This was an open-label, randomized controlled trial in patients with histology-proven NASH. Consecutive patients attending the hepatology clinic of the Prince of Wales Hospital, Hong Kong were screened. Eligible patients were randomized in 1:1 ratio to receive probiotic treatment or usual care for 6 months. Randomization was carried out through the use of computer-generated list of random numbers in blocks of 4. Treatment assignment was kept in sealed envelopes until the time of randomization. All study medications were dispensed by a research nurse. Compliance with the study medication was calculated from the unused sachets of study medications at each visit. The study protocol was approved by the ethics committee of The Chinese University of Hong Kong. All subjects gave informed written consent. While the subjects in both groups did not receive monetary compensation, they received free clinical care and medical tests. The study was registered at ClinicalTrials.gov (NCT00870012).

Patients aged 18 to 70 years had histology-proven NASH, defined as hepatic steatosis of more than 5% and the presence of either hepatocyte ballooning or intralobular hepatocyte necrosis on a liver biopsy performed within 6 months before inclusion. Histological scoring was also performed according to the NASH Clinical Research Network system.22 These patients had elevated alanine aminotransferase (ALT) level of over 30 IU/L in men and 19 IU/L in women. We excluded patients with positive hepatitis B surface antigen, antibody against hepatitis C virus, or anti-nuclear antibody titer above 1/160; significant alcohol consumption (over 20 g per day in men or 10 g per day in women); ALT above 10 times the upper limit of normal; liver decompensation or malignancy; or consumption of systemic corticosteroids or methotrexate in the last 6 months.

At baseline, all NASH patients received lifestyle advice from an investigator. They were instructed to lose weight, reduce fat intake, and exercise at least 3 times per week. Patients randomized to the probiotic group also received one sachet of Lepicol probiotic and prebiotic formula (Healthy Bowels Company Ltd, Birmingham, UK) twice a day for 6 months. The formula contained 5 probiotic cultures including Lactobacillus plantarum (ATCC 14917), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842), Lactobacillus acidophilus (ATCC 4356), Lactobacillus rhamnosus (ATCC 7469) and Bifidobacterium bifidum (ATCC 29521). Each 10 g sachet contained 200 million probiotic cultures. Other ingredients of the formula included 3 g of fructo-oligo-saccharides (prebiotics), cellulose, magnesium stearate, silica and milk.

A research nurse contacted each patient by phone at week 4 to identify new symptoms. Thereafter, the patients were followed up at months 3 and 6. At each visit, new symptoms and concomitant drugs were recorded. Body weight, body height, waist circumference and blood pressure were measured. Waist circumference was measured at a level midway between the lower rib margin and iliac crest with the tape all around the body in the horizontal position. Body mass index (BMI) was calculated as body weight (kg) divided by body height (m) squared. Blood was taken for liver biochemistry, glucose and lipids after fasting for at least 8 h. All biochemical tests were performed using the cobas® 8000 modular analyzer (Roche Diagnostics, West Sussex, England).

Intrahepatic triglyceride content (IHTG) was measured at baseline and month 6 by proton-magnetic resonance spectroscopy using the whole body 3 Tesla scanner (Philips Healthcare, Best, the Netherlands). Whole-body 3.0T scanner with a single voxel point-resolved spectroscopy sequence and an echo time of 40 ms and repetition time of 5,000 ms was used. A survey scan was first performed in the abdominal region to help positioning a volume measuring 20 (AP) x 15 (RL) x 40 (FH) mm within the liver. The scanner’s built-in body coil was used for both signal transmission and reception. No-water-suppressed spectra was acquired using 32 signal averages and the data were exported for offline spectral analysis. Water (4.65 ppm) and lipid (1.3 ppm) peak amplitudes were measured to determine vertebral marrow fat content, which was defined as the relative fat signal amplitude in terms of a percentage of the total signal amplitude (water and fat) and calculated according to the following equation:

Fat content= [Ifat/(Ifat + Iwater)] x 100.

Where Ifat and Iwater are the peak amplitudes of fat and water, respectively. Radiologists performing spectral analysis were blinded to the treatment assignment.

The primary endpoint was the reduction in IHTG from baseline to month 6 as measured by proton-magnetic resonance spectroscopy. Secondary endpoints included changes in ALT, aspartate aminotransferase (AST), BMI, fasting glucose, lipids and liver stiffness measurement by transient elastography. Liver stiffness measurement was performed using the Fibroscan machine (Echosens, Paris, France). The procedure was performed according to the manufacturer’s instructions as described previously.23,24 Liver stiffness measurements were considered reliable only if 10 successful acquisitions were obtained, the success rate was above 60%, and the interquartile range (IQR)-to-median ratio was below 0.3.

Continuous variables were expressed in mean ± standard deviation, or median (IQR) if they were not in normal distribution. Continuous variables were compared between the two treatment arms using unpaired t test or Mann-Whitney U test. Categorical variables were compared using χ2 test or Fisher exact test as appropriate. The change in IHTG within group was tested using paired t test. Statistical significance was taken as a two-sided P value of < 0.05.

This was a pilot study. We considered a reduction of IHTG by 30% to be clinically significant. Assuming that the reduction of IHTG was 30% in the probiotic group and 10% in the usual care group with a standard deviation of 15%, a sample size of 9 patients per arm would achieve 80% power to detect the difference at a 5% significance level. Allowing a 10% dropout rate, a total of 20 patients were required.

At month 6, IHTG by proton-magnetic resonance spectroscopy decreased from 22.6 ± 8.2% to 14.9 ± 7.0% in the probiotic group (mean reduction 7.7%; 95% confidence interval 0.7%, 14.7%; P = 0.034). In contrast, IHTG remained static in the usual care group at 16.9 ± 6.1% at baseline and 16.0 ± 6.6% at month 6 (mean reduction 1.0%; 95% confidence interval -2.5%, 4.5%; P = 0.55). The probiotic group tended to have greater reduction in IHTG (Table 2). Overall, 8 subjects in the probiotic group and 4 subjects in the usual care group had reduced IHTG at month 6 (P = 0.17) (Figure 2). Six subjects in the probiotic group had IHTG reduced by more than 30% from baseline, compared to 2 subjects in the usual care group (P = 0.17).

Figure 2..

Intrahepatic triglyceride content at baseline and month 6.

(0.02MB).

Similarly, the probiotics group had greater reduction in AST level than the usual care group (Table 2). On the other hand, there was no significant effect of probiotic treatment on the body mass index, waist circumference, glucose and lipid levels, and liver stiffness.

Adverse events occurred in 4 patients in each treatment group (Table 3). All reported adverse events were minor and did not differ between treatment groups.