The spectrum of non alcoholic fatty liver disease in morbidly obese patients . Prevalence and associate risk factors 1

PURPOSE: To determine the prevalence of non alcoholic fatty liver disease (NAFLD) and non alcoholic steatohepatitis (NASH) in morbidly obese patients undergoing bariatric surgery and to identify risk factors associated with the disease spectrum. METHODS: Liver biopsy was performed in 60 patients who underwent bariatric surgery, after other causes of liver disease were excluded. Clinical, biochemical and histological features were evaluated. RESULTS: NAFLD was detected in fifty-seven patients (95%) of the sample and forty patients (66.7%) of the total sample met the criteria for NASH. Perisinusoidal fibrosis was only found in three (7.5%) patients with NASH. The γGT was an independent predictive factor associated with the degree of hepatic steatosis. The variables such as dyslipidemia and ALT were independently associated with the presence of Mallory’s corpuscles with the following values, respectively, OR 0, 05, 95% CI 0.002 to 0.75, P = 0.031 and OR 10, 99, 95% CI 1.44 to 83.93, P = 0.021. CONCLUSIONS: Non alcoholic fatty liver disease seems to be an obese-related condition with approximately half of asymptomatic morbidly obese patients having histological NASH. The γGT was an independent predictor of the degree of steatosis.


Introduction
Non alcoholic fatty liver disease (NAFLD) is considered one of the most common liver diseases, affecting a wide spectrum of patients worldwide.The estimated prevalence in the general population is 10 to 24% and 70% in obese patients 1,2 .Given the obesity epidemic, it is thought to become one of the most important liver pathology in the future.Increased levels of insulin, triglycerides, low HDL, type 2 diabetes glucose intolerance are considered important risk factors.
Approximately 60% of patients with NAFLD have metabolic syndrome 3 .There is a wide spectrum of liver histology in NAFLD, ranging from steatosis to steatohepatitis (NASH), advanced fibrosis and cirrhosis 4 .NASH is characterized by zone 3 macrovesicular steatosis, inflammatory changes, fibrosis and may ultimately lead to cirrhosis and hepatocelular carcinoma in the absence of alcohol use.NASH histological findings resolve or remit in approximately 80% of patients after bariatric surgery 5 . Several studies have been conducted to evaluate the real prevalence of NASH and NAFLD in severely obese patients that underwent bariatric surgery, when liver biopsy was performed.
The prevalence varied from 27 to 98%, which may be explained by recent modifications in histological classification 6 .

Methods
The study was approved by the Ethics Committee for Clinical Investigation at Ceara Federal University of Brazil.
Informed written consent was obtained from every patient.
Seventy eight patients from the Nucleus of Obese of state of Ceara scheduled to perform gastric bypass, after consultation with the surgeon, anesthetist and dietitians, were invited to participate in the study.Eighteen failed to meet the selection criteria.The study was composed of sixty consecutive patients with obesity class II or III, defined as a BMI> 35kg/m2 that were submitted to bariatric gastric bypass surgery type.Intraoperative fine-needle liver biopsy was performed in all patients using 16 gauge biopsy needle-22mm (Bard -Max Core).
Exclusion criteria included: daily alcohol consumption > 30 g in men or > 20 g in women 7 at the time of evaluation or for a period longer than two years at any time, hepatitis B or C, previous history or laboratory evidence for a specific liver disease, had undergone extensive small bowel resection or jejuno-ileal or were on parenteral nutrition.Additionally, patients were excluded if on tamoxifen, amiodarone, corticosteroids, high doses of estrogen, methotrexate and cyclins in the last six months or for a prolonged period of time.
Serology for hepatitis C (anti HCV), hepatitis B (HBsAg, anti HBc IgM, anti HBc IgG, anti HBs), autoantibodies (antinuclear antibodies, anti smooth muscle and anti mitochondrial) was performed.Iron study (ferritin and tranferrin saturation index), serum levels of AST, ALT, γGT, alkaline phosphatase, total bilirubin and fractions, protein fractions, and prothrombin time, ceruloplasmin, uric acid and TSH were evaluated.Additionally, lipid profile (HDL, LDL, VLDL and triglycerides) and glycemic profile (fasting glucose, glycated hemoglobin A1c).An assessment of past and present alcohol consumption was done.
Patients were diagnosed with diabetes mellitus (DM) type 2 or impaired fasting glucose, according to previous classitication 8 , with hypertension when they had a resting blood pressure greater than or equal to 140/90 mmHg in two measurements.Patients were considered dyslipedemic based on the criteria of the Brazilian Society of Cardiology.The diagnosis of metabolic syndrome followed the criteria established by the International Diabetes Federation 2005 9 .Alcohol consumption was classified into four categories: 0, no consumption or sporadic, 1, and 10 g / day; 2 between 10-20g/dia; 3 between 20-30g/dia.Waist circumference (WC) was considered at risk when greater than 102cm in men and> 88cm in women and waist-quandril (WHR) was assessed by measuring waist circumference divided by hip circumference 10 .

Histological evaluation
The material was fixed in paraffin and stained: H&E; periodic acid-Schiff after diastase (PAS-d), Perl's and Masson's trichrome, and examined by a single pathologist (RP), without prior knowledge of clinical and laboratory data of patients.All samples were deemed sufficient for the study by the pathologist.
Patients were defined as having NAFLD when they had at least steatosis on biopsy, which was graded on a scale of 0 to 3 according to the criteria of Brunt et al. 11 .NASH diagnosis was defined as the presence of at least three of the four initial histological findings: 1) predominant macrovesicular steatosis in zone 3, 2) neutrophilic lobular inflammation 3), hepatocellular ballooning, 4) perisinusoidal fibrosis, according to the criteria of Brunt et al. 11 .The degree of steatosis was assessed on a scale of 0 to 3. Zero, indicating no: 1, mild (<33% of parenchyma involved) 2, moderate (33% to 66% of parenchyma involved), and 3, severe (> 66% of parenchyma involved).The degree of lobular inflammation was also graded on a scale of 0 to 3. Zero: no inflammation; The presence of portal fibrosis in the absence of perisinusoidal fibrosis was not considered for the stage of NASH but was studied separately.Thus, histological variables described above, constitute the system of grade and stage of NASH: Grades on a scale of 1 to 3 and stage scale of 1 to 4 11 .

Statistical analysis
Results are expressed as mean ± SD and percentages.
Comparisons of quantitative variables were made using the Student's t-test.Categorical variables and proportions were tested by the qui square and Fisher exact test.Multivariate analysis was tested using logistic regression.A p-value <0.05 was considered as statistically significant All variables with a P value of 0.25 or less in univariate analyses were included in the full logistic regression model, according to recommendations of the Hosmer-Lameshow goodness-of-fit-test. Odds ratio (OR) and 95% confidence intervals (CI) were used as an estimated of the risk.Analysis was performed with SPSS computer software (version 11.5).

Results
Seventy-eight patients were invited to participate in the study, one was excluded for presenting positive marker for hepatitis C during the evaluation, four decided not to carry out with surgery, two had inadequate biopsy sample and eleven did not undergo biopsy for various technical reasons.A total of 60 patients, (73% female and 27% male) were considered for analysis.
The mean ratio of AST / ALT was 0.76 (range, 0.27 to 1.61), with the ratio AST / ALT> 1 detected in six (10%).Twenty-one (35%) had slightly elevated levels of ferritin and transferrin saturation index normal in all.

Histological data
Liver biopsy results are summarized in Table 1.Fiftyseven patients (95%) had hepatic steatosis.Twenty-four (42.1%) had moderate to severe steatosis.Forty (66.6%) met all histologic criteria for NASH as described by Brunt et al.

Factors associated with NASH
In the univariable analysis, the factors that were significantly associated with NAFLD were: triglycerides (P = 0.029); group consisting of type 2 diabetes and those with impaired fasting glucose (P = 0.044), fasting glucose greater than 110mg/ dL (p = 0.034), ALT changes (p = 0.044).The logistic regression analysis identified no factor independently associated with NASH.

Factors associated with fibrosis
Zone 3 perisinusoidal fibrosis was detected in three cases (7.5%) while the isolated portal fibrosis was seen in seven (11.6%).Figure 1The univariable analysis for factors associated with perisinusoidal fibrosis was compromised by the low prevalence of perisinusoidal fibrosis in the sample.However, taking into account that in morbid obesity, portal fibrosis alone has received attention 12 , all type of fibrosis (perisinusoidal, portal, perisinusoidal and portal) were analyzed as a group, and portal fibrosis also been analyzed separately.Only the elevation of γ GT showed a significant association with the first group (p = 0.035).After logistic regression analysis, γ GT did not remain as a significant independent factor.However, there was a trend towards significancy (OR 5.69, 95% CI 0.99 to 32.73, P = 0.051) Table 3.
importance to predict and prevent the development of advanced liver diseases in obese patients.Determining the prevalence of NASH in those patients and further understanding the role of certain predictor factors is of interest, since it would aid in identifying patients at high risk.Furthermore, patients undergoing bariatric surgery would greatly benefit from strategies to allow detection of NASH and closer follow-up given they are at increased risk for the development of cirrhosis, hepatocellular and end-stage liver disease.Therefore, the aim of this study was to prospectively determine the prevalence of NASH in obese patients undergoing bariatric surgery from Northeastern of Brazil and to identify risk factors involved in the development of NASH.