methodological concerns about a recent meta-analysis of the influence of the i148m variant of...

CORRESPONDENCE

Liver Transplantation for Hepatocellular Carcinoma: The Impact of HumanImmunodeficiency Virus Infection—21 Plus 13

To the Editor:

We read with interest the article by Vibert et al.1 recently pub-lished in HEPATOLOGY. The authors described their single-centerexperience with liver transplantation for hepatocellular carcinoma(HCC) in human immunodeficiency virus (HIV)–positive patients(21 cases) and compared those patients to HIV-negative patients(61 cases) who were also affected by HCC. Because of the higherdropout rate among the HIV-positive patients (23.8% versus11.4%), HIV infection impaired the results of liver transplantationfor HCC on an intent-to-treat basis but had no significant impacton overall survival and recurrence-free survival after liver transplan-tation. In our center from 2005 to 2010, we performed transplan-tation for 13 HIV-positive patients affected by HCC. The charac-teristics of this cohort are reported in Table 1. Unlike Vibertet al.’s patients, none of our patients were dropped from the wait-ing list. None experienced HCC recurrence, although threepatients were outside the Milan criteria at listing (23%); only oneof those patients (7.7%) had microvascular invasion. Seventy-sevenpercent had grade 2 and 23% had grade 3 HCC according toEdmondson-Steiner.2 The mean number and total diameter of theHCC nodules were 2 6 1 and 46 6 29 mm, respectively, uponpathological analysis. Before transplantation, all patients weretreated with transarterial chemoembolization or combined transar-terial chemoembolization and radio frequency ablation; the meannecrosis value was 67% 6 39% for the HCC nodules upon patho-logical analysis. Finally, the 1-, 3-, and 5-year patient and graft sur-vival rates were 84.6%, 84.6%, and 70.5% and 84.6%, 84.6%,and 84.6%, respectively, with a median follow-up of 35 months(range ¼ 2-73 months). In conclusion, our experience seems to becomparable to the experience reported by Vibert et al. except forthe absence of HCC recurrence, which was present in 23.8% ofthe patients investigated in the French cohort.

UMBERTO BACCARANI, M.D., PH.D., F.E.B.S.1

GIAN LUIGI ADANI, M.D., PH.D.1

MARCELLO TAVIO, M.D.2

PIERLUIGI VIALE, M.D.3

1Liver Transplant Unit, Department of Medical and Biological Sciences,University of Udine, Udine, Italy

2Division of Infectious Disease, Ospedali Riuniti of Ancona, Ancona,Italy

3Institute of Infectious Disease, University of Bologna, Bologna, Italy

References1. Vibert E, Duclos-Vallee JC, Ghigna MR, Hoti E, Salloum C, Guettier

C, et al. Liver transplantation for hepatocellular carcinoma: the impactof human immunodeficiency virus infection. HEPATOLOGY 2011;53:475-482.

2. Edmondson HA, Steiner PE. Primary carcinoma of the liver: a study of100 cases among 48,900 necropsies. Cancer 1954;7:462-503.

CopyrightVC 2011 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.24287Potential conflict of interest: Nothing to report.

Reply:

We read with much interest the letter of Baccarani and Collconcerning our article on transplantation for patients with humanimmunodeficiency virus (HIV) and hepatocellular carcinoma(HCC).1 They provide interesting results for 13 patients withHIV, and they should be congratulated for the absence of drop-out and the excellent survival rate observed after transplantation.However, their patients differed from ours in certainrespects: 1 . The Model for End-Stage Liver Disease scores of theirpatients were low (median ¼ 10, range ¼ 10-13) in comparisonwith the scores of our patients (median ¼ 15, range ¼

Table 1. Characteristics of the Study Cohort

Patient Sex

Age

(Years)

Virological

Status

Pre-LT

MELD

Score

Pre-LT

CD4 Level

(Cells/mL)

Pre-LT

Nodule (n)

Pre-LT

Diameter

(mm)

Pre-LT

Milan

Criteria

Pre-LT AFP

Level (lg/L)

Pre-LT

Therapy

OS After

Listing

(Months)

OS

After LT

(Months)

Last

Known

Status

1 Male 50 HCV 10 431 1 20 þ 18 TACE 78 73 AFOR

2 Male 58 Negative 10 220 1 15 þ 2 TACE 72 72 AFOR



5 Male 49 HBV 10 239 3 45 � 66 TACE/RF 38 37 DFOR

6 Male 52 HCV 10 227 2 45 � 4 TACE 17 2 DFOR

7 Male 42 HCV/HBV 10 206 1 29 þ 7 TACE/RF 47 42 AFOR

8 Male 51 HBV 10 418 3 24 þ 60 TACE 39 35 AFOR

9 Male 47 HCV 10 224 1 80 � 4 TACE/RF 24 24 AFOR

10 Female 44 HCV 12 909 3 22 þ 13 TACE 15 14 AFOR

11 Male 49 HCV 12 522 2 17 þ 67 TACE 2 2 DFOR

12 Female 44 HCV 10 307 1 30 þ 4 TACE/RF 19 10 AFOR


Abbreviations: AFOR, alive free of recurrence; AFP, alpha-fetoprotein; DFOR, died free of recurrence; HBV, hepatitis B virus; HCV, hepatitis C virus; LT, liver trans-

plantation; MELD, Model for End-Stage Liver Disease; OS, overall survival; RF, radio frequency; TACE, transarterial chemoembolization.

2138

8-31). 2 . No patients in their series had a pretransplant alpha-fetoprotein serum level > 100 lg/L, whereas 6 of our 21 listedpatients (28.5%) did. 3 . Also, only one of their patients (7.7%)had microvascular invasion, whereas the proportion was 50% (8/16) for our transplant patients. 4 . Finally, the CD4 cell count was>200/mL for all their patients, but for 6 of the 21 patients(28.5%) listed for liver transplantation in our series, the count was<200 cells/mL.

Therefore, even if the results are comparable except for the ab-sence of recurrence and dropout in their series, both differencescan be explained by the higher HCC stage of our patients, theirmore altered liver function, and the higher impact of the HIVinfection on their CD4 cell count.

With the stricter selection criteria that Baccarani and Collhave applied for listing patients with HIV and HCC, they haveconfirmed that excellent results can be obtained for thesepatients. However, we should be careful about drawing definitiveconclusions about patient selection from these two limited se-ries. Predictive factors for tumor progression and recurrencemust be better defined in this subgroup of patients, and combi-nations of therapies and more rigorous follow-up are advocatedfor improving the post–liver transplant results for HIV-infectedpatients with HCC.

RENE ADAM, M.D., PH.D.1,2,4

ERIC VIBERT, M.D.1,3,5

JEAN-CHARLES DUCLOS-VALLEE, M.D., PH.D.1,3,5

DIDIER SAMUEL, M.D., PH.D.1,3,51Centre Hepato-Biliaire, Hopital Paul Brousse

Assistance Publique-Hopitaux de ParisVillejuif, France

2Unite 776 and 3Unite 785, Institut National de laSante et de la Recherche MedicaleVillejuif, France

4Unites Mixtes de Recherche en Sante 776 and 5Unites Mixtes deRecherche en Sante 785 Universite Paris-SudVillejuif, France

Reference1. Vibert E, Duclos-Vallee JC, Ghigna MR, Hoti E, Salloum C, Guettier C,

et al. Liver transplantation for hepatocellular carcinoma: the impact ofhuman immunodeficiency virus infection. HEPATOLOGY 2011;53:475-482.


More on Vitamin C Deficiency in Mice and Humans

To the Editor:

I read with great interest the article by Park et al.,1 whofound that a vitamin C deficiency ameliorated liver fibrosis viathe up-regulated expression of peroxisome proliferator-activatedreceptor gamma in senescence marker protein 30 (SMP30)knockout mice. The results are interesting and shed light on thepossible mechanisms underlying the attenuated liver fibrosis ofSMP30 knockout mice. Here I propose that if these results inSMP30 knockout mice are to be extended to humans, we willneed more comprehensive studies based on the new and signifi-cant finding that humans employ a unique strategy for adaptingto vitamin C deficiency.

As we know, humans and some other mammals such asguinea pigs and fruit bats have an inborn metabolic error, thatis, an inability to synthesize vitamin C from glucose due to adefective form of the gene for the L-gulonolactone oxidaseenzyme required in the last step of vitamin C synthesis.2 Mon-tel-Hagen et al.3 recently demonstrated that human erythrocytesfrom the vitamin C auxotrophs can dramatically enhance theirvitamin C–transporting ability by switching the preference ofglucose transporter 1 (Glut1) from glucose to the oxidized formof ascorbic acid [L-dehydroascorbic acid (DHA)]. The mem-brane protein stomatin has been shown to regulate the substratepreference switch. More interestingly, erythrocyte Glut1 and itsassociated DHA uptake are specific to species unable to producevitamin C from glucose. Mice can synthesize vitamin C, andfurther experimentation has indicated that their mature erythro-cytes do not harbor Glut1 or transport DHA. Therefore, incomparison with mice, humans possess a compensatory mecha-nism to adapt to a vitamin C deficiency, and this may lead us toreevaluate the human vitamin C deficiency status and potentiallyresult in more complicated reflections on vitamin C deficiencyin human liver tissues. I do not mean to question the rationalityand significance of the findings of Park et al.,1 but I submit thatthis unique compensatory mechanism should be considered in

future studies on the relationships between vitamin C deficiencyand liver fibrosis in humans.

LIANG SHEN, PH.D.Shandong Provincial Research Center for

Bioinformatic Engineering and TechniqueShandong University of TechnologyZibo, People’s Republic of China

References1. Park JK, Ki MR, Lee HR, Hong IH, Ji AR, Ishigami A, et al. Vitamin

C deficiency attenuates liver fibrosis by way of up-regulated peroxisomeproliferator-activated receptor-gamma expression in senescence markerprotein 30 knockout mice. HEPATOLOGY 2010;51:1766-1777.

2. Nishikimi M, Fukuyama R, Minoshima S, Shimizu N, Yagi K. Cloningand chromosomal mapping of the human nonfunctional gene forL-gulono-c-lactone oxidase, the enzyme for L-ascorbic acid biosynthesismissing in man. J Biol Chem 1994;269:13685-13688.

3. Montel-Hagen A, Kinet S, Manel N, Mongellaz C, Prohaska R, BattiniJL, et al. Erythrocyte Glut1 triggers dehydroascorbic acid uptake inmammals unable to synthesize vitamin C. Cell 2008;132:1039-1048.


Reply

We read with great interest Shen’s letter and thank him for his in-terest in our study.1 We agree that the unique compensatory mecha-nism for adapting to vitamin C deficiency should be considered when

HEPATOLOGY, Vol. 53, No. 6, 2011 CORRESPONDENCE 2139

the relationship between vitamin C deficiency and liver fibrosis inhumans is examined. It has previously been established that senes-cence marker protein 30 (SMP30) has gluconolactonase activity,which plays a major role in L-ascorbic acid (vitamin C) biosynthesisin the liver.2 Therefore, vitamin C deficiency is induced in SMP30knockout mice by their inability to synthesize vitamin C; the same istrue for L-gulono-c-lactone oxidase (GLO) knockout mice, humans,primates, guinea pigs, and fruit bats. In our study, we found novel evi-dence showing that liver fibrosis is ameliorated by vitamin C defi-ciency through the up-regulated expression of peroxisome prolifera-tor-activated receptor c (PPAR-c) in SMP30 knockout mice.1

Because PPAR-c plays a pivotal role in cellular senescence,3 it isbelieved that the cellular senescence of liver parenchymal and nonpar-enchymal cells is the main factor in the amelioration of liver fibrosisdue to vitamin C deficiency.

Recently, Montel-Hagen et al.4 demonstrated that erythrocyteglucose transporter 1 (Glut1) triggers dehydroascorbic acid uptake inmammals unable to synthesize vitamin C: the preference of Glut1 isswitched from glucose to the oxidized form of vitamin C, L-dehy-droascorbic acid (DHA). This means that mammals unable to syn-thesize vitamin C have a compensatory mechanism for recycling vita-min C. However, in adult mice, erythrocytes do not harbor Glut1and do not transport DHA, and this means that there is no compen-satory mechanism via Glut1 for vitamin C deficiency in mice. There-fore, the condition of complete vitamin C deficiency can be easilyinduced in SMP30 knockout mice and GLO knockout mice in com-parison with human beings and primates. In our study,1 we showedthat the serum vitamin C level of the SMP30 knockout mice wasalmost undetectable, and this indicated that the SMP30 knockoutmice did not have another compensatory mechanism for their vita-min C deficiency. Consequently, we concluded that their vitamin Cdeficiency ameliorated liver fibrosis through the induction of cellularsenescence by up-regulated PPAR-c.

When we extend our results with SMP30 knockout mice tohuman beings, we should point out a few factors with respect to theunique compensatory mechanism for vitamin C deficiency. First, weneed to note the process of the formation and degradation of DHAin vivo. Although human erythrocytes can recycle DHA by convertingit to ascorbic acid, it is known that mammalian plasma has a systemrapidly promoting the hydrolysis of DHA into diketogulonate(DKG), which is rapidly degraded into 5-carbon and 4-carbon spe-cies.5 Moreover, previous studies have revealed that the hydrolysis ofDHA into DKG is irreversible in vivo.6 These results mean that someDHA can be recycled and that some DHA can be hydrolyzed intoDKG. Eventually, if there is no supply of vitamin C, all the DHAmay be hydrolyzed into DKG, and this would induce vitamin C defi-ciency; however, human beings and primates have a unique compen-satory mechanism for DHA recycling. Therefore, it is strongly

believed that human beings and primates need an appropriate intakeof vitamin C.

Returning to our study1 and the relationship between vitaminC and liver fibrosis in human beings, we strongly expect that ame-liorated liver fibrosis will be observed to cause cellular senescencein human beings with complete vitamin C deficiency, as we havedemonstrated in SMP30 knockout mice. However, in comparisonwith SMP30 knockout mice, inducing the condition of completevitamin C deficiency might take much longer in human beingsand primates because of their compensatory mechanism for adapt-ing to vitamin C deficiency. In future studies on the relationshipbetween the amount of vitamin C intake and liver fibrosis inhumans, we totally agree that the unique compensatory mecha-nism for adapting to vitamin C deficiency should be considered.

JIN-KYU PARK, D.V.M., PH.D.1

KYU-SHIK JEONG, D.V.M., PH.D.1,21College of Veterinary Medicine

Kyungpook National UniversityDaegu, Republic of Korea

2Stem Cell Therapeutic Research InstituteKyungpook National UniversityDaegu, Republic of Korea

References1. Park JK, Ki MR, Lee HR, Hong IH, Ji AR, Ishigami A, et al. Vitamin

C deficiency attenuates liver fibrosis by way of up-regulated peroxisomeproliferator-activated receptor-gamma expression in senescence markerprotein 30 knockout mice. Hepatology 2010;51:1766-1777.

2. Kondo Y, Inai Y, Sato Y, Handa S, Kubo S, Shimokado K, et al. Senes-cence marker protein 30 functions as gluconolactonase in L-ascorbicacid biosynthesis, and its knockout mice are prone to scurvy. Proc NatlAcad Sci U S A 2006;103:5723-5728.

3. Gan Q, Huang J, Zhou R, Niu J, Zhu X, Wang J, et al. PPARc accel-erates cellular senescence by inducing p16INK4a expression in humandiploid fibroblasts. J Cell Sci 2008;121:2235-2245.

4. Montel-Hagen A, Kinet S, Manel N, Mongellaz C, Prohaska R, BattiniJL, et al. Erythrocyte Glut1 triggers dehydroascorbic acid uptake inmammals unable to synthesize vitamin C. Cell 2008;132:1039-1048.

5. Koshiishi I, Mamura Y, Liu J, Imanari T. Degradation of dehydroascor-bate to 2,3-diketogulonate in blood circulation. Biochim Biophys Acta1998;1425:209-214.

6. Deutsch JC. Dehydroascorbic acid. J Chromatogr A 2000;881:299-307.


Is Bacterial DNA a Better Marker than Endotoxin of Bacterial Translocationin Decompensated Cirrhosis?

To the Editor:

Translocation of intestinal bacterial products deteriorates sys-temic and liver hemodynamics in patients with cirrhosis.1

We read with interest the report by Bellot et al.,2 who foundthat the presence of bacterial DNA (bactDNA) in patients withcirrhosis aggravates the systemic circulatory dysfunction and isassociated with a more severe intrahepatic endothelial dysfunction.The same group previously reported that bactDNA is associated

with increased serum inflammatory responses, independently of en-dotoxin. Therefore, the question arises whether bactDNA repre-sents a more global marker of bacterial translocation compared toendotoxin in patients with advanced cirrhosis.

We would like to raise some issues concerning the results ofBellot et al. and share the results of our investigations on the roleof endotoxin and bactDNA in patients with decompensated cirrho-sis. First, although patients with bactDNA had more profound sys-temic vasodilation than patients who were negative for bactDNA,

2140 CORRESPONDENCE HEPATOLOGY, June 2011

baseline hepatic venous pressure gradient (HVPG) was similar inboth groups. Second, plasma bactDNA concentration was not cor-related with systemic hemodynamic parameters, thus calling intoquestion the pivotal role of bactDNA in the hemodynamic distur-bances of cirrhosis.

We conducted a study to determine plasma endotoxin in 30patients with cirrhosis who had ascites and to investigate the effect ofintestinal decontamination on HVPG, through use of rifaximin for28 days.3 Endotoxemia was common in this cohort of patients andwas correlated with the severity of liver disease. Moreover, endotoxinlevels decreased significantly after rifaximin administration, and thedifference was correlated with the difference in HVPG values.

Subsequently, we investigated the presence of bactDNA in thesame blood samples. DNA was extracted with the QIAmpDNABlood Minikit (Qiagen, Germany), and bactDNA was tested bypolymerase chain reaction using specific primers for bacterial 16Sribosomal RNA. BactDNA was not detected in any blood samplefrom systemic or splanchnic circulation on days 0 and 29 afterrifaximin administration. The method was validated in patientswith bacteremia and all samples were positive for bacterial genomicfragments.

We have no clear explanation why bactDNA was not detectedin our patients. We may have missed transient episodes of bacterialtranslocation because we only monitored twice for bactDNA andwith an interval of 28 days. Furthermore, rifaximin administrationmay have achieved elimination of bactDNA, but endotoxin wasstill present. However, regardless of issues of detection ofbactDNA, according to our results, endotoxemia could represent areliable marker of bacterial translocation in patients with decom-pensated cirrhosis. The presence of bacterial products and/orbactDNA might be an additional step in the sequence of eventsthat further deteriorate portal hypertension, especially in the ab-sence of viable bacteria or endotoxemia, but this needs furtherinvestigation.

JIANNIS VLACHOGIANNAKOS, M.D.1

GEORGE DAIKOS, M.D.1

ULRICH THALHEIMER, M.D.2

ANDREW K. BURROUGHS, M.D.2

SPIROS D. LADAS, M.D.11Hepatogastroenterology Unit, First Department of Propaedeutic

Medicine, Medical School, Athens University, Laiko GeneralHospital, Athens, Greece

2The Sheila Sherlock Liver Centre, Royal Free Hospital, London, UK

References1. Thalheimer U, Triantos CK, Samonakis DN, Patch D, Burroughs AK.

Infection, coagulation and variceal bleeding in cirrhosis. Gut 2005;54:556-563.

2. Bellot P, Garcia-Pagan JC, Frances R, Abraldes JG, Navasa M, Perez-Mateo M, et al. Bacterial DNA translocation is associated with systemiccirculatory abnormalities and intrahepatic endothelial dysfunction inpatients with cirrhosis. HEPATOLOGY 2010;52:2044-2052.

3. Vlachogiannakos J, Saveriadis AS, Viazis N, Theodoropoulos I, Fou-doulis K, Manolakopoulos S, et al. Intestinal decontamination improvesliver haemodynamics in patients with alcohol-related decompensatedcirrhosis. Aliment Pharmacol Ther 2009;29:992-999.


Reply:

We thank Vlachogiannakos et al. for their interest in our recent ar-ticle.1 One of the main questions raised in their letter is whether bac-terial DNA detection is a more accurate marker of bacterial transloca-tion than endotoxin. Considering that the detection of endotoxin,which is almost exclusively present in gram-negative microorganisms,or its soluble receptor lipopolysaccharide binding protein may severelyunderestimate the translocation of gram-positive microorganisms, wehave proposed bacterial DNA detection as a broad-range marker ofbacterial translocation in the setting of cirrhosis. In our experience,bacterial DNA is present in approximately 35% of patients with cir-rhosis and ascites, and similar findings have recently been reported byAngeli et al.2 with the same methodology.

Our results show that baseline portal pressures are similar forpatients with ascites independently of the presence of bacterialDNA. This finding is in line with previous clinical studies showingthat elevated serum levels of lipopolysaccharide binding protein,which is a more sensitive marker of endotoxemia than endotoxinitself because of its longer biological half-life, are not associatedwith a greater degree of portal hypertension.3

In our study,1 which included a large series of uninfectedpatients with cirrhosis, we did not find any correlation between thebacterial DNA concentration and systemic hemodynamic parame-ters (mean arterial pressure and systemic vascular resistance). Thisdoes not entirely rule out a role for bacterial translocation becauseother bacterial products besides bacterial DNA can hypotheticallyplay a role in the inflammatory response. Indeed, monocytes frompatients with cirrhosis seem to have an enhanced response to bacte-rial products. Therefore, small or even undetectable amounts ofbacterial products may cause an enhanced release of cytokines bymonocytes and thus further impair systemic hemodynamics.

We cannot offer a clear explanation for the discrepanciesbetween our results and Vlachogiannakos et al.’s results withrespect to the detection of bacterial DNA. Methodological varia-tions are likely responsible for the differences because we wouldexpect to find bacterial DNA in 30% to 35% of the baseline sam-ples from Vlachogiannakos et al.’s patients (they all had ascites,and some had measurable levels of endotoxin). The inability todetect bacterial DNA after a 28-day course of rifaximin does notseem unexpected, although we do not have our own data for this.

Molecular techniques are in continuous development. Thedetection of small amounts of bacterial DNA in blood samplesfrom patients is always challenging, and we must take advantage ofmore powerful methodologies such as pyrosequencing for studyingdifferent issues that still remain controversial with respect to bacte-rial translocation in patients with cirrhosis.

PABLO BELLOT, M.D.2

JOSE SUCH, M.D.2

JAIME BOSCH, M.D.11Hepatic Hemodynamic Laboratory

Hospital ClinicBarcelona, Spain

2Liver UnitHospital General y Universitariode Alicante, Alicante, Spain

References1. Bellot P, Garcıa-Pagan JC, Frances R, Abraldes JG, Navasa M, Perez-

Mateo M, et al. Bacterial DNA translocation is associated with systemiccirculatory abnormalities and intrahepatic endothelial dysfunction inpatients with cirrhosis. HEPATOLOGY 2010;52:2044-2052.

2. Angeli P, Brun P, Cesari M, Fasolato S, Cavallin M, Castagliuolo I,et al. Prevalence of bacterial DNA in patients with cirrhosis and refrac-tory ascites and its role in the development of cardiac and renal dys-functions [Abstract]. HEPATOLOGY 2010;52(Suppl):342A.


3. Albillos A, de la Hera A, Gonzalez M, Moya JL, Calleja JL, MonserratJ, et al. Increased lipopolysaccharide binding protein in cirrhoticpatients with marked immune and hemodynamic derangement. HEPA-

TOLOGY 2003;37:208-217.

CopyrightVC 2011 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.24336Potential conflict of interest: Nothing to report.Address reprint requests to: Jaime Bosch, Calle Villaroel 170, Barcelona08036, Spain. E-mail: [email protected]; fax: (34)-932279856.

Nonalcoholic Fatty Liver Disease Activity Score and Brunt’s Pathologic Criteria for the Diagnosis ofNonalcoholic Steatohepatitis: What Do They Mean and Do They Agree?

To the Editor:

We read with great interest two articles recently published inHEPATOLOGY that compare different histological classifications fornonalcoholic fatty liver disease (NAFLD).1,2

The first study1 confirms the lack of a strict relationship betweenBrunt’s classification3 and Kleiner’s classification4 in a large cohort ofAmerican adults [j statistic ¼ 0.57, 95% confidence interval (CI) ¼0.51-0.62]. The authors report that not all biopsy samples with anonalcoholic fatty liver disease activity score (NAS) � 5 met thediagnostic criteria for definite steatohepatitis (SH), and some caseswith NAS � 4 did; this indicates that an NAS threshold value of �5cannot be used to reliably establish the presence or absence of SH.

The second study2 reports an even lower level of agreement betweenBrunt’s and Kleiner’s classifications (j statistic ¼ 0.178; 95% CI ¼0.117-0.240) and concludes that the diagnosis of SH by NAS, regard-less of its threshold, is inappropriate. Moreover, fibrosis remained an in-dependent predictor of liver-related mortality in a series of 257 NAFLDpatients after a median follow-up of 146 months.

Knowing that fibrosis obviously dictates survival for patients withNAFLD2 and that inflammation is the precursor lesion of fibrosis,5

we thought that it would be interesting to assess whether the findingspublished in the aforementioned studies1,2 could be reproduced in acohort of European patients with NAFLD who were evaluated at asingle tertiary liver center. To this end, we compared the diagnosticyields of the two most widely followed histological classifications forour cohort of 96 NAFLD patients without cirrhosis.

Histological liver samples were evaluated by a single experiencedliver pathologist (L.L.); only biopsy samples at least 15 mm long withat least six portal tracts were considered eligible for analysis.

According to Brunt’s criteria, 31 patients did not have SH, and65 patients did have SH; according to Kleiner’s criteria, 61 patientsdid not have SH (i.e., NAS � 4), and 35 did have SH (i.e., NAS �5). NAS was �5 in 53.8% and �4 in 46.2% of the patients with SHaccording to Brunt’s criteria, whereas NAS was �4 in 100% of those

without SH. All biopsy samples with NAS � 5 fulfilled Brunt’s diag-nostic criteria for SH. NAS � 4 did not indicate benign histologicalfindings; this agreed with Brunt’s most recent study1 because 49.8%of the patients with NAS � 4 had SH according to Brunt’s originalcriteria.3 Table 1 shows the independent predictors of SH accordingto a stepwise multivariate logistic regression analysis.

On the basis of our experience and the findings of recent stud-ies,1,2 we can conclude that both classifications faithfully mirror meta-bolic derangements typical of SH. Moreover, the correlation betweenBrunt’s and Kleiner’s original classifications3,4 is fair to moderate [jstatistic ¼ 0.43, 95% CI ¼ 0.27-0.59 (this study)]. This agreement,however, might be increased up to 0.74 (0.55-0.93) if the SH cutoffwere lowered to NAS ¼ 4 or up to 0.66 (0.47-0.85) if patients witha Brunt grade of 1 were no longer considered to have SH. Finally, wemaintain that, by reflecting both inflammation and fibrosis more ana-lytically, Brunt’s original classification1 provides more substantial infor-mation to the practicing clinical hepatologist.

STEFANO BALLESTRI, M.D.AMEDEOLONARDO, M.D.PAOLA LORIA, M.D.Unit of Internal Medicine, Department of Internal Medicine

Endocrinology, Metabolism, and GeriatricsUniversity of Modena and Reggio EmiliaModena, Italy

References1. Brunt EM, Kleiner DE, Wilson LA, Belt P, Neuschwander-Tetri BA, for

NASH Clinical Research Network (CRN). Nonalcoholic fatty liver disease(NAFLD) activity score and the histopathologic diagnosis in NAFLD: dis-tinct clinicopathologic meanings. HEPATOLOGY 2011;53:810-820.

2. Younossi Z, Stepanova M, Rafiq N, Makhlouf H, Younoszai Z,Agrawal R, et al. Pathologic criteria for non-alcoholic steatohepatitis(NASH): inter-protocol agreement and ability to predict liver-relatedmortality. HPATOLOGY 2011.

Table 1. Independent Predictors of NASH According to Brunt’s Criteria and NAS

Predictor

Brunt’s Criteria NAS

Odds Ratio 95% CI P Value Odds Ratio 95% CI P Value

Age (years) 1.072 0.978-1.176 0.140 1.029 0.979-1.081 0.256

Sex (male) 0.859 0.126-5.869 0.877 0.739 0.199-2.746 0.651

Body mass index (kg/m2) 1.308 0.993-1.772 0.056 — — —

Homeostasis model assessment score 1.817 1.058-3.119 0.030 — — —

Uric acid 2.368 1.208-4.641 0.012 1.518 1.040-2.215 0.031

Glutamic pyruvic transaminase — — — 1.017 1.002-1.032 0.023

Total CH 0.957 0.934-0.981 <0.001 0.981 0.967-0.994 0.005

MS 9.318 1.254-69.219 0.029 — — —

The variables processed in the logistic regression analysis were selected from those predictors of SH found to be significant (P< 0.05) in the univariate analysis

and according to our clinical judgment and the available literature. Age and sex were used as fixed covariates. Dashes indicate that variables were not kept in the

final model. For Brunt’s criteria, see Brunt et al.3; for NAS, see Kleiner et al.4


3. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Ba-con BR. Nonalcoholic steatohepatitis: a proposal for grading and stag-ing the histological lesions. Am J Gastroenterol 1999;94:2467-2474.

4. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cum-mings OW, et al., for Nonalcoholic Steatohepatitis Clinical ResearchNetwork. Design and validation of a histological scoring system fornonalcoholic fatty liver disease. HEPATOLOGY 2005;41:1313-1321.

5. Argo CK, Northup PG, Al-Osaimi AM, Caldwell SH. Systematicreview of risk factors for fibrosis progression in non-alcoholic steatohe-patitis. J Hepatol 2009;51:371-379.


Reply:

We thank Ballestri et al. for their comments about our recentand past publications.1,2 We would like to remind them and ourreading audience that the nonalcoholic fatty liver disease activity score(NAS) of the Nonalcoholic Steatohepatitis (NASH) Clinical ResearchNetwork was developed and validated for numerically describing thespectrum of nonalcoholic fatty liver disease. It was never intended toreplace a pathologist’s diagnosis of steatohepatitis, but this conclusionis implicit in the analysis undertaken by Ballestri et al. Because otherauthors have also misapplied the NAS to diagnose NASH, we under-took the study described in our 2011 publication1 to determine theimplications of doing this in a very large patient sample. Interestingly,we found that a high NAS correlated more with elevated alanineaminotransferase levels, whereas the diagnosis of NASH correlatedmore with features of metabolic syndrome.

A scoring system is meant for just that: scoring relevant lesions ofan injury pattern so that the severity of the disease can be monitoredsemiquantitatively over time and in cohorts of patients, especiallythose in treatment trials. None of the scoring systems for chronichepatitis, liver transplant rejection, and biliary diseases were devel-oped to diagnose these diseases, and neither was the NAS. When we

described the NAS,2 we did note that there was a statistical corre-lation between an NAS value � 5 and an independent diagnosisof steatohepatitis by a pathologist. Unfortunately, this observationhas been misinterpreted by some as a proposal to replace the di-agnosis with the score, even though we clearly stated otherwise inour study. Publications from the NASH Clinical ResearchNetwork have consistently separated the diagnosis from the score,and we have emphasized this separation in our presentations. Wehope that other investigators will find it appropriate to make thisdistinction as well.

ELIZABETH M. BRUNT, M.D.1

DAVID E. KLEINER, M.D., PH.D.2

BRENT A. NEUSCHWANDER-TETRI, M.D.31Department of Pathology and Immunology

Washington UniversitySt. Louis, MO

2Department of Laboratory MedicineNational Cancer InstituteNational Institutes of HealthBethesda, MD

3Division of Gastroenterology and HepatologyDepartment of MedicineSaint Louis UniversitySt. Louis, MO

References1. Brunt EM, Kleiner DE, Wilson L, Belt P, Neuschwander-Tetri BA, for

the NASH Clinical Research Network. Nonalcoholic fatty liver disease(NAFLD) activity score and the histopathologic diagnosis in NAFLD:distinct clinicopathologic meanings. HEPATOLOGY 2011;53:810-820.

2. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cum-mings OW, et al. Design and validation of a histological scoring systemfor nonalcoholic fatty liver disease. HEPATOLOGY 2005;41:1313-1321.


Cyclooxygenase Inhibition Up-Regulates Liver Carnitine Palmitoyltransferase 1AExpression and Improves Fatty Liver

To the Editor:

We read with great interest the article by Orellana-Gavaldaet al. about the ameliorating effects of long-term hepatic genetransfer of carnitine palmitoyltransferase 1A (CPT1A) on obesity-induced hepatic steatosis, diabetes, and insulin resistance.1 Theauthors observed increased lipid oxidation mediated by a signifi-cant up-regulation of liver CPT1A messenger RNA (mRNA). Thiseffect not only improved lipid and glucose metabolism, but alsohad direct impact on liver inflammatory stress triggered by high-fatdiet (HFD) feeding.

Orellana-Gavalda et al. suggest that increasing hepatic CPT1Aexpression is a valid in vivo strategy to reduce obesity-related com-plications. In a rat model of nonalcoholic fatty liver disease(NAFLD), we observed fairly similar results with indomethacin, adual pharmacological inhibitor of cyclooxygenase 1 (COX1) (pros-

taglandin H synthase 1 [PTGS1]) and COX2 (PTGS2). We eval-uated the effect of the drug on reversing fatty liver, and we alsoexplored the impact on liver mRNA expression of several lipogenicand glucogenic genes, and nuclear receptors. Rats were given aHFD2 for 8 weeks, and after this period, animals were randomlydivided into two groups. For 4 weeks, along with access to HFD,one group received indomethacin (n ¼ 5 rats, 1 mg/day) every24 hours, and the second group (n ¼ 5 rats) was fed with HFD; acontrol group (6 rats) was fed with standard chow diet (SCD) for12 weeks. We observed that indomethacin significantly revert fattyliver disease (Fig. 1). The most remarkable effects of COX inhibi-tion by indomethacin in the HFD group in comparison with theSCD group were: a 230% increase of liver expression of CPTA1mRNA, a 100% increase of liver abundance of PCK1 mRNA (phos-phoenolpyruvate carboxykinase, the main control point for the regu-lation of gluconeogenesis), and an increase of 84% of PPARa mRNA


(peroxisome proliferator-activated receptor alpha, a transcription fac-tor that controls the expression of genes encoding fatty acid oxidationenzymes and mitochondrial fatty acid oxidation) (Fig. 1).

As far as we know, we show for the first time that indometh-acin is able to increase liver CPT1A mRNA. We can not explainthe exact mechanism by which the drug influences liver CPT1Aexpression, although an inhibitory effect of a COX product onthe gene expression is an obvious option, but we agree withOrellana-Gavalda et al. that liver CPT1A is a prime target toincrease beta-oxidation of hepatic long-chain fatty acids. Otherexplanations are probable. Indomethacin was regarded as a dualPPARc/PPARa ligand.3 In addition, the 50-flanking region ofCOX2 has several potential transcription regulatory sequences,including CCAAT/enhancer binding protein motif (a gene thatspecifically regulates hepatic gluconeogenesis and lipogenesis4) andtwo nuclear factor-jB sites (a key modulator of liver injury inNAFLD). Hence, these observations may explain the beneficialeffects of indomethacin on NAFLD. In summary, our results rep-resent proof of principle that pharmacological COX inhibitionmay provide a novel approach for reversing fatty liver by modu-lating the liver CPT1A mRNA expression. These results also addsome clues about the potential role of the inducible COX2 andits proinflammatory prostaglandin products in metabolic disor-ders, including NAFLD.

MARIA S. ROSSELLI, M.SC.1

ADRIANA L. BURGUENO, PH.D.2

CARLOS J. PIROLA, PH.D.2

SILVIA SOOKOIAN, M.D., PH.D.1

1Department of Clinical and Molecular Hepatology and2Department of Molecular Genetics and Biology of Complex

Diseases, Institute of Medical Research ‘‘Alfredo Lanari’’Instituto de Investigaciones Medicas, University of BuenosAires–National Council of Scientific and Technological Research(CONICET), Ciudad Autonoma de Buenos Aires, Buenos AiresArgentina

References1. Orellana-Gavalda JM, Herrero L, Malandrino MI, Paneda A, Sol

Rodriguez-Pena M, Petry H, et al. Molecular therapy for obesity anddiabetes based on a long-term increase in hepatic fatty-acid oxidation.HEPATOLOGY 2011;53:821-832.

2. Landa MS, Garcia SI, Schuman ML, Burgueno A, Alvarez AL, SaraviaFE, et al. Knocking down the diencephalic thyrotropin-releasing hor-mone precursor gene normalizes obesity-induced hypertension in therat. Am J Physiol Endocrinol Metab 2007;292:E1388-E1394.

3. Lehmann JM, Lenhard JM, Oliver BB, Ringold GM, Kliewer SA. Per-oxisome proliferator-activated receptors alpha and gamma are activatedby indomethacin and other non-steroidal anti-inflammatory drugs. JBiol Chem 1997;272:3406-3410.

4. Pedersen TA, Bereshchenko O, Garcia-Silva S, Ermakova O, Kurz E,Mandrup S, et al. Distinct C/EBPalpha motifs regulate lipogenic andgluconeogenic gene expression in vivo. EMBO J 2007;26:1081-1093.


Fig. 1. Liver histology score and abundance of liver CPT1A, PCK1, and PPARa mRNA analyzed by quantitative real-time polymerase chainreaction in each experimental group. Quantitative evaluation of steatosis score from hematoxylin and eosin and osmium tetroxide stain of liversections at the end of the experiment in all rats from each experimental group. Data are presented as mean 6 standard error (SE). For testingsteatosis gradation (as a categorical response variable) differences, we used a model with ordinal multinomial distribution and probit as a linkfunction adjusted by body weight as a continuous predictor variable. Liver mRNA expression: Each bar represents mean 6 SE of values. HFD,high-fat diet; HFDþInd, high-fat diet plus indomethacin; SCD, standard chow diet. Real-time polymerase chain reaction was performed for quan-titative assessment of mRNA expression. In each sample, gene expression was normalized by the expression of the housekeeping TATA box bind-ing protein (TBP) gene. The ratio was log-transformed and analyzed using analysis of variance.

This study was partially supported by grants PICT 2006-124 and PICT2008-1521 (Agencia Nacional de Promocion Cientıfica y Tecnologica), andUBACYT M55 (Universidad de Buenos Aires).


Methodological Concerns About a Recent Meta-Analysis of the Influence of the I148M Variant ofPatatin-Like Phospholipase Domain Containing 3 on the Susceptibility and Histological

Severity of Nonalcoholic Fatty Liver Disease

To the Editor:

We read the meta-analysis by Sookoian and Pirola1 with greatinterest. Their meta-analysis suggests that patatin-like phospholi-pase domain containing 3 rs738409 C/G is a strong modifier ofthe natural history of nonalcoholic fatty liver disease. However,several points should be mentioned here.

The perfect searching strategy and the use of more related data-bases allow researchers to include an extensive number of potentiallyeligible studies, and this is crucial for a meta-analysis. Although theMedical Literature Analysis and Retrieval System Online (MEDLINE)database is one of the most comprehensive databases for health careinformation, its coverage is not complete.2 Lemeshow et al.3 andSeminara et al.4 suggested that at least MEDLINE, another elec-tronic database, and hand searching should be used for a thoroughsearch. In this meta-analysis, only the MEDLINE database wassearched for eligible studies. In addition, Sookoian and Pirola1 lim-ited the search to publications written in English. Using thisapproach, they may have neglected some eligible studies, and thismay have resulted in selection or publication bias. Moreover, localdatabases also should have been searched.

The Hardy-Weinberg equilibrium should be evaluated in a con-trol group. The deviation from the Hardy-Weinberg equilibriumpresents the probability of genotyping errors, selection bias, or otherbias.5 However, the Hardy-Weinberg equilibrium test was not per-formed in this meta-analysis. According to the sources of the con-trols, a case-control study is usually categorized as a hospital-basedcase-control (HCC) study (the controls are hospitalized patients) ora population-based case-control study (the controls are healthy peo-ple). A meta-analysis based on an HCC study may be biased becauseHCC controls always have some kind of disease, unhealthy life habit,or risk genotype.6 It is routine in a meta-analysis for a stratified anal-ysis to be performed according to the sources of the controls to con-firm the validity of the results rather than bias.6 Similarly, to retainthe homogeneity and make the results more reliable, Sookoian andPirola1 should perform a subgroup analysis for this meta-analysisand thus confirm the validity of the results.

LIU LIU, M.M.1

JIANG-HUA SHAO, M.D., PHD1

KAI WANG, M.M.1

FU-ZHOU HUA, M.M.21Departments of 1General Surgery and 2Anesthesiology

Second Affliated Hospital of Nanchang UniversityNanchang City, Jiangxi, ChinaE-mail: [email protected]

References1. Sookoian S, Pirola CJ. Meta-analysis of the influence of I148M variant

of patatin-like phospholipase domain containing 3 gene (PNPLA3) onthe susceptibility and histological severity of nonalcoholic fatty liver dis-ease. HEPATOLOGY 2011; Doi: 10.1002/hep.24283.

2. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gotzsche PC, IoannidisJP, et al. The PRISMA statement for reporting systematic reviews andmeta-analyses of studies that evaluate health care interventions: explana-tion and elaboration. PLoS Med 2009;6:e1000100.

3. Lemeshow AR, Blum RE, Berlin JA, Stoto MA, Colditz GA. Searchingone or two databases was insufficient for meta-analysis of observationalstudies. J Clin Epidemiol 2005;58:867-873.

4. Seminara D, Khoury MJ, O’Brien TR, Manolio T, Gwinn ML, LittleJ, et al. The emergence of networks in human genome epidemiology:challenges and opportunities. Epidemiology 2007;18:1-8.

5. Mayo O. A century of Hardy-Weinberg equilibrium. Twin Res HumGenet 2008;11:249-256.

6. Benhamou S, Lee WJ, Alexandrie AK, Boffetta P, Bouchardy C, But-kiewicz D, et al. Meta- and pooled analyses of the effects of glutathioneS-transferase M1 polymorphisms and smoking on lung cancer risk.Carcinogenesis 2002;23:1343-1350.


Reply:

We thank both Rosselli et al. and Arduini et al. for their commentson our article recently published in HEPATOLOGY.1 We reported thatliver-specific overexpression of carnitine palmitoyltransferase 1A(CPT1A) not only reduces obesity-induced hepatic steatosis but alsoameliorates systemic adiposity, hyperglycemia, and insulin resistance. Byshowing that indomethacin reduces hepatic steatosis in rats, Roselliet al.2 confirmed our results. Interestingly, this reversion by indometha-cin treatment seems to be mediated by increased hepatic expression ofCPT1A, phosphoenolpyruvate carboxykinase 1, and peroxisome prolif-erator-activated receptor a. These results provide new evidence for theidea that strategies based on the indirect stimulation of CPT1A bypharmacological treatments, such as those examined by Roselli et al.and others,3,4 or on the direct stimulation of CPT1A and fatty acid ox-idation (FAO), as described in our study, can lead to new ways of treat-ing obesity and its metabolic disorders.

On the other hand, Conti et al.5 recently demonstrated thatteglicar, a specific reversible inhibitor of CPT1A, improves hyper-glycemia and hyperinsulinemia in obese rodents, although it dou-bles the liver triglyceride levels. These results raise the interestingquestion whether CPT1A stimulation or inhibition would be amore effective treatment for obesity and insulin resistance. Arduiniet al. commented that in murine models such as db/db mice, inwhich the gluconeogenic pathway is activated, promoting FAOseems to be in conflict with the antidiabetic effect observed in ourstudies. They argued that increased FAO provides ATP andNADH producing an increase in acetyl coenzyme A, which mightallosterically activate pyruvate carboxylase, a key enzyme in the glu-coneogenic pathway. Our results clearly show that over a long pe-riod (3 months), a permanent increase in FAO promoted by he-patic overexpression of a malonyl coenzyme A–insensitive CPT1Aisoform (CPT1AM) counteracts not only hyperglycemia and hy-perinsulinemia but also hepatic steatosis in db/db mice. We agreewith Arduini et al. that it is necessary to perform new experimentsto discern molecular mechanisms that might explain the antidia-betic effect of increased FAO in the liver. Furthermore, futurestudies with longer follow-up periods are needed to validate thesafety of both strategies in terms of pharmacological feasibility. Themajor challenge for CPT1A inhibition is the prevention of the de-velopment of hepatic steatosis. This side effect has interrupted thedevelopment of other systemic inhibitors, such as etomoxir and 2-tetradecylglycidic acid, as therapeutic tools. For direct hepaticCPT1A stimulation, a major challenge is the delivery system. Forindirect CPT1A stimulation by pharmacological treatments (e.g.,those examined by Roselli et al.), the challenge is the interference with


other metabolic pathways that might produce undesirable side effects.Finally, we thank Arduini et al. for bringing the error on p 825 to ourattention. We will ask the editor of HEPATOLOGY to fix this.

JOSEP MARIA ORELLANA-GAVALDA, PH.D.1,2

LAURA HERRERO, PH.D., M.D.1,2

MARIA IDA MALANDRINO, PH.D., M.D.1,2

GUILLERMINA ASINS, PH.D., M.D.1,2

FAUSTO G. HEGARDT, PH.D.1,2

DOLORES SERRA, PH.D., M.D.1,21Department of Biochemistry and Molecular Biology, Institute of

Biomedicine, University of Barcelona, Barcelona, Spain2Network Center for Biomedical Research in the Physiopathology of

Obesity and Nutrition, CIBERobn, Barcelona, Spain

References1. Orellana-Gavalda JM, Herrero L, Malandrino MI, Paneda A, Sol

Rodriguez-Pena M, Petry H, et al. Molecular therapy for obesity anddiabetes based on a long-term increase in hepatic fatty-acid oxidation.HEPATOLOGY 2011;53:821-832.

2. Rosselli MS, Burgueno AL, Pirola CJ, Sookoian S. Cyclooxygenase inhi-bition upregulates liver carnitine palmitoyltransferase 1A (CPT1A)expression and improves fatty liver. HEPATOLOGY 2011 Mar 23. doi:10.1002/hep.24309.

3. Hwang JH, Kim DW, Jo EJ, Kim YK, Jo YS, Park JH, et al. Pharma-cological stimulation of NADH oxidation ameliorates obesity andrelated phenotypes in mice. Diabetes 2009;58:965-974.

4. Li PP, Shan S, Chen YT, Ning ZQ, Sun SJ, Liu Q, et al. The PPARal-pha/gamma dual agonist chiglitazar improves insulin resistance and dys-lipidemia in MSG obese rats. Br J Pharmacol 2006;148:610-618.

5. Conti R, Mannucci E, Pessotto P, Tassoni E, Carminati P, Giannessi F,et al. Selective reversible inhibition of liver carnitine palmitoyl-transfer-ase 1 by teglicar reduces gluconeogenesis and improves glucose homeo-stasis. Diabetes 2011;60:644-651.


Reply:

We read with great interest the letter by Liu et al., who raise sev-eral methodological concerns about our recently published meta-analy-sis.1 Although their points are theoretically valid for any meta-analysis,we have found them inapplicable to our article. The first concernraised by Liu et al. is the searching strategy, which they claim shouldinclude the use of local databases and manual searching. They shouldhave provided actual proof of missing studies; we are quite confidentthat no other reports with reliable data on the influence of rs738409on nonalcoholic fatty liver disease had been published by the time ofour search. For instance, manual searching with Google Scholar didnot reveal any other eligible studies in English or other languages. Inaddition, local databases do not always include only peer-reviewedarticles, so the data are not necessarily reliable. Moreover, with respectto clinical practice, not all studies in local databases are consideredequally strong, and they can be almost impossible to find. Hence, thisconcern is overcome. Liu et al.’s second concern is the Hardy-Wein-berg equilibrium (HWE) in the control groups. We are very glad toreport that a v2 test was used to determine whether the observed ge-notype frequencies conformed to the HWE expectations. The dataextracted from all the articles (except two) showed no deviations fromthe HWE with very stringent criteria (Pearson’s goodness-of-fit v2 <0.05, degree of freedom ¼ 1). The publications whose results did notfollow the HWE2,3 showed borderline P values. Hence, this concernis overcome. Finally, Liu et al. claim that we did not perform a strati-fied analysis based on the sources of the controls to confirm the valid-ity of our results. To prove that the results are not biased, we haveperformed the suggested analysis, and we have found that the effectsof rs738409 on nonalcoholic fatty liver disease are very similar withcontrols selected from the general population and with controlsselected from a hospital-based setting; the effects are even greater inthe random model with general population controls (Fig. 1). In con-clusion, methodological considerations are always very welcome.Nevertheless, criticisms must be accompanied and justified by actualfacts so that the readers can determine whether they are valid. In thisparticular case, all the concerns raised by the authors are generic con-siderations that are not applicable to our article, and we regret thetitle of their letter.

Fig. 1. Stratified analysis of the controls. References for the listed studies are provided elsewhere.1 Abbreviations: CI, confidence interval; GP,general population; HB, hospital-based setting; T2D, type 2 diabetes.


SILVIA SOOKOIAN, M.D., PH.D.1

CARLOS J. PIROLA, PH.D.2

Departments of 1Clinical and Molecular Hepatology and2Molecular Genetics and Biology of Complex Diseases, Alfredo Lanari

Institute of Medical Research, University of Buenos Aires/NationalCouncil of Scientific and Technological Research, Buenos Aires,Argentina

References1. Sookoian S, Pirola CJ. Meta-analysis of the influence of I148M variant

of patatin-like phospholipase domain containing 3 gene (PNPLA3) onthe susceptibility and histological severity of nonalcoholic fatty liver dis-ease. HEPATOLOGY 2011.

2. Kantartzis K, Peter A, Machicao F, Machann J, Wagner S, KonigsrainerI, et al. Dissociation between fatty liver and insulin resistance inhumans carrying a variant of the patatin-like phospholipase 3 gene. Di-abetes 2009;58:2616-2623.

3. Wagenknecht LE, Palmer ND, Bowden DW, Rotter JI, Norris JM,Ziegler J, et al. Association of PNPLA3 with non-alcoholic fatty liverdisease in a minority cohort: the Insulin Resistance AtherosclerosisFamily Study. Liver Int 2011;31:412-416.


Percutaneous Liver Biopsy in the Clinical Management of Hepatocellular Carcinoma: Back to the Future

To the Editor:

We read with great interest the article by Iavarone et al.,1 whostudied the role of tumor grading in the diagnosis of hepatocellularcarcinoma (HCC) detected during surveillance by dynamic contrastimaging techniques in patients with compensated cirrhosis. Theauthors showed that the tumor grade and size influence the accu-racy of imaging techniques in HCC diagnosis; in fact, accuracywas greater for poorly differentiated (high-grade) nodules > 2 cmversus more differentiated (low-grade) nodules � 2 cm. Theseobservations indirectly confirm the correlation between HCC gradeand vascularization: high-grade HCC is better detected by imaging.2,3

We appreciate the attempt of Iavarone et al.1 to find a correla-tion between diagnostic imaging techniques and HCC gradingbecause the latter greatly influences HCC outcomes and is a strongpredictor of recurrence after surgery.

However, we believe that the only way to obtain preoperativehistological information is needle core biopsy (NCB). We recentlyevaluated the overall accuracy of preoperative NCB in assessing tu-mor grading in patients with cirrhosis undergoing liver resectionfor a single HCC.4 We found that preoperative NCB is a safe pro-cedure (no serious adverse events were observed) and an accuratetool for assessing the tumor grade, particularly for small HCCs. Infact, for HCCs < 3 cm, we found an accuracy of 97%, a sensitiv-ity of 86%, a specificity of 100%, a positive predictive value of100%, and a negative predictive value of 96%, whereas for HCCs< 2 cm, we found an accuracy of 93%, a sensitivity of 100%, aspecificity of 75%, a positive predictive value of 91%, and a nega-tive predictive value of 100%. The overall accuracy in detectinglow-grade HCCs was greater than the overall accuracy in detectinghigh-grade HCCs (98% versus 65%, respectively). NCB is theonly technique also capable of grading small HCCs (�2 cm);dynamic contrast imaging techniques have poorer diagnostic accu-racy. Moreover, we did not observe any correlation between tumorsize and HCC grading; this observation was also made by Iavaroneet al.1 In fact, we found that high-grade HCCs were present to thesame extent (ca. 20%) in nodules � 3 cm and in nodules > 3 cm.

All these findings, together with the inconsistent recent resultsregarding the contrast enhancement ultrasonographic pattern as apredictor of HCC grading,5,6 underscore and elevate the impor-tance of the role of preoperative NCB. However, we believe thatNCB should be performed not only for small nodules present inpatients with cirrhosis, which could be undetected by imagingtechniques, but also for those nodules detected by imaging andthose nodules in the surrounding liver tissue. Preoperative histolog-ical information (mainly HCC grading) and genetic profiling7 rep-resent essential tools for updated HCC clinical management.

ANTONIO COLECCHIA, M.D.LUCIA MONTRONE, M.D.ELEONORA SCAIOLI, M.D.DAVIDE FESTI, M.D.Department of Clinical Medicine

University of BolognaBologna, Italy

References1. Iavarone M, Sangiovanni A, Forzenigo LV, Massironi S, Fraquelli M,

Aghemo A, et al. Diagnosis of hepatocellular carcinoma in cirrhosis bydynamic contrast imaging: the importance of tumor cell differentiation.HEPATOLOGY 2010;52:1723-1730.

2. Sakabe I, Yamamoto T, Kubo S, Hirohashi K, Hamuro M, NakamuraK, et al. Correlation between dynamic computed tomographic and his-topathological findings in the diagnosis of small hepatocellular carci-noma. Dig Surg 2004;21:413-420.

3. Asayama Y, Yoshimitsu K, Nishihara Y, Irie H, Aishima S, Taketomi A,et al. Arterial blood supply of hepatocellular carcinoma and histologicgrading: radiologic-pathologic correlation. AJR Am J Roentgenol 2008;190:W28-W34.

4. Colecchia A, Scaioli E, Montrone L, Vestito A, Di Biase AR, Pieri M,et al. Pre-operative liver biopsy in cirrhotic patients with early hepato-cellular carcinoma represents a safe and accurate diagnostic tool fortumour grading assessment. J Hepatol; doi:10.1016/j.jhep.2010.06.037.

5. Liu GJ, Xu HX, Lu MD, Xie XY, Xu ZF, Zheng YL, et al. Correlationbetween enhancement pattern of hepatocellular carcinoma on real-timecontrast-enhanced ultrasound and tumour cellular differentiation onhistopathology. Br J Radiol 2007;80:321-330.

6. von Herbay A, Vogt C, Westendorff J, Haussinger D, Gregor M. Cor-relation between SonoVue enhancement in CEUS, HCC differentiationand HCC diameter: analysis of 130 patients with hepatocellular carci-noma (HCC). Ultraschall Med 2009;30:544-550.

7. Villanueva A, Hoshida Y, Toffanin S, Lachenmayer A, Alsinet C, SavicR, et al. New strategies in hepatocellular carcinoma: genomic prognos-tic markers. Clin Cancer Res 2010;16:4668-4694.



Two Solutions in Search of a Problem

To the Editor:

Two recent articles in HEPATOLOGY suggest that the problems offructose-induced fibrosis severity in patients with nonalcoholic fattyliver disease (NAFLD)1 or nonalcoholic hepatic steatosis (NASH)2

could be solved if their exposure were limited either directlythrough diet or indirectly through curcumin therapy. However, theevidence for a unique fructose effect is sufficiently weak in bothstudies that the authors appear left with two solutions in search ofa problem.

The evidence of Abdelmalek et al.1 for a fructose-specific rolein NAFLD is not convincing for several reasons. First, in the cate-gorization of subjects by sweetened beverage intake, the designation‘‘no fructose consumers’’ is misleading; fructose intake from nonbe-verage sources was neither calculated nor reported and could havesignificantly altered clinical and histological conclusions. Second,the raw data in Table 1 of their article (before statistical manipula-tion) demonstrate inconsistent fructose dose–dependent trends forall but two of the seven metabolic parameters and for all of thehistological parameters measured. Third, the caloric differencebetween the subject groups [the highest fructose consumers (sevenor more servings per week) had twice the calorie intake of the no-fructose group (zero servings per week)] cannot be ignored as a farsimpler explanation for the few observed differences reported bythe authors. This is especially compelling because the fructose in-crement in moderate consumers versus zero consumers could nothave exceeded 15% of the total energy, whereas that for the highestconsumers could have been as low as 5% (calculated from Table 1of their article). Thus, rather than proposing reduced fructoseintake as the solution for NAFLD patients at risk for liver fibrosis,Abdelmalek et al. should recommend that these patients eat less ofeverything.

The experimental design of Li et al.2 provided rats ad libitumaccess to 10% (wt/vol) fructose in water with or without curcuminor pioglitazone and standard rat chow. The data in Fig. 1 of theirarticle show that the fructose rats consumed approximately 1650mL of fluid per week (236 mL/day), which is equivalent to 94kcal/day from fructose. For perspective, this would be like feedinga 70-kg human more than 5200 g of fructose per day (21,000kcal/day). From the combination of such a highly exaggerated ex-posure and a lack of a suitable nonfructose carbohydrate control,

we can conclude only that (1) this study does not prove a mean-ingful role for fructose in the development of hepatic steatosis attypical intake levels (45 g/day or 180 kcal/day3), and (2) the curcu-min therapy solution proposed by Li et al. has academic interestbut is likely unnecessary in humans.

Because Abdelmalek et al.1 and Li et al.2 have failed to demon-strate a unique effect for fructose in patients with NAFLD orNASH, respectively, they are left with two solutions in search of aproblem.

JOHN S. WHITE, PH.D.1

JOHN P. FOREYT, PH.D.21White Technical Research, Argenta, IL2Baylor College of Medicine, Houston, TX

References1. Abdelmalek MF, Suzuki A, Guy C, Unalp-Arida A, Colvin R, Johnson

RJ, et al. Increased fructose consumption is associated with fibrosis se-verity in patients with nonalcoholic fatty liver disease. HEPATOLOGY

2010;51:1961-1971.2. Li JM, Li YC, Kong LD, Hu QH. Curcumin inhibits hepatic protein-

tyrosine phosphatase 1B and prevents hypertriglyceridemia and hepaticsteatosis in fructose-fed rats. HEPATOLOGY 2010;51:1555-1566.

3. Marriott BP, Cole N, Lee E. National estimates of dietary fructoseintake increased from 1977 to 2004 in the United States. J Nutr 2009;139:1228S-1235S.

CopyrightVC 2011 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.24134Potential conflict of interest: John S. White is the president of White TechnicalResearch and is a consultant to the food and beverage industry in the area ofnutritive sweeteners. His clients include research institutes, food industry coun-cils, trade organizations, and individual companies. John P. Foreyt is a profes-sor of the Department of Medicine at Baylor College of Medicine and is thedirector of the Behavioral Medicine Research Center. He has received researchfunding from the National Institutes of Health and has served as a consultantto the pharmaceutical and food industries, food industry councils, trade organi-zations, and research institutes.

Methodological Quality Assessment for Traditional Chinese Medicine: CONSORT Is Better

To the Editor:

We read the article by Zhang et al.1 with great interest. Theauthors assessed the methodological quality of randomized con-trolled trials (RCTs) of traditional Chinese medicine (TCM)with the Jadad scoring system.2 However, we would like to com-ment on concerns that have been raised about the scoringsystem.

The assessment criterion adopted in the study is less com-prehensive and outdated. Jadad scoring, though widely used invalidating RCTs, has been attacked in recent years. Accumulat-ing evidence suggests that Jadad scoring is flawed and overlysimplistic, places too much emphasis on blinding, and hasdiminishing consistency with different raters.3,4 Nowadays, the

Consolidated Standards of Reporting Trials (CONSORT)statement encompasses various initiatives developed by theCONSORT group to alleviate the problems arising from inad-equate reporting of RCTs.5 It offers a standardized way of pre-paring reports of trial findings that facilitates accurate andtransparent reporting with critical appraisal and interpretation.More and more highly cited academic journals, including HE-

PATOLOGY, have adopted the CONSORT statement for stand-ardization and integrity. In China, CONSORT for TCM hasbeen developed and widely adopted; it provides more specific-ity and sensitivity for the assessment of TCM methodologicalquality.6 Therefore, it would have been much better if theCONSORT standard, instead of Jadad scoring, had been usedin this meta-analysis.


In summary, the methodological quality of the analysis needs tobe reassessed.

MING-HUA ZHENG, M.D.1

YU-CHEN FAN, M.D.2

KE-QING SHI, M.D.1

YONG-PING CHEN, M.D.11Liver Research Center

Department of Infection and Liver DiseasesThe First Affiliated Hospital ofWenzhou Medical College, Wenzhou, China

2Department of HepatologyQilu Hospital ofShandong University, Jinan, China

References1. Zhang L, Wang G, Hou W, Li P, Dulin A, Bonkovsky HL. Contempo-

rary clinical research of traditional Chinese medicines for chronic hepa-titis B in China: an analytical review. HEPATOLOGY 2010;51:690-698.

2. Jadad AR, Moore RA, Carroll D, Jenkinson C, Reynolds DJ, GavaghanDJ, et al. Assessing the quality of reports of randomized clinical trials:is blinding necessary? Control Clin Trials 1996;17:1-12.

3. Berger VW. Is the Jadad score the proper evaluation of trials? J Rheuma-tol 2006;33:1710-1711.

4. Clark HD, Wells GA, Huet C, McAlister FA, Salmi LR, Fergusson D,et al. Assessing the quality of randomized trials: reliability of the Jadadscale. Control Clin Trials 1999;20:448-452.

5. Moher D, Schulz KF, Altman D. The CONSORT statement: revisedrecommendations for improving the quality of reports of parallel-grouprandomized trials. JAMA 2001;285:1987-1991.

6. Wu TX, Li YP, Bian ZX, Li TQ, Li J, Dagenais S, et al. Consolidatedstandards for reporting trials of traditional Chinese medicine (CON-SORT for TCM) [in Chinese]. Chin J Evid Based Med 2007;7:601-605.


Is Hydrophobia Necessary for the Hepatologist Prescribing Nonselective Beta-Blockers in Cirrhosis?

To the Editor:

Recently, Lebrec and colleagues from Clichy, France, reportedan increased mortality in 77 patients with cirrhosis and varices andrefractory ascites in whom propranolol was administered, comparedto 74 patients with refractory ascites but no varices, who were nottaking nonselective beta-blockers (NSBBs).1 During follow-up last-ing a median of 8 months (range 1-47 months), the probability ofdeath was 59% at 1 year and 72% at 2 years: 81% of patients tak-ing propranolol died during the follow-up, and use of propranololwas the third cause of death, with odds ratio ¼ 2.61 (95% confi-dence interval ¼ 1.63-4.19).1

These findings are potentially very important, but are difficultto reconcile with some of the published literature. Although it istrue that most randomized controlled trials (RCTs) comparingbeta-blockers to placebo or other pharmacotherapy for preventionof bleeding from varices excluded patients with advanced cirrhosisand refractory ascites, this was not universal. Moreover, despitemore rebleeding, an increased mortality with propranolol has notbeen reported in comparative trials versus banding ligation. Indeed,the recent trial by Lo et al., with extended follow-up, showed bet-ter survival with beta-blockers than with banding despite morerebleeding.2 Second, when we reviewed the literature to explore thepotential beneficial effect of propranolol in preventing spontaneousbacterial peritonitis (SBP) in patients with cirrhosis, we includedthree RCTs and one prospective study comprising 644 patients,468 with ascites, and 257 receiving propranolol.3 Among these,125 patients had Child C fibrosis (101 were taking NSBBs). Theaverage hepatic venous pressure gradient was comparable to thatdocumented in the study from Clichy. Moreover, in the prospectivestudy, 67 of 134 (50%) patients had tense ascites requiring para-centesis. However, the overall mortality in the four studies was21%, which is significantly lower than in the group in Clichy,despite a much longer follow-up: 8 years in two RCTs and 5 yearsin one RCT3 (Table 1).

In addition, the causes of death were different in the reviewedstudies compared to the present study. Gastrointestinal bleedingwas the most important cause of death, followed by hepatocellularcarcinoma (HCC). Hepatorenal syndrome, which was notdescribed as cause of death in the Clichy study, was described infour patients, all nonhemodynamic responders to beta-blockers. It

is important to note that propranolol appeared to confer a reducedrisk of developing SBP, which is a frequent infection in patientswith refractory ascites. In contrast in the patients reported in theClichy study, infection was the most important cause of death.1

In our review,3 NSBB conferred a statistically significant relativerisk reduction of 12% (95% confidence interval ¼ 5.5%-18.8%)in the future occurrence of SBP, which was not closely related tothe hemodynamic targets of hepatic venous pressure gradientreduction, whether to less than 12 mm Hg or to a 20% reductionfrom baseline. The protective effect of NSBBs could be due to areduction of bacterial translocation, due to an increase in intestinalmotility and/or by a decrease in intestinal permeability consequentto the reduction of portal pressure.4,5 Indeed, a model of splanch-nic sympathectomy in the cirrhotic rat has demonstrated preven-tion of bacterial translocation of E. coli.6 Moreover, a significantdecrease in the incidence of postsurgical infections has been shownin a cohort study of patients with cirrhosis treated with proprano-lol and ciprofloxacin, compared to ciprofloxacin alone after laparo-scopic surgery (14.7% versus 42.4%).7 This effect may be due tothe possible mechanisms described above, but also due possibly toan improvement of host defenses by NSBB, through inhibition ofthe stress-related cyclic adenosine monophosphate protein kinase Apathway, which has an inhibitory effect on the immune system.8

A possible important difference between the studies wereviewed,3 and that from Clichy, is that the mean dose of propran-olol used was below 100 mg in all the former studies, but in thelatter French study, 47% of patients were taking propranolol at adose of 160 mg/day, except for one patient who was taking pro-pranolol at a dose between 100 and 160 mg/day.1

It would have been interesting, after removing the causes ofdeath due to HCC in the Clichy study, to evaluate the incidenceof infections during follow-up, and the deaths related to this,according to whether propranolol was taken or not.

Thus, it is important to understand whether the increased mor-tality attributed to propranolol is solely due to the dose used or tothe specific clinical setting of refractory ascites in patients treated atClichy (or both), or if the increased mortality occurred by chance,or due to an associated factor not captured by the authors. Anurgent survey of patient databases, and of current patients treatedwith NSBBs is needed, to confirm or refute the potential danger ofprescribing or continuing to prescribe NSBBs in patients with


cirrhosis who have or who develop refractory ascites. It is impor-tant to establish if hepatologists need to be hydrophobic when pre-scribing NSBBs for patients with cirrhosis!

MARCO SENZOLO, M.D., PH.D.1

ELENA NADAL, M.D.1

EVANGELOS CHOLONGITAS, M.D.2

ANDREW K. BURROUGHS, M.B., CH.B.21Multivisceral Transplant Unit

Department of Surgical and Gastroenterological SciencesUniversity Hospital of Padua, Padua, Italy

2The Royal Free Sheila Sherlock Liver Centre andDepartment of Surgery UCL and Royal Free HospitalLondon, UK

References1. Serste T, Melot C, Francoz C, Durand F, Rautou PE, Valla D, et al.

Deleterious effects of beta-blockers on survival in patients with cirrhosisand refractory ascites. HEPATOLOGY 2010;52:1017-1022.

2. Lo GH, Chen WC, Lin CK, Tsai WL, Chan HH, Chen TA, et al.Improved survival in patients receiving medical therapy as comparedwith banding ligation for the prevention of esophageal variceal rebleed-ing. HEPATOLOGY 2008;48:580-587.

3. Senzolo M, Cholongitas E, Burra P, Leandro G, Thalheimer U, PatchD, et al. beta-Blockers protect against spontaneous bacterial peritonitisin cirrhotic patients: a meta-analysis. Liver Int 2009;29:1189-1193.

4. Senzolo M, Fries W, Buda A, Pizzuti D, Nadal E, Sturniolo GC, et al.Oral propranolol decreases intestinal permeability in patients with cir-rhosis: another protective mechanism against bleeding? Am J Gastroen-terol 2009;104:3115-3116.

5. Senzolo M, Cholongitas E, Marelli L, Thalheimer U, Patch D, Bur-roughs AK, et al. The low incidence of bacterial infections could be a

protective factor against variceal bleeding per se in hemodynamicresponders to propranolol. Am J Gastroenterol 2006;101:2436-2437.

6. Worlicek M, Knebel K, Linde HJ, Moleda L, Scholmerich J, Straub RH,et al. Splanchnic sympathectomy prevents translocation and spreading of Ecoli but not S aureus in liver cirrhosis. Gut 2010;59:1127-1134.

7. Pardo A, Bartolı R, Lorenzo-Zuniga V, Planas R, Vinado B, Riba J,et al. Effect of cisapride on intestinal bacterial overgrowth and bacterialtranslocation in cirrhosis. HEPATOLOGY 2000;31:858-863.

8. Peuschel KE. Antiviral therapy with non-selective beta-blockers: prelim-inary experimental and clinical corroboration. Med Hypotheses 2005;64:256-260.

9. Turnes J, Garcia-Pagan JC, Abraldes JG, Hernandez-Guerra M, Del-l’Era A, Bosch J. Pharmacological reduction of portal pressure andlong-term risk of first variceal bleeding in patients with cirrhosis. Am JGastroenterol 2006;101:506-512.

10. Gonzales Suarez, Guarner C, Villanueva C, et al. Pharmacologic treatment

of portal hypertension in the prevention of community-acquired spontane-

ous bacterial peritonitis. Eur J Gastroenterol Hepatol 2006;18:49-55.

11. Abraldes JG, Tarantino I, Turnes J, Garcia-Pagan JC, Rodes J, Bosch J. He-

modynamic response to pharmacological treatment of portal hypertension

and long-term prognosis of cirrhosis. HEPATOLOGY 2003;37:902-908.12. Cholongitas E, Papatheodoridis GV, Manesis EK, Burroughs AK,

Archimandritis AJ. Spontaneous bacterial peritonitis in cirrhotic

patients: Is prophylactic propranolol therapy beneficial? J Gastroenterol

Hepatol 2006;21:581-587.


Table 1. Review of Incidence and Causes of Death in Four Studies Evaluating Propanol for the Prevention of SBP in PatientsWith Cirrhosis and Ascites

Parameter

Study

Turnes et al.9 Gonzales Suarez et al.10 Abraldes et al.11 Cholongitas et al.12

Type of study RCT RCT RCT Retrospective

Type of publication Full length Full length Full length Full length

Number of patients 71 230 73 134

Mean age 6 SD (years) 57.8 6 1.7 59 6 1.2 54.5 6 8 66 6 12

Males/females (number) 25/71 149/81 48/25 28/106

Etiology of liver disease (alcohol/nonalcohol) 15/56 110/120 37/36 20/106

Severity of liver disease, Child A/B/C (number) 58/13/0 50/131/49 32/36/9 17/116/67

% of patients with ascites at inclusion 35 64 31 24.6

% of patients treated with propranolol 100 50 100 25

Control group nonresponders sclerotherapy nonresponders not treated

Follow-up (months) 76 23 6 1.4 70 36

Death (number/%) 24/31.5% 86/37.3% 11/15% 16/12%

Death NSBB/ controls (%) 32/34.7 34/41 3.5/31 6/13.8

Causes of death NA 35 gastrointestinal bleeding,

17 infection, 6 HCC,

8 other causes

4 gastrointestinal bleeding,

4 hepatorenal syndrome,

2 EPS, 1 other causes

NA

NA, not available.


Comment on a Systematic Review of Randomized Trials of Peginterferon Alpha-2a VersusPeginterferon Alpha-2b in Chronic Hepatitis C

To the Editor:

We read with interest the letter by Kershenobich et al. inHepatology regarding the meta-analysis of randomized trials com-paring pegylated interferon (PEG-IFN) alpha-2a and alpha-2b inthe treatment of chronic hepatitis C (CHC) by Awad et al.1,2 Weagree regarding the importance of a uniform study population intreatment-naıve patients with CHC. This is especially true for thestudy by Laguno et al., which included patients coinfected withhuman immunodeficiency virus (HIV).3

We performed a meta-analysis of four available studies compar-ing PEG-IFN alpha-2a and peginterferon alpha-2b in the treat-ment of patients with CHC who have concomitant HIV coinfec-tion: one randomized,3 one prospective–retrospective,4 and twoprospective studies5,6 with one of them reported as an abstract.6 Atotal of 1009 patients (581 treated with PEG-IFN alpha-2a; sam-ple size, 63-557; mean age, 41 years; 69%-75% males) weretreated in the four studies.3-6

Pooled analysis of the data showed that the odds of achievingrapid virologic response (RVR), early virologic response (EVR),and sustained virologic response (SVR) were similar with PEG-IFN alpha-2a and PEG-IFN alpha-2b (Table 1). Similarly, theodds of treatment discontinuation due to serious adverse effectswere similar with PEG-IFN alpha-2a and PEG-IFN alpha-2b (Ta-ble 1). The data were homogeneous for all the analyses. There wasno evidence to indicate any publication bias. After excluding thestudy reported as an abstract, the results with the two PEG-IFNcompounds were still similar.

The SVR rates were 36% and 35% with PEG-IFN alpha-2aand PEG-IFN alpha-2b, respectively. Subgroup analyses of theSVR based on the genotype status (genotype 1 or 4 and genotype2 or 3) and viral load showed similar efficacy and safety data forthe two types of PEG-IFN. The data were homogeneous withoutany suggestion of publication bias in all the analyses.

In summary, PEG-IFN alpha-2a and PEG-IFN alpha-2b havesimilar efficacy and safety in the treatment of HCV in HIV-coin-fected patients, based on the literature currently available. We agreewith Kershenobich et al. that further randomized studies areneeded to compare efficacy and safety of the two types of PEG-IFN in the treatment of HCV infection, especially in those indi-viduals coinfected with HIV.

ASHWANI K. SINGAL, M.D.1,2

SARAT C. JAMPANA, M.D.2

BHUPINDERJIT S. ANAND, M.D., PH.D.31Department of Internal Medicine2Division of Gastroenterology University of Texas Medical Branch,

Galveston, TX and 3Department of Gastroenterology and HepatologyMichael E. DeBakey Veterans Affairs Medical CenterBaylor College of Medicine, Houston, TX

References1. Kershenobich D, Munoz L, Male R, Gaytan J, Sanchez F. Proceed with

caution: peginterferon alpha-2a versus peginterferon alfa-2b in chronichepatitis C. A systematic review of randomized trials. HEPATOLOGY

2010;52:2240-2241.2. Awad T, Thorlund K, Hauser G, Stimac D, Mabrouk M, Gluud C.

Peginterferon alpha-2a is associated with higher sustained virologicalresponse than peginterferon alfa-2b in chronic hepatitis C: systematicreview of randomized trials. HEPATOLOGY 2010;51:1176-1184.

3. Laguno M, Cifuentes C, Murillas J, Veloso S, Larrousse M, Payeras A,et al. Randomized trial comparing pegylated interferon alpha-2b versuspegylated interferon alpha-2a, both plus ribavirin, to treat chronic hepatitisC in human immunodeficiency virus patients. HEPATOLOGY 2009;49:22-31.

4. Berenguer J, Gonzalez-Garcia J, Lopez-Aldeguer J, Von-WichmannMA, Quereda C, Hernando A, et al. Pegylated interferon {alpha}2aplus ribavirin versus pegylated interferon {alpha}2b plus ribavirin forthe treatment of chronic hepatitis C in HIV-infected patients. J Anti-microb Chemother 2009;63:1256-1263.

5. Fumaz CR, Munoz-Moreno JA, Ballesteros AL, Paredes R, Ferrer MJ,Salas A, et al. Influence of the type of pegylated interferon on the onsetof depressive and neuropsychiatric symptoms in HIV-HCV coinfectedpatients. AIDS Care 2007;19:138-145.

6. Vispo E, Barriero P, Rodriguez-Novoa S, Labarga P, Martın-CarboneroL, Soriano V, et al. Early hepatitis C virus decay with weight-based rib-avirin plus either PEGIFNa-2a or PEGIFNa-2b in HIV/HCV coin-fected patients [Abstract]. HEPATOLOGY 2007;46:353A.


Dietary Supplementation with Methyl Donor Groups Could Prevent Nonalcoholic Fatty Liver

To the Editor:

We read with great interest the work by Kohly et al. in HEPATO-

LOGY,1 which describes a model of obesity and nonalcoholic fattyliver disease (NAFLD) generated by a diet with high amounts offat and carbohydrates. Another way to implement a model of he-

patic damage is the dietary depletion of methyl donor groups, suchas choline or betaine,2,3 which leads to nonalcoholic steatohepatitis(NASH). Other studies have reported that supplementation withthese kinds of molecules can induce epigenetic changes and regu-late the gene expression profile.4 In this sense, we hypothesizedthat dietary methyl donor supplementation could be able to reverse

Table 1. Pooled Analysis of Studies Comparing PEG-IFNAlfa-2a and Alfa-2b for Efficacy in Achieving Different

Efficacy Outcomes and Safety

Number of

Studies

Number of Patients

(PEG-IFN Alfa-2a

Treated)

Odds

Ratio

95%

Confidence

Interval P Value

RVR 2 381 (231) 1.34 0.86-2.07 0.19

EVR 2 369 (231) 1.19 0.72-1.97 0.50

SVR 2 731 (408) 1.09 0.80-1.48 0.57

Treatment

discontinuation

due to SAE

3 802 (443) 1.09 0.73-1.63 0.68

ETR, end of treatment response; EVR, early virologic response; RVR, rapid viro-

logic response; SAE, serious adverse effects; SVR, sustained virologic response.


the negative effects of a nutritional model of nonalcoholic fattyliver in rats.

To confirm this concept, we performed a study with 48 maleWistar rats that were divided into four dietary groups with 12 ratseach (Fig. 1): control diet (C), methyl donor–supplemented con-trol (Csupl), a diet high in fat and sugar (HFS), and an HFS dietsupplemented with methyl donor groups, including betaine, chol-ine, vitamin B12, and folic acid (HFSsupl). Chow (2014; HarlanTeklad Global Diets) and obesogenic diets (D12451; ResearchDiets) were provided ad libitum, and food intake was not affectedby dietary treatment. The initial and final total fat mass, as well asthe final fat content in the liver, were measured by EchoMRi ana-lyzer.5 After 8 weeks of dietary treatment, the animals were sacri-ficed and tissues and plasma were frozen for later analysis.

The obesogenic model was successfully achieved, showing statis-tical differences (P < 0.01) between control-fed and HFS-fed ratsin different phenotypical variables such as body and fat depotweights and total fat. The analyses of plasma parameters revealedan increase in the atherogenic index. Moreover, the obesogenic dietinduced an increase in liver fat stores when compared to the C

group and, and as hypothesized, this damage was partially reversedwith methyl donor supplementation (Fig. 1).

In conclusion, the HFS diet led to an obese and NAFLD phe-notype characterized by an increase in liver lipid accumulation.Nutritional supplementation with a cocktail of methyl donors par-tially reversed this extra-adipose lipid accumulation. These datasuggest that methyl donor supplementation might prevent theestablishment of NAFLD, a precursor to NASH and cirrhosis, andthat different epigenetic changes altering the expression of genesrelated to liver fat metabolism could be involved.

PAUL CORDERO, PH.D.JAVIER CAMPION, PH.D.FERMIN I. MILAGRO, PH.D.J. ALFREDO MARTINEZ, PH.D.Department of Nutrition and Food Science

Physiology and ToxicologyUniversity of Navarra, Pamplona, Spain

References1. Kohli R, Kirby M, Xanthakos SA, Softic S, Feldstein AE, Saxena V,

et al. High-fructose, medium chain trans fat diet induces liver fibrosisand elevates plasma coenzyme Q9 in a novel murine model of obesityand nonalcoholic steatohepatitis. HEPATOLOGY 2010;32:934-944.

2. Pogribny IP, Tryndyak VP, Bagnyukova TV, Melnyk S, Montgomery B,Ross SA, et al. Hepatic epigenetic phenotype predetermines individualsusceptibility to hepatic steatosis in mice fed a lipogenic methyl-defi-cient diet. J Hepatol 2009;32:176-186.

3. Vetelainen R, van Vliet A, van Gulik TM. Essential pathogenic and meta-bolic differences in steatosis induced by choline or methione-choline defi-cient diets in a rat model. J Gastroenterol Hepatol 2007;32:1526-1533.

4. Waterland RA, Travisano M, Tahiliani KG, Rached MT, Mirza S.Methyl donor supplementation prevents transgenerational amplificationof obesity. Int J Obes 2008;32:1373-1379.

5. Tinsley FC, Taicher GZ, Heiman ML. Evaluation of a quantitativemagnetic resonance method for mouse whole body composition analy-sis. Obes Res 2004;32:150-160.


Fig. 1. Hepatic fat content measured by EchoMRi analyzer. Dataare presented as means 6 standard deviations. Differences were ana-lyzed by two-way analysis of variance (ANOVA) (Diet � Supplementa-tion). Different letters indicate significant differences between groups.C, control diet; Csupl, methyl donor–supplemented control diet; HFS,high-fat, high-sugar diet; HFSsupl, methyl donor–supplemented HFSdiet; ns, nonstatistical significance; *P < 0.05, ***P < 0.001.


methodological concerns about a recent meta-analysis of the influence of the i148m variant of...

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