Received Date : 04-Apr-2013 Accepted Date : 06-Jun-2014 Article type
: Original Paper
VISCERAL LEISHMANIASIS IS ASSOCIATED WITH MARKED CHANGES IN SERUM LIPID PROFILE Evangelos N Liberopoulos1*, Fotini Apostolou1, Irene F Gazi1, Christina Kostara2, Eleni T Bairaktari2, Alexandros D Tselepis3, Moses Elisaf1 1
Department of Internal Medicine, and 2Department of Biochemistry, Medical School, University of Ioannina, 3Atherothrombosis Research Center/Laboratory of Biochemistry, School of Chemistry, University of Ioannina, Ioannina, Greece
*Corresponding author Dr Evangelos N Liberopoulos MD FASA FRSH Department of Internal Medicine University of Ioannina Medical School Ioannina 45 110, Greece Tel.: +30 2651007502; fax: +30 2651007016 E-mail: [email protected]
ABSTRACT Background: Infection is often accompanied by lipid profile alterations. The aim of the study was to evaluate the lipid profile changes in patients with visceral leishmaniasis (VL). Materials and Methods: We included 15 patients [10 men, aged 50 (24-82) years old] with VL and 15 age- and sex-matched controls. The parameters estimated at diagnosis and 4 months after VL resolution were: total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), apolipoproteins (apo) A-Ι, B, E, C-II, C-III, lipoprotein (a) [Lp(a)], activities of lipoprotein-associated phospholipase A2 (Lp-PLA2), HDL-Lp-
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/eci.12288 This article is protected by copyright. All rights reserved.
PLA2, PON1 (paraoxonase 1) and cholesterol ester transfer protein (CETP), cytokines (interleukins 1β and 6 and tumor necrosis factor α), as well as LDL subfraction profile. Results: Patients with VL at diagnosis had lower levels of TC, LDL-C, apoΒ, and Lp(a), and higher TG and apoE concentrations compared with 4 months after VL resolution. The activities of Lp-PLA2, HDL-Lp-PLA2 and ΡΟΝ1 were reduced at diagnosis compared with post-treatment values. VL patients had decreased levels of both large and sdLDL-C at diagnosis; no effect on mean LDL particle size was observed. Patients with VL at diagnosis had decreased HDL-C and apoA-I concentrations; these increased 4 months after VL resolution, but remained lower compared with controls. The activities of HDL-Lp-PLA2 and PON1 remained lower in patients after VL resolution compared with controls. Conclusions: Patients with VL exhibit increased TG levels and decreased cholesterol subclasses at diagnosis. HDL-C, apoA-I and associated enzymes remain lower 4 months after VL resolution compared with controls.
Key words: leishmaniasis, infection, lipid profile, lipoproteins, atherosclerosis
INTRODUCTION Several infections have been linked to an increased atherogenic potential, including those caused by Chlamydia pneumoniae, Cytomegalovirus (CMV), Herpes simplex virus (HSV) and Helicobacter pylori (1). Moreover, regardless of the cause, infection is associated with alterations in lipid and lipoprotein concentrations (1, 2) as well as of associated enzymes, such as lipoprotein-associated phospholipase A2 (Lp-PLA2) (3-5), paraoxonase 1 (PON1) (6) and cholesteryl-ester transfer protein (CETP) (3, 4, 7). For example, periodontitis, human immunodeficiency virus (HIV) infection as well as Brucella melitensis, Leptospira interrogans and Epstein-Barr virus (EBV) infections have been associated with increased small dense low-density lipoprotein cholesterol (sdLDL-C) levels (3, 4, 810). Visceral leishmaniasis (VL) is a vector-borne protozoal infection caused by the replication of Leishmania species in macrophages. There are an estimated 500,000 new cases and more than 50,000 deaths from VL each year, particularly in developing countries (11). The HIV/AIDS pandemic has modified natural history of leishmaniasis. HIV infection increases the risk of developing VL, reduces the likelihood of a therapeutic response, and greatly increases the probability of relapse (12). VL can even be asymptomatic or subclinical. Classic symptoms of VL include fever, abdominal discomfort, weight loss, cough, pallor, splenomegaly and hepatomegaly, while common laboratory findings include pancytopenia and hypergammaglobulinemia (13). The effect of VL on plasma lipid profile has not been thoroughly studied. Experimental studies have shown that both L. Infantum and L. Donovani infections lead to alterations in lipid profile, with the increase in triglyceride (TG) levels being the most prominent (14, 15). Infantile VL is associated with increased TGs and apolipoprotein (apo) E levels and decreased concentrations of total cholesterol (TC) and HDL-C, as well as apo A-I, while apoB remain unchanged (16, 17). Adults with VL exert similar lipid profile alterations (18-20). In a previous case report the prominent lipid
This article is protected by copyright. All rights reserved.
feature in a patient with VL was marked hypocholesterolemia with reduced lipoprotein (a) [Lp(a)] (18). We undertook the present study to evaluate in detail the possible quantitative and qualitative effects of VL on serum lipid parameters and associated enzymes.
MATERIALS AND METHODS Study population Consecutive patients who were diagnosed with VL at the 2nd Department of Internal Medicine, University Hospital of Ioannina, Greece, between May 2003 and June 2012 were considered. The diagnosis of VL was established by the presence of high titers of antileishmania antibodies via indirect immunofluorescence assay and indirect hemagglutination antibodies, as well as demonstration of intracellular parasites in bone marrow aspiration in patients with compatible clinical and laboratory findings in some cases. All patients were negative for HIV infection. No patient was receiving any hypolipidemic agents or had any clinical or laboratory evidence of any disease known to affect lipid metabolism, such as neoplasia, renal or liver dysfunction failure, and hypo- or hyperthyroidism. All patients were examined at diagnosis and 4 months after the resolution of VL (defined as resolution of febrile illness). Fifteen age- and sex-matched healthy volunteers (control population) were also included in the present study. The latter group visited the outpatient clinic of the 2nd Department of Internal Medicine for a regular check-up. All individuals signed an informed consent for the participation in the present study. This observational study was approved by the Ethics Committee of the University Hospital of Ioannina. Reporting of the study conforms to STROBE statement along with references to STROBE and the broader EQUATOR guidelines (21). Laboratory measurements Fasting serum levels of TC, HDL-C and TG were determined enzymatically on an Olympus AU600 Clinical Chemistry analyser (Olympus Diagnostica, Hamburg, Germany). Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula (except for one patient whose TG levels exceeded the cut-off point of 400 mg/dL, for whom LDL-C concentration was not calculated). Apo A-I, apoB, apoE as well as Lp(a) levels were measured with a Behring Nephelometer BN100 using reagents from Date Behring Holding Gmbh (Liederbach, Germany). ApoC-II and apoC-III were determined by an immunoturbidimetric assay provided by Kamiya Biomedical Company (Seattle,WA). LDL subclass analysis was performed electrophoretically using high-resolution 3% polyacrylamide gel tubes and the Lipoprint LDL System (Quantimetrix) according to the manufacturer’s instructions (22). Lp-PLA2 and CETP activities as well as PON1 hydrolysing activities against paraoxon [PON1 (paraoxonase)] and phenyl acetate [PON1 (arylesterase)] were determined as previously described (3, 7). Cytokines [interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα)] were determined by ELISA (Quantikine; R&D Systems, Inc., Minneapolis, MN, USA). Each sample was measured in duplicate with appropriate sensitivities for IL-1β (
: Original Paper
VISCERAL LEISHMANIASIS IS ASSOCIATED WITH MARKED CHANGES IN SERUM LIPID PROFILE Evangelos N Liberopoulos1*, Fotini Apostolou1, Irene F Gazi1, Christina Kostara2, Eleni T Bairaktari2, Alexandros D Tselepis3, Moses Elisaf1 1
Department of Internal Medicine, and 2Department of Biochemistry, Medical School, University of Ioannina, 3Atherothrombosis Research Center/Laboratory of Biochemistry, School of Chemistry, University of Ioannina, Ioannina, Greece
*Corresponding author Dr Evangelos N Liberopoulos MD FASA FRSH Department of Internal Medicine University of Ioannina Medical School Ioannina 45 110, Greece Tel.: +30 2651007502; fax: +30 2651007016 E-mail: [email protected]
ABSTRACT Background: Infection is often accompanied by lipid profile alterations. The aim of the study was to evaluate the lipid profile changes in patients with visceral leishmaniasis (VL). Materials and Methods: We included 15 patients [10 men, aged 50 (24-82) years old] with VL and 15 age- and sex-matched controls. The parameters estimated at diagnosis and 4 months after VL resolution were: total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), apolipoproteins (apo) A-Ι, B, E, C-II, C-III, lipoprotein (a) [Lp(a)], activities of lipoprotein-associated phospholipase A2 (Lp-PLA2), HDL-Lp-
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/eci.12288 This article is protected by copyright. All rights reserved.
PLA2, PON1 (paraoxonase 1) and cholesterol ester transfer protein (CETP), cytokines (interleukins 1β and 6 and tumor necrosis factor α), as well as LDL subfraction profile. Results: Patients with VL at diagnosis had lower levels of TC, LDL-C, apoΒ, and Lp(a), and higher TG and apoE concentrations compared with 4 months after VL resolution. The activities of Lp-PLA2, HDL-Lp-PLA2 and ΡΟΝ1 were reduced at diagnosis compared with post-treatment values. VL patients had decreased levels of both large and sdLDL-C at diagnosis; no effect on mean LDL particle size was observed. Patients with VL at diagnosis had decreased HDL-C and apoA-I concentrations; these increased 4 months after VL resolution, but remained lower compared with controls. The activities of HDL-Lp-PLA2 and PON1 remained lower in patients after VL resolution compared with controls. Conclusions: Patients with VL exhibit increased TG levels and decreased cholesterol subclasses at diagnosis. HDL-C, apoA-I and associated enzymes remain lower 4 months after VL resolution compared with controls.
Key words: leishmaniasis, infection, lipid profile, lipoproteins, atherosclerosis
INTRODUCTION Several infections have been linked to an increased atherogenic potential, including those caused by Chlamydia pneumoniae, Cytomegalovirus (CMV), Herpes simplex virus (HSV) and Helicobacter pylori (1). Moreover, regardless of the cause, infection is associated with alterations in lipid and lipoprotein concentrations (1, 2) as well as of associated enzymes, such as lipoprotein-associated phospholipase A2 (Lp-PLA2) (3-5), paraoxonase 1 (PON1) (6) and cholesteryl-ester transfer protein (CETP) (3, 4, 7). For example, periodontitis, human immunodeficiency virus (HIV) infection as well as Brucella melitensis, Leptospira interrogans and Epstein-Barr virus (EBV) infections have been associated with increased small dense low-density lipoprotein cholesterol (sdLDL-C) levels (3, 4, 810). Visceral leishmaniasis (VL) is a vector-borne protozoal infection caused by the replication of Leishmania species in macrophages. There are an estimated 500,000 new cases and more than 50,000 deaths from VL each year, particularly in developing countries (11). The HIV/AIDS pandemic has modified natural history of leishmaniasis. HIV infection increases the risk of developing VL, reduces the likelihood of a therapeutic response, and greatly increases the probability of relapse (12). VL can even be asymptomatic or subclinical. Classic symptoms of VL include fever, abdominal discomfort, weight loss, cough, pallor, splenomegaly and hepatomegaly, while common laboratory findings include pancytopenia and hypergammaglobulinemia (13). The effect of VL on plasma lipid profile has not been thoroughly studied. Experimental studies have shown that both L. Infantum and L. Donovani infections lead to alterations in lipid profile, with the increase in triglyceride (TG) levels being the most prominent (14, 15). Infantile VL is associated with increased TGs and apolipoprotein (apo) E levels and decreased concentrations of total cholesterol (TC) and HDL-C, as well as apo A-I, while apoB remain unchanged (16, 17). Adults with VL exert similar lipid profile alterations (18-20). In a previous case report the prominent lipid
This article is protected by copyright. All rights reserved.
feature in a patient with VL was marked hypocholesterolemia with reduced lipoprotein (a) [Lp(a)] (18). We undertook the present study to evaluate in detail the possible quantitative and qualitative effects of VL on serum lipid parameters and associated enzymes.
MATERIALS AND METHODS Study population Consecutive patients who were diagnosed with VL at the 2nd Department of Internal Medicine, University Hospital of Ioannina, Greece, between May 2003 and June 2012 were considered. The diagnosis of VL was established by the presence of high titers of antileishmania antibodies via indirect immunofluorescence assay and indirect hemagglutination antibodies, as well as demonstration of intracellular parasites in bone marrow aspiration in patients with compatible clinical and laboratory findings in some cases. All patients were negative for HIV infection. No patient was receiving any hypolipidemic agents or had any clinical or laboratory evidence of any disease known to affect lipid metabolism, such as neoplasia, renal or liver dysfunction failure, and hypo- or hyperthyroidism. All patients were examined at diagnosis and 4 months after the resolution of VL (defined as resolution of febrile illness). Fifteen age- and sex-matched healthy volunteers (control population) were also included in the present study. The latter group visited the outpatient clinic of the 2nd Department of Internal Medicine for a regular check-up. All individuals signed an informed consent for the participation in the present study. This observational study was approved by the Ethics Committee of the University Hospital of Ioannina. Reporting of the study conforms to STROBE statement along with references to STROBE and the broader EQUATOR guidelines (21). Laboratory measurements Fasting serum levels of TC, HDL-C and TG were determined enzymatically on an Olympus AU600 Clinical Chemistry analyser (Olympus Diagnostica, Hamburg, Germany). Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula (except for one patient whose TG levels exceeded the cut-off point of 400 mg/dL, for whom LDL-C concentration was not calculated). Apo A-I, apoB, apoE as well as Lp(a) levels were measured with a Behring Nephelometer BN100 using reagents from Date Behring Holding Gmbh (Liederbach, Germany). ApoC-II and apoC-III were determined by an immunoturbidimetric assay provided by Kamiya Biomedical Company (Seattle,WA). LDL subclass analysis was performed electrophoretically using high-resolution 3% polyacrylamide gel tubes and the Lipoprint LDL System (Quantimetrix) according to the manufacturer’s instructions (22). Lp-PLA2 and CETP activities as well as PON1 hydrolysing activities against paraoxon [PON1 (paraoxonase)] and phenyl acetate [PON1 (arylesterase)] were determined as previously described (3, 7). Cytokines [interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα)] were determined by ELISA (Quantikine; R&D Systems, Inc., Minneapolis, MN, USA). Each sample was measured in duplicate with appropriate sensitivities for IL-1β (
