Serum Carnitine Levels in ChronicHepatitis C Patients Before and AfterLymphoblastoid Interferon-α TreatmentMariano Malaguarnera, Salvatore Restuccia, Ignazio Di Fazio, Anna Maria Zoccolo,Laura Ferlito and Prospera Bentivegna

Department of Internal Medicine and Geriatrics, University of Catania, Catania, Italy

Abstract Objective: Chronic liver disease is often a hypocarnitinaemic condition. Sincecarnitine affects lipid metabolism, modifications of lipid pattern and energy me-tabolism can be expected in patients affected by chronic viral hepatitis. The aimof this study was to assess the relationship between serum carnitine levels andthe grading of chronic hepatitis C, and to evaluate the effects of lymphoblastoidinterferon (IFN)-αn1 on carnitine levels in patients with hepatitis C.Design:Weevaluated carnitine serum levels in a group of 32 patientswith chronichepatitis C before and after treatment with intramuscular IFNα 3MU 3times/week for 6 months, comparing them with levels in 20 healthy controls.Statistical correlations between serum carnitine, histological activity index score,duration of disease and lipid pattern were also evaluated.Results: Serum carnitine levels, which were statistically lower in hepatitis Cpatients than in controls before therapy, increased after IFNα (p = 0.0003 vspretreatment). There were no significant changes in total cholesterol in any pa-tient after treatment, although serum triglyceride levels increased (p = 0.0003).Serum carnitine levels were correlated with age (r = 0.35; p = 0.02), type ofresponse (r = – 03; p = 0.04), duration of disease (r = – 0.8; p = 0.0001) andhigh-density lipoprotein cholesterol levels (r = 0.43; p = 0.005) after completionof IFNα treatment.Conclusion: It is suggested that the post-treatment increase in serum carnitineobserved in this study could be considered a new index of improved liver function.Also, exogenous administration of carnitine may be useful in patients withchronic hepatitis C who have reduced endogenous synthesis of this substance.

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Carnitine plays a key role in the regulation ofenergy metabolism because it is a cofactor requiredfor the transport of long chain fatty acids into themitochondrial membrane.[1]Although the average daily diet contains signif-

icant carnitine concentrations, endogenous synthe-

sis is required to maintain normal plasma carnitinelevels.[2] Carnitine can be found in liver, heart,skeletal muscles, adrenal gland and semen: its con-centration is correlated to the nutritional state ofthese tissues.[3] The human liver and kidney are themajor sites of carnitine production; skeletal mus-

cles and heart cannot synthesise carnitine.[4,5] Livercarnitine levels constitute about 2% of the total car-nitine in the body.[6]Carnitine serum levels may be reduced in pa-

tients affected by chronic hepatitis C.[7] In thesepatients, the decreased carnitine pool reduces theability to oxidise fatty acids in the liver, heart andmuscles, provoking an overload of intramitocondr-ial acyl coenzyme A and, consequently, increasingcellular damage. Since carnitine affects lipoproteinmetabolism in the liver, marked alterations in lipidpatterns can also be expected in patients affectedby chronic viral hepatitis.The aim of this study was to assess the relation-

ship between serum carnitine levels and the gradeof chronic hepatitis C, and to evaluate the effectsof interferon (IFN)-αn1 treatment on carnitine lev-els in patients with hepatitis C.

Materials and Methods

40 patients (26 males, 14 females; mean age58.8 years) affected by chronic hepatitis C and 20healthy volunteers (13 males and 7 females; meanage 51.1 years) were studied. Baseline charac-teristics are provided in table I. Informed consentwas obtained from patients and volunteers. The

study protocol conformed to the ethical guidelinesof the 1975 Declaration of Helsinki as reflected ina priori approval by the institution’s human re-search review committee.Exclusion criteria included positivity for hepa-

titis B virus infection, HIV infection, alcoholicliver disease, liver cirrhosis and impaired nutri-tional status. Adiagnosis of chronic hepatitis C wasbased on clinical, biohumoral and histologicalfindings.Blood samples were withdrawn from patients

and controls after an overnight fasting period of 12hours and were frozen at= −80°C within 2 hours.Hepatitis C virus antibodies were determined usinga second generation enzyme-linked immunosorb-ent assay method (Ortho Diagnostics). Hepatitis Cvirus RNAwas extracted from serum samples, cop-ied into complementary DNAby reverse transcrip-tion, amplified by polymerase chain reaction(PCR) and quantified by competitive reverse tran-scription-PCR assay according to the method ofBecker-Ande Hahlbrock.[8]The following parameters were determined in

patients and controls with hepatitis C both beforetreatment and after intramuscular lymphoblastoidIFNαn1 3 mega units (MU) 3 times per week for 6

Table I. Summary of baseline characteristics of patients with chronic hepatitis C and healthy volunteers

Patients (n = 40) Healthy volunteers (n = 20)

Treatment IM IFNαn1 3MU 3 times/week for 6 months None

Gender (f/m) 14/26 7/13

Age (years) Mean 58.8 ± 8; range 32-69 Mean 51.1 ± 1.3; range 22-75

Duration of disease (years) 2.82 ± 1.2; range 1-48 NA

Mild chronic hepatitis (n) 10 NA

Moderate chronic hepatitis (n) 10 NA

Severe chronic hepatitis (n) 12 NA

Mean HAI score 12.12 ± 3.74; range 4-18; median 12 NA

Serum carnitine (mg/L) Mean 4.75 ± 1.5*; median 4.4 7.71 ± 0.92

Serum HDL cholesterol(mg/dL)

Mean 40.9 ± 6.3*** Mean 44.4 ± 11.5

Total serum cholesterol(mg/dL)

Mean 175.8 ± 27.54**; range 121-220; median 173 Mean 193.6 ± 48.1; range 126-328;median 183

Serum triglycerides (mg/dL) Mean 115.02 ± 33.7; range 44-173; median 108.5 Mean 126 ± 84.4; range 32-342;median 88

ALT (U/L) 134.1 ± 48.1

HAI = histological activity index; HDL = high-density lipoprotein; IM = intramuscular; INF = interferon; NA = not applicable; * p = 0.01; ** p= 0.02; *** p = 0.0001, vs healthy volunteers.

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months: serum carnitine levels (enzymatic UV ul-traviolet test; reactive by Boehringer Mannheim,Germany), total cholesterol levels, high-density li-poprotein (HDL) cholesterol levels, triglyceridelevels and alanine aminotransferase levels.An enzymatic method[9,10] was used to deter-

mine total cholesterol and triglyceride values(Hitachi 704 Analyser – Twin TG/CHO reactive,Boehringer Mannheim Automated Analysis, Ger-many). HDL cholesterol was also determined us-ing an enzymatic method, after precipitation of li-poproteins containing ApoB by phosphotungsticacid/magnesium chloride[11] (Hitachi 704Analyser– HDL cholesterin/cholesterol reactive, BoehringMannheim, Germany).A liver biopsy was obtained from hepatitis pa-

tients using a modified Menghini technique beforeenrolment. The sample was fixed in neutral 4%formaldehyde solution for routine histological pro-cessing and evaluation. Knodell and Ishak’s histo-logical activity index (HAI) score[12] was used toevaluate the grade of the disease. After completionof IFNα treatment, a further liver biopsy was per-formed in complete responders (for definition, seebelow) only.Patients were divided into 3 groups (mild, mod-

erate and severe chronic hepatitis C) depending onthe initial histological findings (table I). The re-sponse to IFNα treatment was evaluated as fol-lows: complete responders were patients whoachieved normalisation of serum alanine amino-transferase levels during and after completion oftreatment. Patients were followed for 12 monthsafter treatment completion. Patients not achievingcomplete response during treatment and those withrecurring disease after initial remission duringtherapy were designated partial responders. Pa-tients who did not present significant biohumoralchanges during treatment were designated non-responders. Statistical analysis on extrapolateddata was performed usingWilcoxon’s nonparamet-ric test and Spearman’s correlation rank test. A pvalue of 0.05 was considered statistically signifi-cant.

Results

Response to Treatment

After completion of lymphoblastoid alpha in-terferon treatment 5 complete responders, 15 par-tial responders and 20 nonresponders were ob-served. Mean alanine aminotransferase levelsdecreased from 134.1 to 95.8 U/L (table II).

Effect of Interferon-α on Serum Carnitine Levels

Before IFNα treatment, the mean serum carnit-ine level was 4.75 ± 1.5 mg/L in chronic hepatitisC patients and 7.71 ± 0.92 mg/L in healthy volun-teers (p = 0.01). Serum carnitine increased in pa-tients with chronic hepatitis C after 6 months’treat-ment with IFNα to 5.9 ± 1.3 mg/L (p = 0.0003 vspretreatment; p = 0.01 vs volunteers; table II).

Effect of Interferon-α on Lipid Profile

There was a statistically significant differencebetween hepatitis C patients and healthy controlsin both pre- and post-treatment levels of total cho-lesterol (p = 0.02 and 0.007, respectively) andHDLcholesterol (p = 0.0001) but not in triglyceride se-rum levels.The mean pretreatment total cholesterol level

was 175.8 ± 27.54 mg/dL. We observed a slightdecrease in total cholesterol mean serum levels af-

Table II. Summary of results after intramuscular interferon-αn13MU 3 times/week for 6 months in patients with chronic hepatitisC

No. patients responding 5 CR; 15 PR; 20 NR

Serum carnitine (mg/L) 5.9 ± 1.3*; median 5.8

Serum HDL cholesterol (mg/dL) 40.5 ± 8.2††

Total serum cholesterol (mg/dL) 175.4 ± 30.5†

Serum trygliceride (mg/dL) 117.4 ± 32.5*

Mean HAI score in CR 4.74 ± 0.88; range 3.7-6;median 4.5

ALT (U/L) 95.8 ± 55.8

CR = complete response; HAI = histological activity index; HDL =high-density lipoprotein; NR = nonresponse; PR = partial response;* p = 0.0003 vs pretreatment; † p = 0.007 vs healthy volunteers; ††p = 0.0001 vs healthy volunteers.

Serum Carnitine after Interferon-α Treatment 67

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ter completion of IFNα treatment (175.4 ± 30.5mg/dL) but the difference was not significant.The mean pretreatment triglyceride level was

115.02 ± 33.7 mg/dL, increasing after IFNα treat-ment to 117.4 ± 32.5 mg/dL (p = 0.0003).No significant differences were observed be-

tween pre and post-treatment HDL cholesterol se-rum levels (40.9 ± 6.3 mg/dL vs 40.5 ± 8.2 mg/dLrespectively).

Correlations Between Carnitine Levels andOther Parameters

There were statistically significant correlationsbetween post-treatment serum carnitine levels andHDL cholesterol values (r = +0.43; p = 0.005), ageof the patients (r = −0.35; p = 0.02), and responseto treatment (r = +0.3; p = 0.04). There was a sta-tistically significant correlation between pre-treat-ment serum carnitine levels and duration of disease(r ==−0.85; p = 0.00001). No correlation was foundbetween gender and serum carnitine levels.There were no statistical correlations between

pre- and post-treatment serum carnitine levels andtotal cholesterol levels or between pre- and post-treatment serum carnitine levels and HAI score.

Discussion

Our data confirmed that liver disease markedlyreduces endogenous synthesis of carnitine. Thisfinding is in contrast to a previous report[13] thatfound elevated carnitine concentrations in theplasma of patients affected by advanced liver dis-ease, apparently refuting the concept that impairedliver functions are associated with a hypocarni-tinaemic condition.IFNα therapy resulted in a statistically signifi-

cant increase in carnitine serum levels, althoughlevels remained lower than those observed inhealthy subjects.It is known that IFNαn1, along with other cyto-

kines, can cause an increase in lipolysis throughactivation of endothelial lipoprotein-lipase.[14-16]The lipolysis process delivers free fatty acids andglycerol from adipocytes, which provides the sub-strate for enhancing endogenous triglyceride syn-

thesis. On the other hand, this increase in substratelevels determines, at a mitochondrial level, en-hancement of beta-oxidation, and this may be animportant stimulus for carnitine synthesis.Furthermore, it is possible that the enhanced

levels of serum carnitine are related to improvedliver function, as suggested by the positive rela-tionship found between serum carnitine levels andthe response to IFNα treatment. These findings aresupported by the negative relationship observedbetween serum carnitine levels and duration of dis-ease before IFNα treatment.Thus, increased serum carnitine levels may rep-

resent a protective tool against the IFN-induced ac-tion on blood lipids. After treatment we observed astriking correlation between HDL cholesterol val-ues and carnitine serum levels. This effect has notbeen reported frequently and was not obviouslypredictable from the known biochemical functionsof carnitine. In fact, other studies[17] found thatthere is frequently a relationship between exoge-nous administration of carnitine, decreases in tri-glyceride levels and increases in HDL cholesterollevels.However, in our study series there was no sig-

nificant correlation between serum carnitine andtriglyceride levels, suggesting a possible influenceof carnitine on HDL cholesterol levels only. Thisfinding intimates that carnitine can influence thecatabolism of HDLcholesterol independently fromtriglyceride by decreasing the rate of HDL choles-terol breakdown. Havel et al.[18] postulated that thisaction may be due to a carnitine-induced effect onlipoprotein lipase activity, although a consequentreduction of triglyceride levels was not seen in ourstudy series. We postulate that this may be becauseof increased liver synthesis of very low density li-poprotein (VLDL), as a result of the improved liverfunction after IFNα therapy. Interferon-induced ef-fects on the lipid profile could also play a key role(albeit not well understood) in this process.The favourable effects of increased serum car-

nitine on liver steatosis are still debated, althoughincreased serum carnitine was associated with re-

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duced liver steatosis in complete responders, as ob-served in post-treatment liver biopsy.Our data suggest that the post-treatment in-

creases in serum carnitine observed in our studyseries may be considered a new index of improvedliver function. Exogenous administration of carnit-ine may be required in patients with chronic hepa-titis C who have reduced endogenous synthesis ofthis substance.

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Correspondence and reprints: Dr Mariano Malaguarnera,Istituto di Medicina Interna e Geriatria, Universita diCatania, Via Messina 829 – 95126 Catania, Italy.E-mail: malaguar@mbox.unict.it

Serum Carnitine after Interferon-α Treatment 69

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