European Journal of Internal Medicine 26 (2015) 152–153
Contents lists available at ScienceDirect
European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Letter to the Editor Circulating homocysteine in nonalcoholic fatty liver disease Keywords: Cysteine Homocysteine Glutathione Methionine Non-alcoholic fatty liver disease
Dear Editor, We read with interest the study of Leach et al. [1], showing higher serum homocysteine (Hcy) levels in patients with biopsy-proven nonalcoholic steatohepatitis (NASH) than apparently healthy controls of lower body mass index (BMI) and waist circumference [1]. We had previously reported on serum Hcy levels in biopsy-proven patients with nonalcoholic fatty liver disease (NAFLD) [2]; our preliminary data showed that Hcy levels were similar between NAFLD patients and age-, gender- and BMI-matched controls. Unexpectedly, we had also observed that, within NAFLD patients, Hcy was lower in NASH patients than those with nonalcoholic simple steatosis (SS), and that lower Hcy was associated with NASH independently of potential cofounders [2]. Notably, Hcy was lower by increasing the severity of fibrosis and portal inflammation, but not steatosis, lobular inflammation or ballooning. In that study, we had not separately compared NASH patients and controls, as it was inadequately reported by the authors [1]. We had not performed this comparison because, in the clinical setting, there is greater need for noninvasive indices differentiating NASH from SS patients rather than NAFLD from non-NAFLD individuals [3]; the latter is more easily achieved with ultrasonography and liver function tests. Extending our preliminary series, we introduced the noninvasive marker HSENSI (acronym of Homocysteine, Serum glutamic oxaloacetic transaminase [SGOT/AST], Erythrocyte sedimentation rate [ESR], Nonalcoholic Steatohepatitis Index), as a low cost index that may accurately predict NASH [4]: within NAFLD patients, those with higher SGOT and ESR, but lower Hcy were at higher risk for NASH [4]. Following the publication of the Leach et al. study [1], we performed a post-hoc analysis of our data [2], in which NASH patients (n = 16) and apparently healthy controls (n = 22) had similar serum Hcy levels (12.3 ± 2.5 vs. 12.6 ± 4.6 μmol/L, respectively; p = 0.806). Notably, Hcy was also similar between SS patients (n = 15) and controls (14.7 ± 2.1 vs. 12.6 ± 4.6 μmol/L, respectively; p = 0.105). Similar to the Leach et al. study [1], Hcy was correlated with vitamin B12, folic acid and HOMA-IR; contrary to the Leach et al. study [1], Hcy was not correlated with BMI, liver function tests, glucose and lipid levels. Population and methodological differences may partly explain the controversy between Leach et al. and our study. Patients and controls were not BMI-matched, as in our study. There were gender differences (males 68.8% and 24.5%, respectively) and age differences
(approximately 45 and 55 years, respectively). Vitamin B12 levels were lower in the control than NASH group in the Leach et al. study [1], whereas similar in our study. Furthermore, Hcy levels were measured with different methods (high performance liquid chromatography and immuno-chemiluminescence, respectively). Importantly, our findings are in line with relative data of a recent study in biopsy-proven pediatric NAFLD; patients with NASH (n = 30) had lower Hcy levels compared to non-NASH (n = 34) [5]. Although the sum of NAFLD patients (n = 64) had higher Hcy than age- and BMI-matched controls, Hcy levels were not different between NASH patients and controls, similarly to our study [5]. Other authors have also reported on Hcy between NAFLD patients and controls, as previously summarized in detail [1,2,5], but the issue remains largely controversial. To explain the seemingly paradoxical trend of Hcy in our study, we had speculated that lower Hcy in NASH than SS patients may be a counterbalancing effect; Hcy may be depleted in NASH in order to increase: a) the synthesis of the antioxidant glutathione via the transsulfuration pathway, because of the increasing oxidative stress by increasing the disease severity; and b) the assembly and export of very low density lipoprotein-cholesterol from the liver by increasing methionine remethylation, and thus methyl group availability via the remethylation pathway [2]. Despite this speculation, we had not measure either glutathione or methionine in our study. Regarding glutathione, Pastore et al. [5] observed lower cysteine (together with Hcy) in NASH than non-NASH children, whereas glutathione was similar between groups, an observation that strengthens our previous speculation (i.e., in order to keep sufficient glutathione levels, Hcy is depleted via the transsulfuration pathway). However, glutathione was lower in NAFLD patients than controls in this study [5], and in NASH patients than controls in the Leach et al. study [1]. Although the design of these studies do not allow drawing conclusions for causative relationships, it could be hypothesized that glutathione is well-balanced in the controls, whereas is depleted in NAFLD patients, which contributes to NAFLD pathogenesis. Within the NAFLD spectrum, when SS progresses to NASH, Hcy declines in a counterbalancing effort to replenish glutathione, thereby limiting the progression to more advanced disease. Regarding methionine, it seems that methyl-depleted diets promote NASH, whereas replenishing methyl stores prevents NAFLD progression in experimental models [6]. In a more recent study, dietary methyldonor supplementation in mice, being on a high-fat diet, prevented the progression of lipid accumulation (steatosis) in the liver, although it did not reverse NAFLD [7]. More importantly, mice fed with methionine- and choline-deficient (MCD) diet develop severe hyperhomocysteinemia and activation of the hepatic unfolded protein response (UPR), which is implicated in NAFLD pathogenesis [8]. Supplementing the MCD diet with Hcy, it attenuates the MCD diet-induced hepatic UPR activation and other adverse effects of the MCD diet, including hepatic lipid accumulation and plasma alanine transaminase elevation. Notably, Hcy supplementation replenished the MCD diet-induced depletion of hepatic S-adenosylmethionine [9], a finding supporting our previous speculation.
http://dx.doi.org/10.1016/j.ejim.2015.01.015 0953-6205/© 2015 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
Letter to the Editor
The cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) system regulates Hcy and cysteine metabolism, and contributes to endogenous biosynthesis of hydrogen sulfide (H2S); H2S, a gaseous transmitter endogenously produced by CBS/CSE, seems to improve mitochondrial function, which plays a critical role in NAFLD pathogenesis. The CBS/CSE system is highly expressed and active in the liver and it seems to interplay with insulin resistance, lipid accumulation, inflammation, oxidative and endoplasmic reticulum stress, as reviewed in detail elsewhere [10]. Its dysregulation, presenting as changes in circulating Hcy and/or H2S levels, has been reported in NAFLD patients. Delving into the hepatic CBS/CSE system will improve our understanding of NAFLD pathogenesis and give rise to new therapeutic perspectives [10]. However, much relative research is needed to elucidate its potential diagnostic and/or therapeutic advantages. Conflict of interest SAP, JK and MAT: No conflict of interest. Acknowledgments Grants or other financial support: None. References [1] 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. [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, Mantzoros CS. Necessity for timely noninvasive diagnosis of nonalcoholic fatty liver disease. Metabolism 2014;63:161–7. [4] 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.
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[5] 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. [6] Oz HS, Chen TS, Neuman M. Methionine deficiency and hepatic injury in a dietary steatohepatitis model. Dig Dis Sci 2008;53:767–76. [7] Dahlhoff C, Worsch S, Sailer M, Hummel BA, Fiamoncini J, Uebel K, et al. Methyldonor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levels. Mol Metab 2014;3:565–80. [8] Henkel A, Green RM. The unfolded protein response in fatty liver disease. Semin Liver Dis 2013;33:321–9. [9] Henkel AS, Elias MS, Green RM. Homocysteine supplementation attenuates the unfolded protein response in a murine nutritional model of steatohepatitis. J Biol Chem 2009;284:31807–16. [10] Sarna LK, Siow YL, Karmin O. The CBS/CSE system: a potential therapeutic target in NAFLD? Can J Physiol Pharmacol 2015;93:1–11.
Stergios A. Polyzos Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Corresponding author at: Simou Lianidi 13, 55134 Thessaloníki, Macedonia, Greece. Tel./fax: +30 2310424710. E-mail address: [email protected]. Jannis Kountouras Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece Michael A. Tsoukas Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 22 January 2015
Contents lists available at ScienceDirect
European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Letter to the Editor Circulating homocysteine in nonalcoholic fatty liver disease Keywords: Cysteine Homocysteine Glutathione Methionine Non-alcoholic fatty liver disease
Dear Editor, We read with interest the study of Leach et al. [1], showing higher serum homocysteine (Hcy) levels in patients with biopsy-proven nonalcoholic steatohepatitis (NASH) than apparently healthy controls of lower body mass index (BMI) and waist circumference [1]. We had previously reported on serum Hcy levels in biopsy-proven patients with nonalcoholic fatty liver disease (NAFLD) [2]; our preliminary data showed that Hcy levels were similar between NAFLD patients and age-, gender- and BMI-matched controls. Unexpectedly, we had also observed that, within NAFLD patients, Hcy was lower in NASH patients than those with nonalcoholic simple steatosis (SS), and that lower Hcy was associated with NASH independently of potential cofounders [2]. Notably, Hcy was lower by increasing the severity of fibrosis and portal inflammation, but not steatosis, lobular inflammation or ballooning. In that study, we had not separately compared NASH patients and controls, as it was inadequately reported by the authors [1]. We had not performed this comparison because, in the clinical setting, there is greater need for noninvasive indices differentiating NASH from SS patients rather than NAFLD from non-NAFLD individuals [3]; the latter is more easily achieved with ultrasonography and liver function tests. Extending our preliminary series, we introduced the noninvasive marker HSENSI (acronym of Homocysteine, Serum glutamic oxaloacetic transaminase [SGOT/AST], Erythrocyte sedimentation rate [ESR], Nonalcoholic Steatohepatitis Index), as a low cost index that may accurately predict NASH [4]: within NAFLD patients, those with higher SGOT and ESR, but lower Hcy were at higher risk for NASH [4]. Following the publication of the Leach et al. study [1], we performed a post-hoc analysis of our data [2], in which NASH patients (n = 16) and apparently healthy controls (n = 22) had similar serum Hcy levels (12.3 ± 2.5 vs. 12.6 ± 4.6 μmol/L, respectively; p = 0.806). Notably, Hcy was also similar between SS patients (n = 15) and controls (14.7 ± 2.1 vs. 12.6 ± 4.6 μmol/L, respectively; p = 0.105). Similar to the Leach et al. study [1], Hcy was correlated with vitamin B12, folic acid and HOMA-IR; contrary to the Leach et al. study [1], Hcy was not correlated with BMI, liver function tests, glucose and lipid levels. Population and methodological differences may partly explain the controversy between Leach et al. and our study. Patients and controls were not BMI-matched, as in our study. There were gender differences (males 68.8% and 24.5%, respectively) and age differences
(approximately 45 and 55 years, respectively). Vitamin B12 levels were lower in the control than NASH group in the Leach et al. study [1], whereas similar in our study. Furthermore, Hcy levels were measured with different methods (high performance liquid chromatography and immuno-chemiluminescence, respectively). Importantly, our findings are in line with relative data of a recent study in biopsy-proven pediatric NAFLD; patients with NASH (n = 30) had lower Hcy levels compared to non-NASH (n = 34) [5]. Although the sum of NAFLD patients (n = 64) had higher Hcy than age- and BMI-matched controls, Hcy levels were not different between NASH patients and controls, similarly to our study [5]. Other authors have also reported on Hcy between NAFLD patients and controls, as previously summarized in detail [1,2,5], but the issue remains largely controversial. To explain the seemingly paradoxical trend of Hcy in our study, we had speculated that lower Hcy in NASH than SS patients may be a counterbalancing effect; Hcy may be depleted in NASH in order to increase: a) the synthesis of the antioxidant glutathione via the transsulfuration pathway, because of the increasing oxidative stress by increasing the disease severity; and b) the assembly and export of very low density lipoprotein-cholesterol from the liver by increasing methionine remethylation, and thus methyl group availability via the remethylation pathway [2]. Despite this speculation, we had not measure either glutathione or methionine in our study. Regarding glutathione, Pastore et al. [5] observed lower cysteine (together with Hcy) in NASH than non-NASH children, whereas glutathione was similar between groups, an observation that strengthens our previous speculation (i.e., in order to keep sufficient glutathione levels, Hcy is depleted via the transsulfuration pathway). However, glutathione was lower in NAFLD patients than controls in this study [5], and in NASH patients than controls in the Leach et al. study [1]. Although the design of these studies do not allow drawing conclusions for causative relationships, it could be hypothesized that glutathione is well-balanced in the controls, whereas is depleted in NAFLD patients, which contributes to NAFLD pathogenesis. Within the NAFLD spectrum, when SS progresses to NASH, Hcy declines in a counterbalancing effort to replenish glutathione, thereby limiting the progression to more advanced disease. Regarding methionine, it seems that methyl-depleted diets promote NASH, whereas replenishing methyl stores prevents NAFLD progression in experimental models [6]. In a more recent study, dietary methyldonor supplementation in mice, being on a high-fat diet, prevented the progression of lipid accumulation (steatosis) in the liver, although it did not reverse NAFLD [7]. More importantly, mice fed with methionine- and choline-deficient (MCD) diet develop severe hyperhomocysteinemia and activation of the hepatic unfolded protein response (UPR), which is implicated in NAFLD pathogenesis [8]. Supplementing the MCD diet with Hcy, it attenuates the MCD diet-induced hepatic UPR activation and other adverse effects of the MCD diet, including hepatic lipid accumulation and plasma alanine transaminase elevation. Notably, Hcy supplementation replenished the MCD diet-induced depletion of hepatic S-adenosylmethionine [9], a finding supporting our previous speculation.
http://dx.doi.org/10.1016/j.ejim.2015.01.015 0953-6205/© 2015 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
Letter to the Editor
The cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) system regulates Hcy and cysteine metabolism, and contributes to endogenous biosynthesis of hydrogen sulfide (H2S); H2S, a gaseous transmitter endogenously produced by CBS/CSE, seems to improve mitochondrial function, which plays a critical role in NAFLD pathogenesis. The CBS/CSE system is highly expressed and active in the liver and it seems to interplay with insulin resistance, lipid accumulation, inflammation, oxidative and endoplasmic reticulum stress, as reviewed in detail elsewhere [10]. Its dysregulation, presenting as changes in circulating Hcy and/or H2S levels, has been reported in NAFLD patients. Delving into the hepatic CBS/CSE system will improve our understanding of NAFLD pathogenesis and give rise to new therapeutic perspectives [10]. However, much relative research is needed to elucidate its potential diagnostic and/or therapeutic advantages. Conflict of interest SAP, JK and MAT: No conflict of interest. Acknowledgments Grants or other financial support: None. References [1] 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. [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, Mantzoros CS. Necessity for timely noninvasive diagnosis of nonalcoholic fatty liver disease. Metabolism 2014;63:161–7. [4] 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.
153
[5] 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. [6] Oz HS, Chen TS, Neuman M. Methionine deficiency and hepatic injury in a dietary steatohepatitis model. Dig Dis Sci 2008;53:767–76. [7] Dahlhoff C, Worsch S, Sailer M, Hummel BA, Fiamoncini J, Uebel K, et al. Methyldonor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levels. Mol Metab 2014;3:565–80. [8] Henkel A, Green RM. The unfolded protein response in fatty liver disease. Semin Liver Dis 2013;33:321–9. [9] Henkel AS, Elias MS, Green RM. Homocysteine supplementation attenuates the unfolded protein response in a murine nutritional model of steatohepatitis. J Biol Chem 2009;284:31807–16. [10] Sarna LK, Siow YL, Karmin O. The CBS/CSE system: a potential therapeutic target in NAFLD? Can J Physiol Pharmacol 2015;93:1–11.
Stergios A. Polyzos Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Corresponding author at: Simou Lianidi 13, 55134 Thessaloníki, Macedonia, Greece. Tel./fax: +30 2310424710. E-mail address: [email protected]. Jannis Kountouras Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece Michael A. Tsoukas Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 22 January 2015
