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Homocysteine in nonalcoholic steatohepatitis: A reply
EJINME-03274; No of Pages 2 European Journal of Internal Medicine xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Letter to the Editor Homocysteine in nonalcoholic steatohepatitis: A reply Keywords: Cysteine Homocysteine Glutathione Methionine Nonalcoholic fatty liver disease Nonalcoholic steatohepatitis
Dear Editor, We read with interest the letter-to-the-Editor of Leach et al. [1], commenting on our [2–4] and their [5] previous works and considerations. We hereby highlight some considerations raised by Leach et al. [1]. Briefly, we previously observed lower serum homocysteine (Hcy) levels in patients with nonalcoholic steatohepatitis (NASH) than nonalcoholic simple steatosis (SS), and lower Hcy was independently associated with NASH [2]. Importantly, Hcy was lower by increasing the severity of fibrosis and portal inflammation, but not steatosis, lobular inflammation or ballooning. Following Leach et al. study [5], we performed a post-hoc analysis, in which NASH patients and controls had similar serum Hcy levels [4]. Leach et al. recruited 50 NASH patients, but no patients with simple steatosis [5], whereas we recruited 31 nonalcoholic fatty liver disease (NAFLD) patients almost equally divided into NASH and SS for the requirements of our preliminary study [2]. It is necessary to clarify that the number of our NASH patients was similar to that of SS, and not lower in NASH than SS, as Leach et al. reported [1] as a potential source of discrepancy in our results. The two studies had a different orientation. Our study was focused on Hcy levels between SS and NASH patients, because of the urgent need of noninvasive fatty liver markers to differentiate SS from NASH in the clinical setting. Our patients and controls were matched to gender, age and adiposity (for both body mass index [BMI] and waist circumference), factors playing crucial role in the pathogenesis of NAFLD and distorting the results, when they are different between the comparing groups. Although statistical methods could help to adjust for unequally distributed parameters between groups, statistics could not replace the careful pre-selection of populations. The aim of Leach et al. study was to compare Hcy levels and oxidative stress markers between NASH patients and controls and evaluate their association with carotid intima-media thickness [5]. Their control group, however, had lower adiposity (both BMI and waist circumference) than NASH
Abbreviations: BMI, body mass index; Hcy, homocysteine; HSENSI, Homocysteine, Serum glutamic oxaloacetic transaminase, Erythrocyte sedimentation rate, Nonalcoholic Steatohepatitis Index; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; SS, simple steatosis.
group, critical parameters that may affect Hcy, oxidative markers and carotid intima-media thickness. Furthermore, although comparisons between NASH and controls (or SS and controls) may be useful to clarify certain associations in NASH (or SS, respectively), they have minimal clinical implication, since the differentiation between NAFLD and nonNAFLD can be simply assessed by ultrasonography in clinical practice [6], as the preferred first-line diagnostic procedure in the management of NAFLD by the current guidelines [7]. Moreover, Leach et al. characterized HSENSI (acronym of Homocysteine, Serum glutamic oxaloacetic transaminase [SGOT/AST], Erythrocyte sedimentation rate [ESR], Nonalcoholic Steatohepatitis Index) “inadequate”. We introduced HSENSI as a noninvasive index of NASH based on a dataset of extensive anthropometric and laboratory data [3]. In brief, we observed that NAFLD patients with higher SGOT and ESR, but lower Hcy were at higher risk for NASH [3]. We acknowledged the small sample size in the limitations of this pilot study, and we aim to validate HSENSI in a large cohort of NAFLD patients. On the other hand, we could not agree with the consideration of “insufficient” implementation of logistic regression; indeed, because of the limitation of small sample size, we implement a rigorous statistical analysis of three levels rather than two levels (univariate and multivariate), which are usually used. Important to note, that HSENSI is a simple (it requires Hcy, SGOT and ESR) and inexpensive index, thereby warranting internal and external validation before its potential clinical use. The design of our [2] and Leach et al. studies [5] was observational, thereby no causative association could be proven per se. Therefore, the claim of Leach et al. “if the altered metabolism of Hcy may contribute to the appearance of oxidative stress and cardiovascular risk in patients with NASH” [1] cannot be shown in an observational study. They simply observed higher Hcy levels in NASH patients than controls [5] and we simply observed similar levels between NASH patients and controls, but lower in NASH than SS patients [2]. The observation of lower serum glutathione and higher malondialdehyde in NASH patients than controls (of lower BMI and waist circumference) [5] cannot prove a causative association between oxidative stress, Hcy and NASH, which needs mechanistic rather than observational studies to be shown. Whereas oxidative stress plays a critical role in the pathogenesis of NAFLD [8], to the best of our knowledge, no mechanistic study has to-date revealed the entire mechanism, i.e., whether: 1) Hcy is depleted in NASH as a counterbalancing mechanism to increase (via transsulfuration pathway) the synthesis of the antioxidant glutathione, which is depleted due to higher oxidative stress observed in NASH, as we previously hypothesized [2]; or 2) transsulfuration pathway is generally impaired in NASH, which results in higher Hcy and lower glutathione levels, as Leach et al. hypothesized [5]. Leach et al. also refer to the recent meta-analysis by Dai et al. summarizing data of eight relevant studies [9]. Although, this metaanalysis favors higher circulating Hcy levels in SS or NASH patients than controls and adds to literature, the authors did not explain the high heterogeneity between studies (i.e., with a meta-regression). Furthermore, the comparison between SS and NASH patients was not performed, which, as aforementioned, has the highest clinical implication.
http://dx.doi.org/10.1016/j.ejim.2016.06.034 0953-6205/© 2016 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Polyzos SA, Kountouras J, Homocysteine in nonalcoholic steatohepatitis: A reply, Eur J Intern Med (2016), http:// dx.doi.org/10.1016/j.ejim.2016.06.034
2
Letter to the Editor
Besides, the finding that some of Pastore et al. data in a pediatric population are in line with our findings and speculations, including the observation of higher Hcy levels in not-NASH than NASH [10], does not render the comparison “improper”, due to any differences between pediatric and adult NAFLD. By looking into Dai et al. meta-analysis, other authors reported that Hcy was not different between adult NASH or SS patients and controls [9]. Lastly, we believe that the credibility of a study is not judged by the degree of agreement with previously published data, but by future studies. In this regard, there are studies initially considered to be heretical and difficult to publish, but finally changed our view to the world. Conflict of interests SAP and JK have no conflict of interest. Acknowledgments Grants or other financial support: This work did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. References [1] Leach NV, Dronca E, Craciun EC, Crisan D. High levels of serum homocysteine in nonalcoholic steatohepatitis. Eur J Intern Med 2016. http://dx.doi.org/10.1016/j.ejim. 2016.06.018. [2] Polyzos SA, Kountouras J, Patsiaoura K, Katsiki E, Zafeiriadou E, Deretzi G, et al. Serum homocysteine levels in patients with nonalcoholic fatty liver disease. Ann Hepatol 2012;11:68–76.
[3] Polyzos SA, Kountouras J, Slavakis A, Zafeiriadou E, Patsiaoura K, Katsiki E, et al. A novel noninvasive index for nonalcoholic steatohepatitis: a pilot study. Biomarkers 2013;18:607–13. [4] Polyzos SA, Kountouras J, Tsoukas MA. Circulating homocysteine in nonalcoholic fatty liver disease. Eur J Intern Med 2015;26:152–3. [5] Leach NV, Dronca E, Vesa SC, Sampelean DP, Craciun EC, Lupsor M, et al. Serum homocysteine levels, oxidative stress and cardiovascular risk in non-alcoholic steatohepatitis. Eur J Intern Med 2014;25:762–7. [6] Polyzos SA, Mantzoros CS. Necessity for timely noninvasive diagnosis of nonalcoholic fatty liver disease. Metabolism 2014;63:161–7. [7] EASL–EASD–EASO. EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. Diabetologia 2016;59:1121–40. [8] Polyzos SA, Kountouras J, Zavos C. Nonalcoholic fatty liver disease: the pathogenetic roles of insulin resistance and adipocytokines. Curr Mol Med 2009;72:299–314. [9] Dai Y, Zhu J, Meng D, Yu C, Li Y. Association of homocysteine level with biopsyproven non-alcoholic fatty liver disease: a meta-analysis. J Clin Biochem Nutr 2016;58:76–83. [10] Pastore A, Alisi A, di Giovamberardino G, Crudele A, Ceccarelli S, Panera N, et al. Plasma levels of homocysteine and cysteine increased in pediatric NAFLD and strongly correlated with severity of liver damage. Int J Mol Sci 2014;15:21202–14.
Stergios A. Polyzos ⁎ Jannis Kountouras Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece ⁎ Corresponding author at: Simou Lianidi 13, 55134 Thessaloníki, Macedonia, Greece. Tel./fax: +30 2310424710. E-mail address: [email protected] (S.A. Polyzos). 26 June 2016 Available online xxxx
Please cite this article as: Polyzos SA, Kountouras J, Homocysteine in nonalcoholic steatohepatitis: A reply, Eur J Intern Med (2016), http:// dx.doi.org/10.1016/j.ejim.2016.06.034
Contents lists available at ScienceDirect
European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Letter to the Editor Homocysteine in nonalcoholic steatohepatitis: A reply Keywords: Cysteine Homocysteine Glutathione Methionine Nonalcoholic fatty liver disease Nonalcoholic steatohepatitis
Dear Editor, We read with interest the letter-to-the-Editor of Leach et al. [1], commenting on our [2–4] and their [5] previous works and considerations. We hereby highlight some considerations raised by Leach et al. [1]. Briefly, we previously observed lower serum homocysteine (Hcy) levels in patients with nonalcoholic steatohepatitis (NASH) than nonalcoholic simple steatosis (SS), and lower Hcy was independently associated with NASH [2]. Importantly, Hcy was lower by increasing the severity of fibrosis and portal inflammation, but not steatosis, lobular inflammation or ballooning. Following Leach et al. study [5], we performed a post-hoc analysis, in which NASH patients and controls had similar serum Hcy levels [4]. Leach et al. recruited 50 NASH patients, but no patients with simple steatosis [5], whereas we recruited 31 nonalcoholic fatty liver disease (NAFLD) patients almost equally divided into NASH and SS for the requirements of our preliminary study [2]. It is necessary to clarify that the number of our NASH patients was similar to that of SS, and not lower in NASH than SS, as Leach et al. reported [1] as a potential source of discrepancy in our results. The two studies had a different orientation. Our study was focused on Hcy levels between SS and NASH patients, because of the urgent need of noninvasive fatty liver markers to differentiate SS from NASH in the clinical setting. Our patients and controls were matched to gender, age and adiposity (for both body mass index [BMI] and waist circumference), factors playing crucial role in the pathogenesis of NAFLD and distorting the results, when they are different between the comparing groups. Although statistical methods could help to adjust for unequally distributed parameters between groups, statistics could not replace the careful pre-selection of populations. The aim of Leach et al. study was to compare Hcy levels and oxidative stress markers between NASH patients and controls and evaluate their association with carotid intima-media thickness [5]. Their control group, however, had lower adiposity (both BMI and waist circumference) than NASH
Abbreviations: BMI, body mass index; Hcy, homocysteine; HSENSI, Homocysteine, Serum glutamic oxaloacetic transaminase, Erythrocyte sedimentation rate, Nonalcoholic Steatohepatitis Index; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; SS, simple steatosis.
group, critical parameters that may affect Hcy, oxidative markers and carotid intima-media thickness. Furthermore, although comparisons between NASH and controls (or SS and controls) may be useful to clarify certain associations in NASH (or SS, respectively), they have minimal clinical implication, since the differentiation between NAFLD and nonNAFLD can be simply assessed by ultrasonography in clinical practice [6], as the preferred first-line diagnostic procedure in the management of NAFLD by the current guidelines [7]. Moreover, Leach et al. characterized HSENSI (acronym of Homocysteine, Serum glutamic oxaloacetic transaminase [SGOT/AST], Erythrocyte sedimentation rate [ESR], Nonalcoholic Steatohepatitis Index) “inadequate”. We introduced HSENSI as a noninvasive index of NASH based on a dataset of extensive anthropometric and laboratory data [3]. In brief, we observed that NAFLD patients with higher SGOT and ESR, but lower Hcy were at higher risk for NASH [3]. We acknowledged the small sample size in the limitations of this pilot study, and we aim to validate HSENSI in a large cohort of NAFLD patients. On the other hand, we could not agree with the consideration of “insufficient” implementation of logistic regression; indeed, because of the limitation of small sample size, we implement a rigorous statistical analysis of three levels rather than two levels (univariate and multivariate), which are usually used. Important to note, that HSENSI is a simple (it requires Hcy, SGOT and ESR) and inexpensive index, thereby warranting internal and external validation before its potential clinical use. The design of our [2] and Leach et al. studies [5] was observational, thereby no causative association could be proven per se. Therefore, the claim of Leach et al. “if the altered metabolism of Hcy may contribute to the appearance of oxidative stress and cardiovascular risk in patients with NASH” [1] cannot be shown in an observational study. They simply observed higher Hcy levels in NASH patients than controls [5] and we simply observed similar levels between NASH patients and controls, but lower in NASH than SS patients [2]. The observation of lower serum glutathione and higher malondialdehyde in NASH patients than controls (of lower BMI and waist circumference) [5] cannot prove a causative association between oxidative stress, Hcy and NASH, which needs mechanistic rather than observational studies to be shown. Whereas oxidative stress plays a critical role in the pathogenesis of NAFLD [8], to the best of our knowledge, no mechanistic study has to-date revealed the entire mechanism, i.e., whether: 1) Hcy is depleted in NASH as a counterbalancing mechanism to increase (via transsulfuration pathway) the synthesis of the antioxidant glutathione, which is depleted due to higher oxidative stress observed in NASH, as we previously hypothesized [2]; or 2) transsulfuration pathway is generally impaired in NASH, which results in higher Hcy and lower glutathione levels, as Leach et al. hypothesized [5]. Leach et al. also refer to the recent meta-analysis by Dai et al. summarizing data of eight relevant studies [9]. Although, this metaanalysis favors higher circulating Hcy levels in SS or NASH patients than controls and adds to literature, the authors did not explain the high heterogeneity between studies (i.e., with a meta-regression). Furthermore, the comparison between SS and NASH patients was not performed, which, as aforementioned, has the highest clinical implication.
http://dx.doi.org/10.1016/j.ejim.2016.06.034 0953-6205/© 2016 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Polyzos SA, Kountouras J, Homocysteine in nonalcoholic steatohepatitis: A reply, Eur J Intern Med (2016), http:// dx.doi.org/10.1016/j.ejim.2016.06.034
2
Letter to the Editor
Besides, the finding that some of Pastore et al. data in a pediatric population are in line with our findings and speculations, including the observation of higher Hcy levels in not-NASH than NASH [10], does not render the comparison “improper”, due to any differences between pediatric and adult NAFLD. By looking into Dai et al. meta-analysis, other authors reported that Hcy was not different between adult NASH or SS patients and controls [9]. Lastly, we believe that the credibility of a study is not judged by the degree of agreement with previously published data, but by future studies. In this regard, there are studies initially considered to be heretical and difficult to publish, but finally changed our view to the world. Conflict of interests SAP and JK have no conflict of interest. Acknowledgments Grants or other financial support: This work did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. References [1] Leach NV, Dronca E, Craciun EC, Crisan D. High levels of serum homocysteine in nonalcoholic steatohepatitis. Eur J Intern Med 2016. http://dx.doi.org/10.1016/j.ejim. 2016.06.018. [2] Polyzos SA, Kountouras J, Patsiaoura K, Katsiki E, Zafeiriadou E, Deretzi G, et al. Serum homocysteine levels in patients with nonalcoholic fatty liver disease. Ann Hepatol 2012;11:68–76.
[3] Polyzos SA, Kountouras J, Slavakis A, Zafeiriadou E, Patsiaoura K, Katsiki E, et al. A novel noninvasive index for nonalcoholic steatohepatitis: a pilot study. Biomarkers 2013;18:607–13. [4] Polyzos SA, Kountouras J, Tsoukas MA. Circulating homocysteine in nonalcoholic fatty liver disease. Eur J Intern Med 2015;26:152–3. [5] Leach NV, Dronca E, Vesa SC, Sampelean DP, Craciun EC, Lupsor M, et al. Serum homocysteine levels, oxidative stress and cardiovascular risk in non-alcoholic steatohepatitis. Eur J Intern Med 2014;25:762–7. [6] Polyzos SA, Mantzoros CS. Necessity for timely noninvasive diagnosis of nonalcoholic fatty liver disease. Metabolism 2014;63:161–7. [7] EASL–EASD–EASO. EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. Diabetologia 2016;59:1121–40. [8] Polyzos SA, Kountouras J, Zavos C. Nonalcoholic fatty liver disease: the pathogenetic roles of insulin resistance and adipocytokines. Curr Mol Med 2009;72:299–314. [9] Dai Y, Zhu J, Meng D, Yu C, Li Y. Association of homocysteine level with biopsyproven non-alcoholic fatty liver disease: a meta-analysis. J Clin Biochem Nutr 2016;58:76–83. [10] Pastore A, Alisi A, di Giovamberardino G, Crudele A, Ceccarelli S, Panera N, et al. Plasma levels of homocysteine and cysteine increased in pediatric NAFLD and strongly correlated with severity of liver damage. Int J Mol Sci 2014;15:21202–14.
Stergios A. Polyzos ⁎ Jannis Kountouras Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece ⁎ Corresponding author at: Simou Lianidi 13, 55134 Thessaloníki, Macedonia, Greece. Tel./fax: +30 2310424710. E-mail address: [email protected] (S.A. Polyzos). 26 June 2016 Available online xxxx
Please cite this article as: Polyzos SA, Kountouras J, Homocysteine in nonalcoholic steatohepatitis: A reply, Eur J Intern Med (2016), http:// dx.doi.org/10.1016/j.ejim.2016.06.034