Material and methods [626898]

Material and methods
The intent of this planned prospective and uncontrolled study was to evaluate the efficacy of
Atorvastatin and Pentoxifylline in treating NAFLD.
The study included 98 patients with histologically confirmed NAFLD, admitted between December 2012
and January 2016 at the Department of Internal Medicine at Filantropia University Hospital in Craiova .
The study was in accordance with the Helsinki Declarat ion of 1975, and approved by the Review Ethics
Board of the University Medicine and Pharmacy of Craiova and of the Filantropia University Hospital. All
patients had to give full informed consent.
Taking into consideration ethical reasons and the poor conse nt to hepatic biopsies, we decided not to
use placebos or controls in this study, this is primarily because it would involve a large number of
patients having to undergo two liver biopsies whilst receiving no active treatment.

Two therapeutic groups were formed after dividing the patients: Group I – 57 dyslipidemic patients,
receiving Atorvastatin 20 mg/day and Group II – 41 patients treated with Pentoxifylline, 800 mg/day (400
mg twice daily).

The average duration of treatment administration was approxi mately 30 weeks. The study design
previewed one screening visit at admission (T0), two regular visits (T1 and T2) at 10, and 20 weeks after
admission and one end -of treatment visit at 30 weeks (T3).
All patients included in the study (51 males/47 females) were Caucasians; their mean age was 54/52
years and had no previous history of drug abuse or alcohol dependence.
The study group was selected using the admission and exclusion criteria. Patients were included in the
study if they were able to give written informed consent, had histologically confirmed NAFLD and had no
history of drug and/or alcohol abuse. Patients with history of alcohol abuse or chronic intake (assumed
or confirmed by their family) were not included in the study.
In monitoring alcohol inta ke we utilized a questionnaire adapted from Behavioral Risk Factor
Surveillance System 2006 Questionnaire [10] administered at each visit. Small amounts of alcohol were
infrequently allowed, but no more than 2 drinks/week, 1 drink being defined as one stan dard US
alcoholic drinks (approximately 14 g ethanol i.e. 12 oz. of beer, 5 oz. of wine, or 1.5 oz. of liquor; 1 US oz.
= approx. 30 ml).
No restriction or modifications in lifestyle or diet were enforced on any patient, besides any current
recommendations made by their endocrinologist or cardiologist. Body mass index (BMI), serum level of
alanine aminotransferase, aspartate aminotransferase, gamma -glutamyl transpeptidase, alkaline

phosphatase, total cholesterol, triglycerides and blood glucos levels were t aken in all patients at
admission, at each visit and at the end of treatment.

Patients underwent liver biopsy at the start and end of the study, we deemed 2 weeks before or after
the first and last visit to be a respectable and acceptable time interval fo r biopsy. Hepatic biopsy was
done using the Menghini technique, with Braun Melsungen Sonocan 20 G needles, 0.8×160 mm and 21
G, 0.8×160 mm.
Biopsy fragments with a length of a minimum of 12 mm were considered adequate and delivered for
histopathological an alysis.
Histological colorations were done to determine inflammation, steatosis and hepatic fibrosis (the Brunt
score) for each case, at inclusion in study and at the end of the treatment [9].
NASH recognized lesion evaluation system developed by Brunt and colleagues, 1999 [9]. Using these
criteria, the NASH Clinical Research Network Pathology Committee designed and validated a histological
system of scoring which addresses the entire spectrum of NAFLD lesions and proposed a NAFLD activity
score (NAS) for c linical trials use [11]. The scoring system is made up of 14 histological features, of which
4 were evaluated semi -quantitatively: lobular inflammation (0 -2), steatosis (0 -3), fibrosis (0 -4) and
hepatocellular ballooning (0 -2). NAS is the sum of lobular in flammation, steatosis and hepatocellular
ballooning scores, a NAS greater than 5 correlating with the NASH diagnosis and scores of 3 or less
considered as “not NASH.” The NAS inclusion scores (Score 1) and study termination scores (Score 2)
were database stored and compared between therapeutic groups.

The results were calculated as mean +/ – standard deviation. For data processing Microsoft Excel was
used (Microsoft Corp., Redmond, WA, USA), along with the XLSTAT suite for MS Excel (Addinsoft SARL,
Paris, France).
For statistical comparison of the two groups with each other at inclusion and at the end of the
treatment variance analysis (one way ANOVA) for comparison within the groups themselves variant
analysis, Kruskall -Wallis and Wilcoxon tests were used. A p value of <0.05 was considered significant.