Abstracts

INTRODUCTION

Obesity is increasing its prevalence at an alarming rate, reaching approximately 300 million people worldwide. Said prevalence is estimated at 20%(⁹9. Marquezine GF, Oliveira CM, Pereira AC, Krieger JE, Mill JG. Metabolic syndrome determinants in an urban population from Brazil: social class and gender-specific interaction. Int J Cardiol. 2008;129:259-65.,¹²12. Misra A, Khurana L. Obesity and the metabolic syndrome in developing countries. J Clin Endocrinol Metab. 2008;93:S9-30.). Nonalcoholic fatty liver disease (NAFLD) is among the biggest complications of obesity. NAFLD encompasses a wide spectrum of histopatological changes ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH), which can evolve to cirrhosis and hepatocellular carcinoma(²¹21. White DL, Kanwal F, El-Serag HB. Association Between Nonalcoholic Fatty Liver Disease and Risk for Hepatocellular Cancer, Based on Systematic Review. Clin Gastroenterol Hepatol. 2012;10:1342-59 e2.). SS is defined as the presence of fatty deposits without hepatocellular damage, while NASH is defined as the presence of steatosis associated with hepatocellular lesion with or without fibrosis(²2. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol. 1999;94:2467-74.,³3. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ; American Gastroenterological Association; American Association for the Study of Liver Diseases; American College of Gastroenterologyh. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology. 2012;55:2005-23.). Some risk factors have been associated with the development of NASH, such as metabolic syndrome, age, obesity and diabetes mellitus (DM)(⁷7. Harrison SA, Kadakia S, Lang KA, Schenker S. Nonalcoholic steatohepatitis: what we know in the new millennium. Am J Gastroenterol. 2002;97:2714-24.,²²22. Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med. 2001;7:941-6.). The role of the presence of Fe in liver tissue and its relation with NASH has been the target of countless studies. Small quantities of Fe, found in liver tissue, can act as factor enhancing oxidative stress in the hepatocyte determined by fats, virus and alcohol, increasing toxicity and leading to fibrogenesis and carcinogenesis(¹⁷17. Pietrangelo A. Iron in NASH, chronic liver diseases and HCC: how much iron is too much? J Hepatol. 2009;50:249-51.). Mendler et al. established a relationship between the resistance to insulin and the presence of Fe in liver biopsies of obese individuals(¹¹11. Mendler MH, Turlin B, Moirand R, Jouanolle AM, Sapey T, Guyader D, Le Gall JY, Brissot P, David V, Deugnier Y. Insulin resistance-associated hepatic iron overload. Gastroenterology. 1999;117:1155-63.).

Patients with morbid obesity (MO) underwent to bariatric surgery have been a population in which NAFLD has been much studied, thanks to the possibility of conducting transoperative liver biopsy with low morbidity. The results of this surgery have determined an improvement in the hepatic histopathology of these patients(¹⁰10. Mathurin P, Hollebecque A, Arnalsteen L, Buob D, Leteurtre E, Caiazzo R, Pigeyre M, Verkindt H, Dharancy S, Louvet A, Romon M, Pattou F. Prospective study of the long-term effects of bariatric surgery on liver injury in patients without advanced disease. Gastroenterology. 2009;137:532-40.,¹³13. Moretto M, Kupski C, da Silva VD, Padoin AV, Mottin CC. Effect of bariatric surgery on liver fibrosis. Obesity Surgery. 2012;22:1044-9.). On the other hand, the need for liver biopsy in pre-operative evaluation has been the object of discussion, in some selected cases, considering that patients with advanced fibrosis in pre-operation have increased risk of decompensation in the post-operative, increasing morbimortability of this surgical modality(³3. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ; American Gastroenterological Association; American Association for the Study of Liver Diseases; American College of Gastroenterologyh. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology. 2012;55:2005-23.,¹⁴14. Mosko JD, Nguyen GC. Increased perioperative mortality following bariatric surgery among patients with cirrhosis. Clin Gastroenterol Hepatol. 2011;9:897-901.,²⁰20. Vyberg M, Ravn V, Andersen B. Pattern of progression in liver injury following jejunoileal bypass for morbid obesity. Liver. 1987;7:271-6.).

The aim of this study was to determine the prevalence of SS and NASH in patients with MO, subjected to bariatric surgery in the Obesity Treatment Center of the Santa Casa de Porto Alegre Hospital Complex, and to establish a correlation of the anatomopathological findings (steatosis, inflammatory activity and iron deposits) with the presence of liver fibrosis.

METHODS

In this retrospective study, a total of 250 liver biopsies were analyzed, conducted in the transoperation of patients subjected to bariatric surgery from 2007 to 2012, in the Obesity Treatment Center of the Santa Casa de Porto Alegre Hospital Complex. Patients aged less than 18 years were excluded, as well as those who presented serological markers for viral hepatitis and those with alcohol intake >20 g/day.

The liver biopsies were evaluated by the same pathologist. The histochemical techniques used to analyze the specimen were hematoxylin/eosin (HE), Masson's trichrome (to evaluate fibrosis) and Perls (to evaluate iron deposits). Steatosis was considered to be present on reaching over 5% of the sample studied. The percentage presence of 5% to 33% was considered to be mild steatosis (G1); of 33% to 66% as moderate steatosis (G2) and greater than 66% as severe steatosis (G3)(²2. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol. 1999;94:2467-74.).

To diagnose NASH, the presence of steatosis associated with hepatocyte ballooning and/or inflammatory infiltrate was deemed necessary. The activity of NASH was classified as mild (A1), moderate (A2) and severe (A3), as described by the Pathology Committee of the NASH Clinical Research Network. The degree of fibrosis was classified as stage 1A, when sinusoidal/discrete cellular fibrosis was identified; degree 1B if sinusoidal/dense and diffuse cellular fibrosis; and 1C in case portal fibrosis was identified. In stage 2, pericellular/perisinusoidal fibrosis was considered, associated with periportal fibrosis; in stage 3, the anterior changes associated with bridging fibrosis and in stage 4, cirrhosis was diagnosed(¹1. Brunt EM. Nonalcoholic steatohepatitis. Semin Liver Dis. 2004;24:3-20.). For statistical analysis, the degree of fibrosis was classified as mild (stage 1A, 1B, 1C), moderate (stage 2), severe (stage 3) and cirrhosis (stage 4).

A semi-quantitative analysis of the iron deposits in the hepatocytes and in the Kupffer cells was conducted, according to the classification used by Sebastiani et al.(¹⁸18. Sebastiani G, Vario A, Ferrari A, Pistis R, Noventa F, Alberti A. Hepatic iron, liver steatosis and viral genotypes in patients with chronic hepatitis C. J Viral Hepat. 2006;13:199-205.) in which degree 0 corresponds to the total absence of Fe; degree 1 to the minimum quantity; degree 2 to mild quantity; degree 3 to moderate quantity and degree 4 to great quantity of Fe.

Statistical analysis

The data analysis was conducted using the SPSS (Statistical Package for the Social Sciences) software, version 18.0. This sample size supports a minimum difference between groups of 20%, with power of 85% and significance level of 5%. The continuous variables were described with use of mean and standard deviation and the categorical variables by means of absolute and relative frequencies.

To evaluate the association between the categorical variables, the Pearson chi-square test was applied. For the continuous or ordinal variables, the Spearman correlation test was used.

The statistical significance level considered was 5% (P≤0.05).

This study was approved by the Institutional Review Board of Irmandade da Santa Casa de Misericórdia de Porto Alegre - ISCMPA, with number 70375.

RESULTS

Out of the 250 biopsies analyzed, 200 (80%) were of women, and 50 (20%) of men, with an average age of 36.8 ± 10.2 years. The average body mass index (BMI) was 43.6 ± 5.2 Kg/m².

Steatosis was present in 226 (90.4%) samples. With regard to its intensity, it was observed that 76 (30.4%) were classified as mild; 71 (28.4%) as moderate and 79 (31.6%) as intense (Figure 1).

NASH was diagnosed in 176 (70.4%) cases. Mild degree was found in 120 (48.4%) cases; moderate in 50 (20%) cases, and severe in 6 (2.4%) cases (Figure 2).

Fibrosis was referred to in 108 (43.2%) biopsies, 95 of which (38%) were mild; 2 (0.8%) were moderate; 7 were intense (2.8%), and cirrhosis was diagnosed in 4 (1.6%) cases (Figure 3).

There was a moderate correlation between the degree of steatosis and the level of inflammatory activity/ballooning (r_s = 0.460;P<0.001) and also a regular correlation between the degree of activity and the degree of fibrosis (r_s = 0.583;P<0.001) (Figures 4 and 5).

Iron deposits were found in mild concentration in 13 (5.2%) samples, being that in 9 the iron was located in the Kupffer cells and in 7 in the hepatocytes.

DISCUSSION

The results of this study show a prevalence of 90.4% of NAFLD in patients with MO who underwent bariatric surgery. This result is consistent with the literature that reports a prevalence of 84% and 96% of NAFLD in these patients(⁴4. Clark JM. The epidemiology of nonalcoholic fatty liver disease in adults. Journal of clinical gastroenterology. 2006;40:S5-10.).

NAFLD has a wide spectrum of alterations that range from SS to fibrosis and eventually to cirrhosis. Within these alterations, this analysis shows that the degree of steatosis was distributed almost uniformly in the samples (30.4%, 28.4% and 31.6%), for mild, moderate and severe degree respectively. Concomitantly, NASH was found in approximately 70% of the sample, there being a moderate correlation with the degree of steatosis. This finding is consistent with the physiopathology of NAFLD, in which excess fat in the hepatocyte triggers the inflammation process, leading to NASH, which can result in fibrosis(²⁴24. Zangar RC, Novak RF. Effects of fatty acids and ketone bodies on cytochromes P450 2B, 4A, and 2E1 expression in primary cultured rat hepatocytes. Arch Biochem Biophys. 1997;337:217-24.). However, this correlation was not strong, since some cases showed intense degree of steatosis with mild or no inflammation, a fact that shows that the liver biopsy, before providing a definitive diagnosis, mainly reflects a stage of evolution of NAFLD.

On analyzing the degree of fibrosis of the samples of this investigation, it was noticed that fibrosis was already present in 48.3% of them; out of these, 38% were mild and only 4.4.% (11 cases) were considered of intense degree. In recent years, bariatric surgery has become an important therapeutic option for MO. Some studies document a significant reduction of fibrosis in liver biopsies conducted during the surgery and after weight loss in these patients(⁵5. de Andrade AR, Cotrim HP, Alves E, Soares D, Rocha R, Almeida A, Almeida CG, de Freitas LA. Nonalcoholic fatty liver disease in severely obese individuals: the influence of bariatric surgery. Ann Hepatol. 2008;7:364-8.,¹⁰10. Mathurin P, Hollebecque A, Arnalsteen L, Buob D, Leteurtre E, Caiazzo R, Pigeyre M, Verkindt H, Dharancy S, Louvet A, Romon M, Pattou F. Prospective study of the long-term effects of bariatric surgery on liver injury in patients without advanced disease. Gastroenterology. 2009;137:532-40.,¹³13. Moretto M, Kupski C, da Silva VD, Padoin AV, Mottin CC. Effect of bariatric surgery on liver fibrosis. Obesity Surgery. 2012;22:1044-9.,¹⁵15. Mummadi RR, Kasturi KS, Chennareddygari S, Sood GK. Effect of bariatric surgery on nonalcoholic fatty liver disease: systematic review and meta-analysis. Clin Gastroenterol Hepatol. 2008;6:1396-402.). However, the same works report that, if the degree of fibrosis is advanced, the chances of regression reduce, moreover, there is an increase in morbimortality after bariatric surgery in patients with advanced fibrosis.

Some studies have been conducted to evaluate clinical lab parameters with histopathological findings. Liew et al. presented retrospective results of 152 patients subjected to laparoscopic bariatric surgery, in which the insulin resistance (IR), evaluated by the HOMA-IR index, was correlated with the diagnosis of NASH(⁸8. Liew PL, Lee WJ, Lee YC, Wang HH, Wang W, Lin YC. Hepatic histopathology of morbid obesity: concurrence of other forms of chronic liver disease. Obes Surg. 2006;16:1584-93.). In a prospective study, with sequential biopsies, Mathurin et al. observed the effects of bariatric surgery on NAFLD and NASH. These authors showed an improvement in all histological and lab parameters in NAFLD in 5 years. It is interesting to note that the improvement in IR, which occurs in the first year after surgery, is a predictive factor of regression of the histological and lab parameters of NAFLD(¹⁰10. Mathurin P, Hollebecque A, Arnalsteen L, Buob D, Leteurtre E, Caiazzo R, Pigeyre M, Verkindt H, Dharancy S, Louvet A, Romon M, Pattou F. Prospective study of the long-term effects of bariatric surgery on liver injury in patients without advanced disease. Gastroenterology. 2009;137:532-40.). In fact, the hepatic lesion and Fb cannot be detected by image or lab tests. Some works have sought to identify clinical/lab markers of NASH, like age, obesity, diabetes mellitus (DM), systemic arterial hypertension, inflammation markers, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio >1 and triglyceride (TG) levels >150 mg/dL. The studies that try to correlate inflammatory markers, like some cytokines and others of oxidative stress present various results(⁷7. Harrison SA, Kadakia S, Lang KA, Schenker S. Nonalcoholic steatohepatitis: what we know in the new millennium. Am J Gastroenterol. 2002;97:2714-24.,¹⁹19. Uysal S, Armutcu F, Aydogan T, Akin K, Ikizek M, Yigitoglu MR. Some inflammatory cytokine levels, iron metabolism and oxidan stress markers in subjects with nonalcoholic steatohepatitis. Clin Biochem. 2011;44:1375-9.,¹⁶16. Musso G, Gambino R, Cassader M, Pagano G. Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity. Ann Med. 2011;43:617-49.).

To date, there is no lab marker for diagnosis and gravity of NASH. In this wise, liver biopsy should perhaps be indicated, in select cases, in the pre-operative evaluation of patients with MO and indication of bariatric surgery. Although this indication is already described, to patients with NAFLD and with risk factors to NASH, in the last consensus of the American Association for Study of Liver Diseases, it is important to develop protocols with specific outlining, aiming at shedding light on this controversy(³3. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ; American Gastroenterological Association; American Association for the Study of Liver Diseases; American College of Gastroenterologyh. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology. 2012;55:2005-23.).

Some authors support that the presence of Fe in the liver tissue is a possible factor involved in the genesis of the inflammation(¹⁷17. Pietrangelo A. Iron in NASH, chronic liver diseases and HCC: how much iron is too much? J Hepatol. 2009;50:249-51.). The excess Fe not linked to transferrin is removed from circulation by the hepatocyte by operation of a peptide called hepcidin, which regulates the homeostasis of Fe(⁶6. Fleming RE, Ponka P. Iron overload in human disease. N Engl J Med. 2012;366:348-59.). The Kupffer cells (KC) also play an important role in the elimination of iron, since they accumulate this ion when the hepatocytes are saturated. It is possible that the KC, when saturated with Fe, lose control over other aggressions suffered by the liver (virus, alcohol, etc.), favoring the progression of fibrosis(¹⁷17. Pietrangelo A. Iron in NASH, chronic liver diseases and HCC: how much iron is too much? J Hepatol. 2009;50:249-51.). In the casuistic of this study, mild deposits of Fe were found in only 10 (4%) cases, without predominance in hepatocytes or in KC. It does not seem, however, that Fe has an important role in the physiopathogeny of NASH. Corroborating our results, the study by Zamin et al. did not establish correlation between mutation of the C282Y and H63D gene (mutation involved in the genesis of hemochromatosis), in 20 patients with NASH. This reinforces the nonparticipation of Fe deposits in the physiopathology of NASH(²³23. Zamin I Jr, Mattos AA, Mattos AZ, Migon E, Bica C, Alexandre CO. Prevalence of the hemochromatosis gene mutation in patients with nonalcoholic steatohepatitis and correlation with degree of liver fibrosis. Arq Gastroenterol. 2006;43:224-8.).

In conclusion, the findings of this investigation confirm a high prevalence of SS and NASH in patients with MO. The correlation between NASH and fibrosis reinforce the indication of bariatric surgery in the treatment of MO and the importance of liver biopsy in the pre-operative evaluation of some patients. Moreover, the low prevalence of iron deposits in the samples analyzed herein makes the participation of Fe in the physiopathogeny of NAFLD questionable.

REFERENCES

Publication Dates

History