the treatment of

obese children with NASH. The results showed that, at the end of 6 months of treatment and 6

months of follow-up, the DHA, choline and vitamin E mixture improved steatosis, ALT activity and

fasting glucose and was not associated with any adverse events. The effect on the histologic pattern

of NASH was evaluated only in patients undergoing DHA-CHO-VE treatment, who at 12 months

showed a significant improvement in steatosis, ballooning and NAS.

To the best of our knowledge, this is the first study that investigates the effects of a mix of

three different nutritional supplements combined with diet and exercise on paediatric NAFLD.

Previous clinical studies, in fact, were based on a single treatment combined with lifestyle

intervention (Nobili et al. 2008; Lavine et al. 2011; Nobili et al. 2013). We previously reported that

vitamin E alone improved liver function and promoted normalisation of ALT levels but it had no

effect on hepatic histopathologic features of injury associated with paediatric NAFLD (Nobili et al.

2008; Nobili et al. 2006). By contrast, treatment of NAFLD In Children study reported that vitamin

E administration, though not reducing ALT levels, improved hepatocellular ballooning in children

with NASH (Lavine et al. 2011). We have demonstrated that the omega-3 DHA improved liver

steatosis, ALT levels, triglyceride levels and IR in paediatric NAFLD in the absence of significant

changes in BMI (Nobili et al. 2013). Furthermore, treatment of NASH children with 250 mg/day

DHA after 18 months ameliorated hepatocyte steatosis, ballooning and NAS. Based on these

findings we hypothesised that the combination between vitamin E and DHA could be more

effective than a single therapy. Furthermore, we decided to add choline since its supplementation

may increase PC-DHA plasma levels (da Costa et al. 2011) and hepatic concentrations of vitamin E

(Borges et al. 2015).

Our results showed that DHA-CHO-VE combination therapy was able to induce a partial

recovery of both steatosis and ballooning, a reduction in NAS and amelioration of ALT and fasting

glucose levels. These results are similar to those previously observed with DHA alone (Nobili et al.

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2013). However, without a parallel single-agent treatment group we cannot exclude that a dose of

vitamin E lower than in previous studies (39IU vs. 600-800IU), along with great variation in dietary

intake of choline, may partially have hidden the effect of supplemental vitamin E and choline. An

additional hypothesis for the apparent lack of an additive effect of choline could be the presence of

single nucleotide polymorphisms in PEMT that impair enzyme activity (da Costa et al. 2011), as it

occurs in subjects with patatin-like phospholipase domain-containing protein 3 gene variants who

are not responders to DHA (Nobili et al. 2014b). In Pemt−/−

/Ldlr−/−

mice, choline supplementation

restored normal levels of circulating levels of BA and prevented NASH progression (Al Rajabi et

al. 2014). We found that circulating BA levels in both the placebo and the DHA-CHO-VE groups,

at baseline as well as at 12 months, were lower than levels in healthy children (Jahnel et al. 2015).

However, BA levels remain unchanged in both placebo and DHA-CHO-VE groups.

BAs are complex regulatory molecules (Fiorucci et al. 2009; Chiang 2013). In the liver, both

free and conjugated BA bind to the ligand-binding domain of farnesoid X receptor (FXR),

inhibiting transcription of CYP7A1, which encodes the rate-limiting enzyme in BA synthesis

(Chiang 2009; de Aguiar Vallim et al. 2013). In the endocrine pathway, intestinal BA activate FXR,

inducing FGF19 synthesis/release, which, in turn, may be transported to the liver to activate the

FGFR4/Klotho-β receptor, inhibiting BA de novo synthesis (Holt et al. 2003; Wu et al. 2007; Lin et

al. 2007). Studies in humans and mice revealed a link between impaired glucose metabolism and

disruption of BA signalling (Lefebvre et al. 2009). Additionally, FXR deficiency is associated with

IR and impaired glucose tolerance in mice (Cariou et al. 2006; Zhang et al. 2006). Human and

animal studies found an association between impaired glucose metabolism and disruption of BA

signalling in diabetes (Duran-Sandoval et al. 2004). Activation of FXR in insulin-resistant ob/ob

mice by the FXR agonist GW4064 led to reduced hyperinsulinemia and improved glucose tolerance

(Cariou et al. 2006). Moreover, diabetic fa/fa rats receiving the FXR agonist 6-ethyl-CDCA showed

an improvement in insulin resistance in liver and skeletal muscle (Fiorucci et al. 2011). In patients

treated with DHA-CHO-VE, we found an improvement in fasting glucose without any association

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between BA levels and fasting glucose or insulin. However, we found that UDCA positively

correlated with circulating levels of FGF19. Furthermore, our results demonstrated that FGF19

levels were significantly higher in the DHA-CHO-VE group after 12 months. These data accord

with our previous studies reporting diminished FGF19 levels in paediatric NAFLD and FGF19

levels inversely correlated with progression to liver fibrosis but positively correlated with UDCA

levels (Alisi et al. 2013; Jahnel et al. 2015). By contrast, high FGF19 levels in the treatment group

may suppress BA de novo synthesis via different pathways (Wu et al. 2011), and short term UDCA

administration may stimulate BA synthesis by reducing circulating FGF19 and FXR activation

(Mueller et al. 2015). Therefore, the recurrent positive association between UDCA and FGF19

levels that we found in children with NAFLD is still far from being explained.

This study displays some limitations: i) the lack of an end-of-study liver biopsy in the

placebo group (avoided for ethical reasons); ii) inability to separate the individual effects of DHA,

vitamin E and choline; iii) inability to perform sample size calculation. However, it is the first

clinical trial to combine choline with two agents known to be efficacious (DHA and vitamin E) in a

paediatric setting (Lavine et al. 2011; Nobili et al. 2013) and its results demonstrate that DHA-

CHO-VE combination therapy is well-tolerated and is able to improve steatosis, ALT and fasting

glucose levels in children with NASH. Further long-term studies, as well as studies addressing

genetic contributions to individual variation in drug response, are needed to assess the impact of a

DHA, vitamin E and choline combination on repair of liver damage and hepato-metabolic

alterations in paediatric NASH.

Funding Source: VN is supported by the Italian Ministry of Health (Fondi di Ricerca Corrente

2015).

Financial Disclosure: Nothing to disclose.

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Conflict of Interest: The authors have no conflict of interest to disclose.

Contributions

V.N. designed the study; E.Z., A.A., J.J. and A.C. performed analyses; A.M. and C.D.C. collected

and analysed patient data; EZ, A.A., G.F. and V.N. wrote and reviewed the manuscript. All authors

approved the final version of the article, including the authorship list.

Acknowledgement

We thank DMF Dietetic Metabolic Food (Italy) who provided Pro DHA Steatolip Plus® with

verified composition and indistinguishable placebo.

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Table 1: Histology in the treatment group vs. placebo group at baseline.

PLACEBO DHA-CHO-VE

Histological parameters N % N %

Steatosis

0 0 - 0 -

1 8 40 7 35

2 7 35 9 45

3 5 25 4 20

Lobular Inflammation

0 1 5 1 5

1 12 60 11 55

2 7 35 8 40

Portal Inflammation 0 1 5 0 -

1 14 70 13 65

2 5 25 7 35

Ballooning

0 1 5 1 5

1 12 60 11 55

2 7 35 8 40

Fibrosis

0 0 - 0 -

1 8 40 4 20

2 12 60 16 80

NAFLD Activity Score (NAS)

1 0 - 0 -

2 1 5 1 5

3 4 20 5 25

4 3 15 2 10

5 8 40 10 50

6 3 15 2 10

7 1 5 0 -

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Table 2. Improvement in histologic features, DHA-CHO-VE group.

Histological parameters Baseline 12 months p

Steatosis 1.85 (0.72) 1.05 (0.97) 0.01

Lobular Inflammation 1.15 (0.48) 1.00 (0.32) 0.26

Portal Inflammation 1.35 (0.48) 1.20 (0.60) 0.40

Ballooning 1.35 (0.57) 0.60 (60.67) 0.001

Fibrosis 2.00 (0.80) 1.83 (0.91) 0.10

NAS 4.35 (1.10) 2.65 (1.49) 0.0003

Data are shown with mean ± standard deviation (SD). p<0.05 was considered significant. NAS = NAFLD Activity

Score.

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Table 3. Liver steatosis by ultrasound at baseline and after 12 months in both treatment

groups.

*Chi-square test, p<0.05 was considered significant.

PLACEBO DHA-CHO-VE

% Baseline 12 months p Baseline 12 months p

No steatosis 0 0 - 0 35 -

Mild 25 45 0.18 15 35 0.14

Moderate 40 45 0.75 40 25 0.31

Severe 35 15 0.14 50 5 0.001*

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Table 4. Clinical and laboratory variables in placebo and treatment groups.

PLACEBO TREATMENT

Baseline 12 months p Baseline 12 months p

Sex (M/F) 10/10 10/10 - 14/6 14/6 -

Age (years) 13.2 (2.1) 14.2 (2.1) 0.15 13.2 (2.3) 14.2 (2.3) 0.19

Weight (kg) 66.3 (15.1) 66.4 (15.3) 0.97 68.0 (18.7) 67.3 (14.8) 0.90

BMI (kg/m2) 28.3 (5.3) 28.1 (5.2) 0.90 27.6 (4.0) 27.7 (4.9) 0.71

WC, cm 89.9 (9.7) 89.1 (9.5) 0.79 86.7 (10.3) 90.9 (10.0) 0.22

AST, UI/L 33.1 (18.3) 34.6 (36.3) 0.87 36.2 (13.0) 31.2 (15.0) 0.28

ALT (UI/L) 51.2 (51.6) 32.5 (17.8) 0.14 53.5 (32.6) 35.3 (20.7) 0.04

Uric acid (mg/dL) 6.5 (3.0) 10.5 (17.4) 0.33 5.3 (0.9) 5.2 (1.0) 0.80

Total cholesterol (mg/dL) 154.5 (30.1) 143.4 (17.9) 0.17 150.1 (31.5) 146.5 (28.4) 0.71

LDL cholesterol (mg/dL) 100.8 (37.6) 94.5 (29.7) 0.57 84.0 (29.4) 76.1 (23.7) 0.37

HDL cholesterol (mg/dL) 46.6 (8.3) 48.6 (8.2) 0.46 47.3 (7.9) 49.65 (11.9) 0.48

Triglycerides (mg/dL) 87.2 (46.2) 81.5 (37.2) 0.68 101.2 (51.3) 82.1 (47.6) 0.24

Glucose (mg/dL) 82.5 (7.2) 80.1 (6.5) 0.27 85.40 (7.5) 81.0 (5.3) 0.04

Insulin (mU/L) 22.3 (14.4) 26.2 (21.6) 0.51 21.9 (14.4) 20.7 (12.7) 0.78

HOMA-IR 4.5 (3.0) 5.1 (3.9) 0.64 4.6 (3.1) 4.1 (2.4) 0.53

Except for sex (actual patient numbers), data are shown with mean ± standard deviation (SD). p<0.05 was considered

significant. AST = aspartate aminotransferase; ALT = alanine aminotransferase; HDL = high-density lipoprotein; LDL

= low-density lipoprotein; HOMA-IR = homeostasis model assessment of insulin resistance.

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Figure Legends

Figure 1: Total BA values in treatment vs. placebo groups. Box plot shows the mean effective

serum levels of BA in the placebo and treatment groups. Horizontal line in each box shows mean

and top and bottom lines of boxes show interquartile range (IQR). Circles represent outliers.

Figure 2. FGF19 values in treatment vs. placebo groups. Box plot shows the mean effective

plasma levels of FGF19 in the placebo and treatment groups. Horizontal line in each box shows

mean and top and bottom lines of boxes show interquartile range (IQR). Circles represent outliers.*

= p < 0.01.

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efficacy of docosahexaenoic acid -choline -vitamin e (dha ... · draft 1 efficacy of...

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