Lack of anti-lipoprotein lipase in Behçet's disease

Abstracts

OBJETIVO: A recente descrição de anticorpos antilipoproteína lipase (anti-LPL) associados à dislipoproteinemia levounos a analisar sua presença e possível associação com achados clínicos e laboratoriais em pacientes com doença de Behçet. PACIENTES E MÉTODOS: Trinta e oito pacientes consecutivos com doença de Behçet [critérios do Grupo de Estudos Internacional em Doença de Behçet (International Study Group on Behçet's Disease - ISGBD)] foram testados para a presença de anticorpos anti-LPL através da técnica de ELISA. Foi realizada avaliação clínica e laboratorial, incluindo perfil lipídico de jejum, pesquisa de autoanticorpos e marcadores inflamatórios (PCR, VHS) na inclusão dos pacientes. Os critérios de exclusão foram quaisquer condição que afetassem o perfil lipídico. RESULTADOS: A média de idade foi de 42 ± 9 anos, sendo 68% do sexo feminino e 68% da cor branca. O tempo médio de duração da doença foi de 9,8 ± 7,5 anos. Vinte e nove por cento dos pacientes apresentaram história de trombose. Os níveis de PCR estavam elevados em 31% dos pacientes e VHS aumentada foi detectada em 31%, com níveis médios de 5,95 ± 10,3 mcg/mL e 14,5 ± 13,2 mm/1ªhora, respectivamente. Cerca de 47% dos pacientes estavam tomando prednisona, com dose média de 7,6 ± 10,8 mg/dia. Quanto aos níveis de risco cardiovascular da NCEP/ATPIII, colesterol elevado foi verificado em 26%, triglicerídeos em 18%, HDL baixo em 15% e elevado LDL em 25% dos pacientes com Behçet. Os níveis médios de CT foram 198 ± 48 mg/dL, de triglicerídeos 121 ± 61 mg/dL, HDL 52.4 ± 14.7 mg/dL e LDL 119 ± 35 mg/dL. Anticorpos anti-LPL to subtipo IgG foram detectados em 0/30 pacientes com Behçet. CONCLUSÃO: Esses dados aqui apresentados corroboram uma ausência de ligação entre inflamação, resposta imunológica e dislipoproteinemia nos pacientes com doença de Behçet e sugerem que outros mecanismos estão associados à dislipidemia vista nestes pacientes.

doença de Behçet; vasculites; lipoproteína lipase; autoanticorpos; aterosclerose; lipoproteínas; inflamação

ORIGINAL ARTICLE

Lack of anti-lipoprotein lipase in Behçet's disease

Jozélio Freire de Carvalho^I; Vilma Santos Trindade Viana^I; Cleonice Bueno^II; Célio Roberto Gonçalves^III; Eloísa Bonfá^IV

^IPh.D., Chief of the Humoral Immunology Laboratory-LIM17-FMUSP

^IIResearcher of LIM17-FMUSP

^IIIAssistant Physician

^IVPh.D., Professor

^{Corresponding to}

ABSTRACT

OBJECTIVE: The recent description of anti-lipoprotein lipase antibodies (anti-LPL) associated with dislipoproteinemia led us to analyze its presence and possible association with clinical and laboratorial findings in patients with Behçet's disease.

PATIENTS AND METHODS: Thirty-eight consecutive patients with Behçet's disease [International Study Group for Behçet's Disease (ISGBD) criteria] were tested for the presence of anti-LPL antibodies by ELISA. Patients underwent clinical and laboratorial evaluation, including fasting lipid profile, determination of autoantibodies, and inflammatory markers (CRP, ESR) before inclusion in the study. Exclusion criteria were as follows: any conditions that affected the lipid profile.

RESULTS: Patients had a mean age of 42 ± 9 years, 68% were females, and 68% were Caucasian. Mean disease duration was 9.8 ± 7.5 years. Twenty-nine percent of the patients had a history of thrombosis. C-reactive protein levels were elevated in 31% of the patients, and ESR was increased in 31% of the patients, with mean levels of 5.95 ± 10.3 mcg/mL and 14.5 ± 13.2 mm/1^st hour, respectively. Approximately 47% of the patients were taking prednisone, with a mean dose of 7.6 ± 10.8 mg/day. As for NCEP/ATPIII cardiovascular risk levels, cholesterol levels were elevated in 26% of the patients, triglycerides in 18%, low HDL in 15%, and elevated LDL in 25% of the patients with Behçet's disease. Mean total cholesterol levels were 198 ± 48 mg/dL, triglycerides 121 ± 61 mg/dL, HDL 52.4 ± 14.7 mg/dL, and LDL 119 ± 35 mf/dL. IgG anti-LPL antibodies were detected in 0/30 patients with Behçet's disease.

CONCLUSION: The data presented here indicates the lack of correlation among inflammation, immune response, and dislipoproteinemia in patients with Behçet's disease and suggests that other mechanisms are associated with the dyslipedemia seen in those patients.

Keywords: Behçet's disease, vasculitis, lipoprotein lipase, autoantibodies, atherosclerosis, lipoproteins, inflammation.

INTRODUCTION

Lipoprotein lipase (LPL) is a component of the lipase family, which hydrolyzes triglyceride molecules found in lipoprotein particles.¹ Physiological regulators of LPL include triglycerides and apolipoprotein (apo) CII, which increase the enzymatic activity of LPL, apo C-III, which inhibits its activity.¹

The report of a significant reduction in LPL activity in SLE patients² led to the hypothesis that it resulted from an inhibitory inflammatory condition or modulation by an autoantibody. Indeed, this last hypothesis was strengthened by a recent report of an increased incidence of anti-lipoprotein lipase antibodies in SLE, which showed a positive correlation with triglyceride levels.^2,3 Those antibodies have also been reported in association with other rheumatologic disorders, including rheumatoid arthritis and systemic sclerosis.⁴

It is reasonable to speculate that the same risk factors seen in SLE patients can also be present in individuals with Behçet's disease. The objective of the present study was to evaluate the presence of anti-LPL antibodies and the possible association with clinical and laboratorial parameters of this disorder.

PATIENTS AND METHODS

Patients: Thirty-eight patients with Behçet's disease (BD) were randomly selected among those followed-up at the Behçet's Disease Outpatient Clinic of the Rheumatology Department of Hospital das Clínicas at the Medical School at Universidade de São Paulo from June 2001 to December 2003. All patients fulfilled the International Study Group for Behçet's Disease⁵ (ISGBD) criteria. Patients were clinically evaluated and their medical records were carefully reviewed. Exclusion criteria were as follows: clinical and/or laboratorial evidence of diabetes mellitus, liver and thyroid disorders, renal failure, nephritic syndrome, history of alcohol abuse, use of hypolipidemic agents, and menopause. All patients were younger than 50 years, had serum creatinine < 1.5 mg/dL, and proteinuria lower than 1.0 g/L/day.^6-8 Individuals taking medications that could interfere with the lipid profile, such as statins and fibrates, were also excluded. Female patients were not pregnant or menopausal at the time of the study. The local Ethics Committee approved the study # 814/01 and patients signed an informed consent.

Laboratorial evaluation: Serum obtained from patients after a 12-hour fasting period underwent immunological and biochemical analysis. All patients were on a regular diet.

Assay for the detection of anti-lipoprotein lipase (LPL) antibodies: The reactivity of anti-LPL IgG was measured by ELISA (Enzyme-Linked Immunosorbent Assay). Briefly, the wells of Costar^® polystyrene plates were sensitized overnight with commercially available LPL from bovine milk (5 µg/mL) (Sigma Chem Co., St. Louis, MO, USA). Sera samples diluted to 1/100 in Tris buffered NS with adult bovine serum were used for the assays. IgG anti-LPL antibodies were detected with anti-human IgG goat antibodies conjugated with alkaline phosphatase (Sigma Chem Co., St. Louis, MO, USA). The reaction was developed with p-nitrophenylphosphate and the optical density (OD) was read at a 405 nm wavelength be a Labsystems Multiskan MS spectrophotometer (Helsinki, Finland). Positive results were defined as values > 3 standard deviation (SD) above the mean OD of the serum samples from 20 healthy individuals included in each assay.²

Lipid profile: Total cholesterol (TC) and triglyceride (TG) levels were measured enzymatically (Boehringer-Mannhein, Argentina, and Merck, Germany, respectively) in a RA 1000 Analyzer (Technicon Instruments Corp.).^9,10 High-density lipoprotein cholesterol (HDL-c) was obtained after precipitation of very low density lipoprotein cholesterol (VLDL-c), and low density lipoprotein cholesterol (LDL-c) using phosphotungstic acid and magnesium chloride.¹¹ Very low density lipoprotein cholesterol and LDL-c were estimated, since triglyceride (TG) levels in all samples were below 400 mg/dL.¹² The levels of VLDL-c were calculated using the triglyceride levels/5 (TG/5)(34) correlation, and LDL-c levels were estimated using the following equation:¹² TC = HDL-c+TG/5+LDL-c. Cardiovascular risk was determined according to the criteria of the National Cholesterol Education Program - Adult Treatment Panel III (NCEP/ATPIII), as follows: TC greater than 200 mg/dL, TG greater than 150 mg/dL, HDL-c lower than 40 mg/dL, and LDL-c greater than 130 mg/dL.¹³

Inflammatory Markers: The levels of C-reactive protein (CRP) and total gamma globulin of all patients were determined by nephelometry and protein electrophoresis, respectively. Erythrocyte sedimentation rate (ESR) was determined by the Westergren method.

Statistical analysis: Descriptive analysis was used in the present study. Results are presented as mean ± SD and percentages.

RESULTS

Patients with BD (n = 38) had a mean age of 42 ± 9 years, 68% were females, and 68% Caucasian. Mean disease duration was 9.8 ± 7.5 years. Approximately 47% of the patients were taking prednisone, but at a low daily dose (7.6 ± 10.8 mg/day) (Table I).

Mean cholesterol levels were 198 ± 48 mg/dL, triglycerides 121 ± 61 mg/dL, HDL 52.4 ± 14.7 mg/dL, and LDL 119 ± 35 mg/dL. Moderate/high cardiovascular risk, according to NCEP/ ATPIII criteria, were observed in 26% of the patients (n = 14) due to high cholesterol, low HDL-c in 15% (n = 5) of the patients, high LDL-c in 25% (n = 16) of the patients, and high triglyceride levels in 18% (n = 13) of the patients (Table 2).

ELISA did not detect anti-PLP antibodies in patients with Behçet's disease.

Patients with BD showed altered inflammatory markers, with mean levels of CRP of 5.95 ± 10.3 mcg/mL, and ESR within normal limits with mean levels of 14.5 ± 13.2 mm/1^st hour. Additionally, 31% of the patients showed elevated CRP, and the ESR was also increased in 31% of the patients (Table 3).

DISCUSSION

The results of the present study demonstrate that patients with Behçet's disease do not have anti-lipoprotein lipase antibodies, although some of those individuals have at least one lipid at high risk levels for cardiovascular disease.

Only patients who fulfilled international criteria for BD⁵ were included in this study. It is important that the strict inclusion and exclusion criteria used to select patients with BD provided a unique opportunity to define more accurately that the changes in lipid profile should be secondary to their disease.

Therefore, drugs and comorbidities, such as diabetes, obesity, thyroid disorders, menopause, renal failure, and nephrotic syndrome represented exclusion criteria, since those factors are well known to interfere with lipid metabolism.^6-8

Analysis of the lipid profile showed that, in patients with Behçet's disease, lipid levels represented high risk for cardiovascular diseases due to elevated levels of total cholesterol, LDL-c, and triglycerides, and low HDL-c levels. Those findings were similar to those observed in chronic inflammatory diseases associated with atherosclerosis,^14,15 such as systemic lupus erythematosus. In this context, lipoprotein lipase (LPL), the target-antigen for the antibody studied here, is known to facilitate the metabolism of plasma lipoproteins and it is the main enzyme responsible for the hydrolysis of circulating triglycerides.¹ Its hindered enzymatic activity is directly involved with the development of hypertriglyceridemia, which is known to have a role in the atherosclerotic process.¹

Anti-LPL auto-antibodies have been implicated in the mechanisms of atherosclerosis in SLE and in other autoimmune inflammatory diseases, such as rheumatoid arthritis and systemic sclerosis.^2-4 Those antibodies have been strictly associated with the increase in triglyceride levels in SLE and scleroderma, and a putative anti-LPL functional role in those diseases derived from the observation that the anti-LPL IgG fraction of scleroderma patients with elevated serum triglyceride levels was capable of inhibiting significantly enzymatic activity in vitro.⁴ Besides, prior studies have demonstrated that patients with active lupus show a strong correlation between circulating anti-LPL and inflammatory markers, especially CRP.²

Despite the presence of elevated levels of inflammatory markers, as well as altered lipid profile, in BD patients, the absence of reactivity to LPL suggests that anti-LPL antibodies are not implicated in the active, permanent, complex vascular inflammatory process that causes damages in those patients. In this aspect, it is reasonable to speculate that the inflammatory processes and dyslipidemia seen in BD and systemic connective tissue disorders (SLE and systemic sclerosis) can reflect several pathogenic mechanisms.¹⁶ Indeed, systemic vasculitis is known to be pauci-immune conditions.

The data of the present study are conclusive in showing that anti-LPL antibodies do not seem to be implicated in the pathophysiology of the atherosclerotic inflammatory process in Behçet's disease.

REFERENCES

Received on 02/28/2009.

Approved on 05/19/2009.

The present study received grant from remaining funds of Brazilian Society of Rheumatology, from Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP#02/03867-0, Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq# 304756/2003-2 (for Eloísa Bonfá), and from Federico Foundation (for Jozélio Freire de Carvalho and Eloísa Bonfá). Department of Rheumatology, Medical School of Universidade de São Paulo, São Paulo, Brazil.

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Publication Dates

• Publication in this collection
  16 Dec 2009
• Date of issue
  Dec 2009

History

• Received
  28 Feb 2009
• Accepted
  19 May 2009