abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC São Paulo, SP, Brazil pmywCHFwqRKVjKQDK3MHDyw 00008 S0066-782X2007000200008 S0066-782X(07)08800208 10.1590/S0066-782X2007000200008 Artigos Originais Atividade da enzima acetil-hidrolase do fator ativador de plaquetas (PAF-AH) em pacientes com diabete melito tipo 1 Castro Simone Henriques de Faria Neto Hugo Caire de Castro Gomes Marilia de Brito Rio de Janeiro RJ FIOCRUZ Laboratório de Imunofarmacologia Rio de Janeiro Universidade do Estado do Rio de Janeiro Hospital Pedro Ernesto Serviço de Diabetes Brazil 02 2007 88 2 179 184 11 04 2006 16 03 2006 15 12 2005 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

OBJETIVO: Avaliar a atividade da acetil-hidrolase do fator ativador de plaquetas (PAF-AH) e sua relação com variáveis clinicodemográficas, com o controle metabólico, os níveis de apolipoproteínas A e B e a suscetibilidade da lipoproteína de baixa densidade (LDL) à oxidação in vitro em pacientes com DM tipo 1 (DM 1). MÉTODOS: Foram avaliados 42 pacientes com DM1 (27 mulheres) e 48 não-diabéticos (16 mulheres), pareados por sexo, idade e índice de massa corporal (IMC). Os exames realizados foram: glicemia de jejum (GJ) e pós-prandial (GPP), lipidograma, ácido úrico (AU), hemoglobina glicosilada (HbA1c) e coeficiente de oxidação da lipoproteína de baixa densidade (LDL) por espectrofotometria. A análise da atividade da PAF-AH foi realizada por espectrofotometria (Cayman Chemical). RESULTADOS: A análise da atividade da PAF-AH mostrou haver maior atividade enzimática nos pacientes com DM 1 do que nos não-diabéticos (0,0150 ± 0,0051 versus 0,0116 ± 0,0041; p < 0,001). Nos pacientes com DM 1, encontramos correlação direta entre a atividade da PAF-AH e a idade e a LDL, e inversa entre a PAF-AH e a HbA1c e a lipoproteína de alta densidade (HDL). CONCLUSÃO: A PAF-AH, na amostra estudada, apresentou maior atividade nos pacientes com DM 1, fator que pode estar relacionado ao maior risco de desenvolver doenças cardiovasculares apresentados por portadores dessa doença. Ainda são necessários novos estudos para avaliar a real participação dessa enzima no risco de desenvolvimento das doenças ateroscleróticas nos pacientes com DM 1.

 PAF-acetilhidrolase diabete tipo 1 doenças ateroscleróticas 20 03 2007

ARTIGO ORIGINAL

Atividade da enzima acetil-hidrolase do fator ativador de plaquetas (PAF-AH) em pacientes com diabete melito tipo 1

Simone Henriques de CastroI; Hugo Caire de Castro Faria NetoII; Marilia de Brito GomesI

IServiço de Diabetes do Hospital Pedro Ernesto - UERJ

IILaboratório de Imunofarmacologia da FIOCRUZ – Rio de Janeiro, RJ

Correspondência

RESUMO

OBJETIVO: Avaliar a atividade da acetil-hidrolase do fator ativador de plaquetas (PAF-AH) e sua relação com variáveis clinicodemográficas, com o controle metabólico, os níveis de apolipoproteínas A e B e a suscetibilidade da lipoproteína de baixa densidade (LDL) à oxidação in vitro em pacientes com DM tipo 1 (DM 1).

MÉTODOS: Foram avaliados 42 pacientes com DM1 (27 mulheres) e 48 não-diabéticos (16 mulheres), pareados por sexo, idade e índice de massa corporal (IMC). Os exames realizados foram: glicemia de jejum (GJ) e pós-prandial (GPP), lipidograma, ácido úrico (AU), hemoglobina glicosilada (HbA1c) e coeficiente de oxidação da lipoproteína de baixa densidade (LDL) por espectrofotometria. A análise da atividade da PAF-AH foi realizada por espectrofotometria (Cayman Chemical).

RESULTADOS: A análise da atividade da PAF-AH mostrou haver maior atividade enzimática nos pacientes com DM 1 do que nos não-diabéticos (0,0150 ± 0,0051 versus 0,0116 ± 0,0041; p < 0,001). Nos pacientes com DM 1, encontramos correlação direta entre a atividade da PAF-AH e a idade e a LDL, e inversa entre a PAF-AH e a HbA1c e a lipoproteína de alta densidade (HDL).

CONCLUSÃO: A PAF-AH, na amostra estudada, apresentou maior atividade nos pacientes com DM 1, fator que pode estar relacionado ao maior risco de desenvolver doenças cardiovasculares apresentados por portadores dessa doença. Ainda são necessários novos estudos para avaliar a real participação dessa enzima no risco de desenvolvimento das doenças ateroscleróticas nos pacientes com DM 1.

Palavras-chave: PAF-acetilhidrolase, diabete tipo 1, doenças ateroscleróticas.

Introdução

A doença cardiovascular é a principal causa de morbidade e mortalidade no diabete melito tipo 1 (DM tipo 1), sendo responsável por 44% de todas as mortes de pacientes com DM tipo 11,2. Diversos métodos foram utilizados para avaliar a presença da doença aterosclerótica nos pacientes com DM tipo 1, evidenciando que a doença coronariana já pode estar presente mesmo em pacientes jovens3-5.

O processo oxidativo é considerado um componente importante no estágio inicial e na progressão da doença aterosclerótica. A atividade da enzima PAF-AH pode ser protetora nesse processo, já que a associação dessa enzima com a LDL e a HDL previne a formação da molécula de LDL minimamente oxidada in vitro e sua internalização pelos macrófagos6-8.

Recentemente, a aterosclerose tem sido considerada uma doença inflamatória crônica. O processo inflamatório contribui significativamente para o início, a progressão e a ruptura das placas ateroscleróticas ricas em lipídios9,10. As células responsáveis pelo dano tecidual associado à inflamação são recrutadas e ativadas por uma série de mediadores, sendo o fator ativador de plaquetas (PAF) um fosfolipídio envolvido nesse processo. Pode haver geração de fosfolipídios com atividade pró-inflamatória por meio da fragmentação oxidativa do ácido graxo fosfatidilcolina, o qual é chamado de PAF-like. A atividade biológica do PAF e dos fosfolipídios PAF-like é atenuada pelas enzimas fosfolipases A2 (FLA2)11.

Estudos mostram que as FLA2 estão envolvidas no processo inflamatório e na aterogênese, e que tanto a do tipo II secretória quanto a associada à lipoproteína (PAF-acetilhidrolase/PAF-AH) podem estar relacionadas com risco de doença coronariana12,13. Teoricamente, essa enzima pode promover a aterogênese, se os produtos que ela liberar dos fosfolipídeos da LDL tiverem efeito deletério na parede arterial14; ou pode ser protetora, se ao hidrolisar o PAF reduzir a tendência inflamatória e trombótica do sangue15. A PAF-AH no plasma está principalmente associada com a LDL (preferencialmente subfração 5); entretanto, uma pequena parcela (< 20% da atividade enzimática) está associada com a HDL (preferencialmente subfração 1)16,17.

O presente estudo tem como objetivo avaliar a atividade da PAF-AH e sua relação com variáveis clinicodemográficas, com o controle metabólico, os níveis de apolipoproteínas A e B e a suscetibilidade da lipoproteína de baixa densidade (LDL) à oxidação in vitro em pacientes com DM tipo 1, a fim de observar a interferência de variáveis classicamente relacionadas com maior risco de doença cardiovascular nos pacientes com diabetes e esse novo fator estudado.

Métodos

Pacientes - Foram avaliados 50 pacientes com DM1, acompanhados regularmente no ambulatório de Diabetes do Hospital Universitário Pedro Ernesto (HUPE) – UERJ, e 48 não-diabéticos, pareados por sexo, idade e índice de massa corporal (IMC), após assinatura do termo de consentimento previamente aprovado pelo comitê de ética do HUPE. As características clinicodemográficas das duas amostras no momento da coleta dos exames são apresentadas na tabela 1.

Os critérios de exclusão para os diabéticos foram: tabagismo, etilismo, infecção sistêmica e/ou uso de medicamentos que pudessem alterar os resultados da determinação da suscetibilidade do LDL à oxidação, como os inibidores da ECA, sulfato ferroso e complexos vitamínicos e de sais minerais contendo vitaminas C e E e Zn, Se, Fe e Cu, e presença de eventos cardiovasculares prévios, retinopatia e nefropatia diabéticas e neuropatia diabética clínica. No grupo dos não-diabéticos, esses critérios incluíam também a presença de familiares diretos portadores de diabete melito.

De acordo com os critérios de exclusão, 8 pacientes com diabete tipo 1 foram excluídos do estudo por apresentarem nefropatia diabética incipiente e/ou retinopatia diabética.

Os participantes foram submetidos a coleta de sangue após 12 horas de jejum e 2 horas após café da manhã, com 400 kcal padronizado em relação à concentração de carboidrato, proteína e gordura. Os exames realizados foram: glicemia de jejum (GJ) e pós-prandial (GPP) (glicose oxidase); colesterol total (CT), colesterol HDL, triglicerídeos (TG) e ácido úrico (AU) por meio de reações colorimétricas com leitura pelo aparelho Cobas-Mira (Roche); hemoglobina glicosilada (HbA1c – HPLC com leitura pelo aparelho Merck Hitachi 9100 – VR 2,6% a 6,2%, e coeficientes de variação intra- e interensaio menor que 1%). A LDL foi calculada pela fórmula de Friedwald18. Foram calculados os índices CT/HDL e LDL/HDL.

Todos os pacientes coletaram 3 amostras de urina noturna em um período de 3 meses, sendo o intervalo mínimo entre elas de 2 semanas. Eles foram orientados a desprezar a urina das 20 horas e coletar toda a urina até 6 horas do dia seguinte para a determinação da taxa de excreção de albumina (EUA). O volume urinário foi aliquotado e estocado em frascos de vidro a 70º C até a análise. A concentração urinária de albumina foi determinada por radioimunoensaio (Diagnostic, California, Estados Unidos, sensibilidade de 0,3 µg/ml) com coeficientes de variação intra- e interensaio de 8,7% and 8,3%, respectivamente. Baseado na EUA, somente os pacientes com normoalbuminúria (EUA < 20 µg/min em duas das três amostras de urina)19 foram incluídos. Os pacientes com DM 1 foram submetidos a fundoscopia com dilatação das pupilas através de oftalmoscopia pelo mesmo oftalmologista.

Para a análise da suscetibilidade da LDL à oxidação in vitro, foram coletados 20 ml de sangue a vácuo em tubos com EDTA, os quais foram centrifugados a 4ºC por 20 minutos a 800 x g. Após a separação do plasma, esse foi imediatamente processado para o isolamento da LDL seguindo as etapas descritas: ajuste da densidade para 1,3 g/ml pela adição de brometo de potássio (4,5g de KBr a cada 9 ml de plasma), com posterior preparo dos tubos de ultracentrifugação com 20 ml de solução salina a 0,9% e os 9 ml de plasma anteriormente preparados. Esses tubos foram então centrifugados a 4ºC por 3 horas a 150.00 x g. Após a ultracentrifugação, a banda de lipoproteínas com densidade entre 1,019 e 1,063 g/ml, compatível com a da LDL, foi coletada20,21. Esse material teve sua concentração protéica dosada pelo método do Biureto22 e ajustada para 0,2 mg/ml. Ao material obtido foi adicionado sulfato de cobre (CuSO4) 20 mM na proporção de 1 µl para cada 1 ml de LDL, e essa solução foi colocada em banho a 37ºC por 24 horas.

A avaliação da LDL oxidada foi feita de forma indireta mediante o cálculo do coeficiente de oxidação dessa partícula23,24, o qual utiliza em sua fórmula a absorvância em 3 comprimentos de onda da luz UV, a saber: 205 nm, 232 nm e 280 nm. O coeficiente de oxidação foi calculado por meio da seguinte fórmula: Abs 205 – Abs 280 / Abs 232 – Abs 280, onde Abs 205 = leitura das duplas ligações dos ácidos graxos poliinsaturados dos fosfolipídeos do LDL; Abs 232 = leitura dos dieno conjugados; e Abs 280 = leitura da fração protéica da LDL.

Resultados

O controle glicêmico dos pacientes estudados estava inadequado e não houve diferença estatisticamente significativa nos níveis de colesterol total e frações dos dois grupos avaliados (tab. 2). Os níveis de ácido úrico e triglicerídeos foram menores nos diabéticos do que nos não-diabéticos [3,65 ± 0,98 mg/dl versus 4,69 ± 1,34 mg/dl; p < 0,001 e 69,00 (26,00-201,00) versus 87,50 (20,00-278,00); p = 0,027 respectivamente].

Os pacientes com DM 1 apresentaram média de coeficiente de oxidação basal da LDL semelhante à dos não-diabéticos. Entretanto, 3h após a adição de CuSO4 esse coeficiente foi menor no grupo dos diabéticos (6,98 ± 1,36 versus 7,91 ± 1,48; p = 0,007) (fig. 1; tab. 3).

De acordo com o Código de Minnesota, nenhum paciente apresentava doença coronariana possível ou definida ou infarto do miocárdio possível ou definido.

A análise da atividade da PAF-AH revelou haver maior atividade enzimática nos pacientes com DM 1 do que nos não-diabéticos (0,0150 ± 0,0051 versus 0,0116 ± 0,0041; p < 0,001 µmol/min/ml) (fig. 2).

Ao avaliarmos a correlação entre a atividade da PAF-AH e as demais variáveis nos pacientes com diabete tipo 1, observamos dados significativos como: idade (r = 0,328; p = 0,034), HbA1c (r = -0,319; p = 0,039), HDL (r = -0,348; p = 0,028) e LDL (r = 0,324; p = 0,041) (fig. 3). A regressão múltipla em stepwise utilizando a atividade da PAF-AH com variável dependente e a idade, sexo, IMC, HbA1c, HDL e LDL como variáveis independentes mostrou HDL e LDL como as variáveis independentes explicativas da variação da atividade desta enzima (step 1: r = 0,231, r2 = 0,053, beta = -0,231, p = 0,039; step 2: r = 0,316, r2 = 0,100, beta = - 0,252, p = 0,017).

Discussão

A literatura é controversa sobre a importância da concentração e atividade da PAF-AH no processo aterosclerótico27-33. Existem estudos demonstrando que pacientes com DM tipo 1 apresentam níveis diminuídos de PAF-AH, diferentemente do que ocorre nos pacientes com DM tipo 227,29. A literatura correlaciona ainda mudanças na atividade dessa enzima com várias doenças inflamatórias, incluindo a aterosclerose, e mostrando que essas alterações podem refletir aumento ou diminuição discretos da atividade enzimática. Contudo, ainda não está definido se essas alterações têm impacto na progressão, gravidade e resolução das doenças associadas com elas11.

Com relação aos pacientes com DM tipo 1, Cavallo-Perin e cols.28 não encontraram diferença na atividade da PAF-AH entre grupos de pacientes com DM tipo 1 sem microalbuminúria, com microalbuminúria e não-diabéticos. Apesar de não haver encontrado diferença na atividade da PAF-AH entre os grupos estudados, Cavallo-Perin e cols.28 e Nathan e cols.27 evidenciaram que os níveis de PAF estão elevados nos pacientes com DM tipo 1, e que isso estaria associado basicamente à maior produção dessa partícula. A divergência entre os dados desses autores e os nossos pode refletir as características da população estudada – sendo a nossa amostra de menor faixa etária, sem complicações microvasculares e com menor índice de massa corporal, bem como o tamanho da amostra – 42 diabéticos versus 7 pacientes com microalbuminúria e 7 sem microalbuminúria avaliados por Cavallo-Perin e cols. Essa maior atividade da PAF-AH nos pacientes com DM 1 pode representar uma tentativa de proteção contra os mecanismos fisiopatológicos induzidos pelos níveis aumentados de PAF encontrados nesses pacientes34 ou exercer ações pró-inflamatórias e pró-aterogênicas30,32,33.

A análise do coeficiente de oxidação evidenciou uma maior oxidação da LDL nos diabéticos tipo 1 após 3 horas de adição do agente oxidante do que nos não-diabéticos. Esses dados também são controversos na literatura35-38. As diferenças podem ser decorrentes do número de pacientes estudados e da diferença nas características clinicodemográficas desses pacientes. Não houve correlação entre a atividade da PAF-AH e o coeficiente de oxidação, não havendo aparentemente, na amostra estudada, relação entre a maior suscetibilidade da LDL dos pacientes com diabetes à oxidação e o nível de atividade da PAF-AH.

A avaliação das correlações da atividade da PAF-AH nos pacientes com diabete tipo 1 foi positiva com a idade e com a LDL, e negativa com HbA1c e HDL. Vários estudos demonstraram aumento da atividade dessa enzima com a idade39-42. A causa desse aumento talvez esteja relacionada com a elevação dos níveis de LDL que acompanha o envelhecimento43, já que há uma significativa correlação entre os níveis de LDL e a atividade da PAF-AH42,44-47. Os dados na literatura confirmam a correlação da atividade desta enzima com o HDL e LDL encontradas em nossa amostra16,17.

Nosso trabalho apresentou limitações no que diz respeito ao estudo de outras variáveis associadas com a doença aterosclerótica, como as proteínas de fase aguda, e a realização de outros métodos para avaliação dessa enfermidade em pacientes com DM tipo 1, como a medida do complexo íntima-média da carótida comum5.

Considerando-se a multifatoriedade da doença aterosclerótica e a controvérsia a respeito da real função da PAF-AH, a maior atividade dessa enzima nos pacientes com DM 1 poderia refletir tanto um mecanismo protetor como um marcador de maior risco de doença aterosclerótica. Estudos prospectivos são necessários para definir a associação da PAF-AH com o risco cardiovascular em pacientes com diabete tipo 1.

Potencial Conflito de Interesses

Declaro não haver conflitos de interesses pertinentes.

Simone Henriques de Castro

Rua Maestro Vila Lobos 01/202

20260-220 – Rio de Janeiro, RJ

E-mail:

sh.castro@uol.com.br

Artigo recebido em 15/12/05; artigo revisado recebido em 16/03/06; aceito em 11/04/06.

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Kim BS, LaBella FS. Comparison of analytical methods for monitoring autoxidation profiles of authentic lipids. J Lipid Res. 1987; 28: 1110-7. Comparison of analytical methods for monitoring autoxidation profiles of authentic lipids J Lipid Res. 1987 1110 7 28 Kim BS LaBella FS 24. Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radical Biol Med. 1992; 13: 341-90. The role of lipid peroxidation and antioxidants in oxidative modification of LDL Free Radical Biol Med. 1992 341 90 13 Esterbauer H Gebicki J Puhl H Jurgens G 25. Rose GA, Blackburn H, Gillum RF, Prineas RJ. Cardiovascular survey methods. 2nd ed. Geneva: World Health Organization; 1982. Cardiovascular survey methods 1982 Rose GA Blackburn H Gillum RF Prineas RJ. 26. Howard BV, Lee ET, Cowan LD, Fabsitz RR, Howard WJ, Oopik AJ, et al. Coronary heart disease prevalence and its relation to heart disease in American Indians: the Strong Heart Study. Am J Epidemiol. 1995; 142: 254-68. Coronary heart disease prevalence and its relation to heart disease in American Indians: the Strong Heart Study Am J Epidemiol. 1995 254 68 142 Howard BV Lee ET Cowan LD Fabsitz RR Howard WJ Oopik AJ 27. Nathan N, Denizot Y, Huc MC, Claverie C, Laubie B, Benveniste J, et al. Elevated levels of paf-acether in blodd of patients with type 1 diabetes mellitus. Diabete Metab. 1992; 18: 59-62. Elevated levels of paf-acether in blodd of patients with type 1 diabetes mellitus Diabete Metab. 1992 59 62 18 Nathan N Denizot Y Huc MC Claverie C Laubie B Benveniste J 28. Cavallo-Perin P, Lupia E, Gruden G, Olivetti C, De Martino A, Cassader M, et al. Increased blood levels of platelet-activanting factor in insulin-dependent diabetic patients with microalbuminuria. Nephrol Dial Transplant. 2000; 15: 994-9. Increased blood levels of platelet-activanting factor in insulin-dependent diabetic patients with microalbuminuria Nephrol Dial Transplant. 2000 994 9 15 Cavallo-Perin P Lupia E Gruden G Olivetti C De Martino A Cassader M 29. Memon RA, Saeed SA, Jabbar A, Jafri A, Gilani AH, Saleem S, et al. Altered platelet activating factor metabolism in insulin dependent diabetes mellitus. J Pak Med Assoc. 1995; 45: 122-5. Altered platelet activating factor metabolism in insulin dependent diabetes mellitus J Pak Med Assoc. 1995 122 5 45 Memon RA Saeed SA Jabbar A Jafri A Gilani AH Saleem S 30. Macphee CH, Moores KE, Boyd HF, Dhanak D, Ife RJ, Leach CA, et al. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor. Biochem J. 1999; 338: 479-89. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor Biochem J. 1999 479 89 338 Macphee CH Moores KE Boyd HF Dhanak D Ife RJ Leach CA 31. Itabe H. Oxidized phospholipids as a new landmark in atherosclerosis. Prog Lipid Res. 1998; 37: 181-207. Oxidized phospholipids as a new landmark in atherosclerosis Prog Lipid Res. 1998 181 207 37 Itabe H 32. Karabina SA, Elisaf M, Bairaktari E, Tzallas C, Sianopoulos KC, Tselepis AD. Increased activity of platelet-activating factor acetylhydrolase in low-density lipoprotein subfractions induces enhanced lysophosphatidylcholine production during oxidation in patients with heterozygous familial hypercholesterolaemia. Eur J Clin Invest. 1997; 27: 595-602. Increased activity of platelet-activating factor acetylhydrolase in low-density lipoprotein subfractions induces enhanced lysophosphatidylcholine production during oxidation in patients with heterozygous familial hypercholesterolaemia Eur J Clin Invest. 1997 595 602 27 Karabina SA Elisaf M Bairaktari E Tzallas C Sianopoulos KC Tselepis AD 33. Murakami M, Kudo I. Phospholipase A2. J Biochem. 2002; 131: 285-92. Phospholipase A2 J Biochem. 2002 285 92 131 Murakami M Kudo I 34. Howard KM, Miller JE, Miwa M, Olson MS. Cell-specific regulation of expression of plasma-type platelet-activating factor acetylhydrolase in the liver. J Biol Chem. 1997; 272: 27543-8. Cell-specific regulation of expression of plasma-type platelet-activating factor acetylhydrolase in the liver J Biol Chem. 1997 27543 8 272 Howard KM Miller JE Miwa M Olson MS 35. Tsai EC, Hirsch IB, Brunzell JD, Chait A. Reduced plasma peroxyl radical trpping capacity and increased susceptibility of LDL to oxidation in poorly controlled IDDM. Diabetes. 1994; 43: 1010-4. Reduced plasma peroxyl radical trpping capacity and increased susceptibility of LDL to oxidation in poorly controlled IDDM Diabetes. 1994 1010 4 43 Tsai EC Hirsch IB Brunzell JD Chait A 36. Liguori A, Abete P, Hayden JM, Cacciatore F, Rengo F, Ambrosio G, et al. Effect of glycaemic control and age on low-density lipoprotein susceptibility to oxidation in diabetes mellitus type 1. Eur Heart J. 2001; 22: 2075-84. Effect of glycaemic control and age on low-density lipoprotein susceptibility to oxidation in diabetes mellitus type 1 Eur Heart J. 2001 2075 84 22 Liguori A Abete P Hayden JM Cacciatore F Rengo F Ambrosio G 37. Jenkins AJ, Klein RL, Chassereau CN, Hermayer KL, Lopes-Virella MF. LDL from patients with well-controlled IDDM is not more susceptible to in vitro oxidation. Diabetes. 1996; 45: 762-7. LDL from patients with well-controlled IDDM is not more susceptible to in vitro oxidation Diabetes. 1996 762 7 45 Jenkins AJ Klein RL Chassereau CN Hermayer KL Lopes-Virella MF 38. Castro SH, Castro-Faria-Neto HC, Clemente ELS, Gomes MB. Avaliação da suscetibilidade do LDL de pacientes com diabetes tipo 1 à oxidação in vitro e sua relação com o controle glicêmico. Arq Bras Endocrinol Metab. 2004; 48: 513-7. Avaliação da suscetibilidade do LDL de pacientes com diabetes tipo 1 à oxidação in vitro e sua relação com o controle glicêmico Arq Bras Endocrinol Metab. 2004 513 7 48 Castro SH Castro-Faria-Neto HC Clemente ELS Gomes MB 39. Satoh K, Imaizumi T, Yoshida H, Kawamura Y, Takamatsu S, Takamatsu M, et al. Platelet-activating factor acetylhydrolase in plasma lipoproteins of healthy men and women. Clin Chim Acta. 1991; 202: 95-103. Platelet-activating factor acetylhydrolase in plasma lipoproteins of healthy men and women Clin Chim Acta. 1991 95 103 202 Satoh K Imaizumi T Yoshida H Kawamura Y Takamatsu S Takamatsu M 40. Kosaka T, Yamagushi M, Miyanaga K, Mizumo K. Serum platelet-activating factor acetylhydrolase (PAF-AH) activity in more than 3000 healthy Japanese. Clin Chim Acta. 2001; 312: 179-83. Serum platelet-activating factor acetylhydrolase (PAF-AH) activity in more than 3000 healthy Japanese Clin Chim Acta. 2001 179 83 312 Kosaka T Yamagushi M Miyanaga K Mizumo K 41. Stafforini DM, Numao T, Tsodikov A, Vaitkus D, Fukuda T, Watanabe N, et al. Deficiency of platelet-activating factor acetylhydrolase is a severity factor for asthma. J Clin Invest. 1999; 103: 989-97. Deficiency of platelet-activating factor acetylhydrolase is a severity factor for asthma J Clin Invest. 1999 989 97 103 Stafforini DM Numao T Tsodikov A Vaitkus D Fukuda T Watanabe N 42. Guerra R, Zhao B, Mooser V, Stafforini DM, Johnston JM, Cohen JC. Determinants of plasma platelet-activating factor acetylhydrolase: heritability and relationship to plasma lipoproteins. J Lipid Res. 1997; 38: 2281-8. Determinants of plasma platelet-activating factor acetylhydrolase: heritability and relationship to plasma lipoproteins J Lipid Res. 1997 2281 8 38 Guerra R Zhao B Mooser V Stafforini DM Johnston JM Cohen JC 43. Mormando RM. Lipid levels: applying the second National Cholesterol Education Program report to geriatric medicine. Geriatrics. 2000; 55: 48-53. Lipid levels: applying the second National Cholesterol Education Program report to geriatric medicine Geriatrics. 2000 48 53 55 Mormando RM 44. Tew DG, Southan SQ, Rice MP, Lawrence MP, Li H, Boyd HF, et al. Purification, properties, sequencing, and cloning of a lipoprotein, associated, serine-dependent phospholipase involved in the oxidative modification of low-density lipoproteins. Arterioscler Thromb Vasc Biol. 1996; 16: 591-9. Purification, properties, sequencing, and cloning of a lipoprotein, associated, serine-dependent phospholipase involved in the oxidative modification of low-density lipoproteins Arterioscler Thromb Vasc Biol. 1996 591 9 16 Tew DG Southan SQ Rice MP Lawrence MP Li H Boyd HF 45. Satoh K, Imaizumi TA, Kawamura Y, Yoshida H, Takamatsu S, Takamatsu M. Increased activity of the platelet-activating factor acetylhydrolase in plasma low density lipoprotein from patients with essential hypertension. Prostaglandins. 1989; 37: 673-82. Increased activity of the platelet-activating factor acetylhydrolase in plasma low density lipoprotein from patients with essential hypertension Prostaglandins. 1989 673 82 37 Satoh K Imaizumi TA Kawamura Y Yoshida H Takamatsu S Takamatsu M 46. Satoh K, Yoshida H, Iamazumi TA, Takamatsu S, Mizuno S. Platelet-activating factor acetylhydrolase in plasma lipoproteins from patients with ischemic stroke. Stroke. 1992; 23: 1090-2. Platelet-activating factor acetylhydrolase in plasma lipoproteins from patients with ischemic stroke Stroke. 1992 1090 2 23 Satoh K Yoshida H Iamazumi TA Takamatsu S Mizuno S 47. Graham RM, Stephens CJ, Sturm MJ, Taylor RR. Plasma platelet-activating factor degradation in patients with severe coronary artery disease. Clin Sci. 1992; 82: 535-41. Plasma platelet-activating factor degradation in patients with severe coronary artery disease Clin Sci. 1992 535 41 82 Graham RM Stephens CJ Sturm MJ Taylor RR Original Articles Platelet-activating factor acetylhydrolase (PAF-AH) activity in patients with type 1 diabetes mellitus

OBJECTIVE: To evaluate platelet-activating factor acetylhydrolase (PAF-AH) activity and its relationship with clinical and demographic variables, metabolic control, apolipoprotein A and B levels and the susceptibility of low-density lipoprotein (LDL) to in vitro oxidation in patients with type 1 diabetes mellitus (DM 1). METHODS: Forty two patients with DM 1 (27 females) and 48 control subjects (16 females) matched for gender, age and body mass index (BMI) were evaluated. The following tests were performed: fast plasma glucose (FG) and postprandial plasma glucose (PPG), lipid profile, uric acid (UA), glycosylated hemoglobin (HbA1c), and low-density lipoprotein (LDL) oxidation rate using colorimetric assay. The PAF-AH activity was analyzed using colorimetric assay (Cayman Chemical). RESULTS: The analysis of PAF-AH activity showed a higher enzyme activity in patients with DM 1 than in control subjects (0.0150 ± 0.0051 vs. 0.0116 ± 0.0041; p < 0.001). In patients with DM 1, a direct correlation between PAF-AH activity and age and LDL, and an inverse correlation between PAF-AH and HbA1c and high-density lipoprotein (HDL) were found. CONCLUSION: In the sample studied, PAF-AH showed a higher activity in patients with DM 1, a factor that may be related to the higher risk of developing cardiovascular diseases observed in these patients. Further studies are necessary to evaluate the real participation of this enzyme in the risk of development of atherosclerotic diseases in patients with DM 1.

 PAF-acetylhydrolase type 1 diabetes atherosclerotic diseases

ORIGINAL ARTICLE

Platelet-activating factor acetylhydrolase (PAF-AH) activity in patients with type 1 diabetes mellitus

Simone Henriques de CastroI; Hugo Caire de Castro Faria NetoII; Marilia de Brito GomesI

IServiço de Diabetes do Hospital Pedro Ernesto - UERJ

IILaboratório de Imunofarmacologia da FIOCRUZ – Rio de Janeiro, RJ - Brazil

Mailing Address

SUMMARY

OBJECTIVE: To evaluate platelet-activating factor acetylhydrolase (PAF-AH) activity and its relationship with clinical and demographic variables, metabolic control, apolipoprotein A and B levels and the susceptibility of low-density lipoprotein (LDL) to in vitro oxidation in patients with type 1 diabetes mellitus (DM 1).

METHODS: Forty two patients with DM 1 (27 females) and 48 control subjects (16 females) matched for gender, age and body mass index (BMI) were evaluated. The following tests were performed: fast plasma glucose (FG) and postprandial plasma glucose (PPG), lipid profile, uric acid (UA), glycosylated hemoglobin (HbA1c), and low-density lipoprotein (LDL) oxidation rate using colorimetric assay. The PAF-AH activity was analyzed using colorimetric assay (Cayman Chemical).

RESULTS: The analysis of PAF-AH activity showed a higher enzyme activity in patients with DM 1 than in control subjects (0.0150 ± 0.0051 vs. 0.0116 ± 0.0041; p < 0.001). In patients with DM 1, a direct correlation between PAF-AH activity and age and LDL, and an inverse correlation between PAF-AH and HbA1c and high-density lipoprotein (HDL) were found.

CONCLUSION: In the sample studied, PAF-AH showed a higher activity in patients with DM 1, a factor that may be related to the higher risk of developing cardiovascular diseases observed in these patients. Further studies are necessary to evaluate the real participation of this enzyme in the risk of development of atherosclerotic diseases in patients with DM 1.

Key words: PAF-acetylhydrolase, type 1 diabetes, atherosclerotic diseases.

Introduction

Cardiovascular disease is the major cause of morbidity and mortality in type 1 diabetes mellitus (DM 1), accounting for 44% of all deaths in patients with DM 11,2. Several methods have been used to evaluate the presence of atherosclerotic disease in patients with DM 1, showing that coronary heart disease may already be present in young patients3-5.

The oxidation process is considered an important component in the initial phase and progression of the atherosclerotic disease. The PAH-AH enzyme activity may be protective in this process, since the association of this enzyme with LDL and HDL prevents the in vitro formation of minimally oxidized LDL molecules and its internalization by macrophages6-8.

Recently, atherosclerosis has been considered a chronic inflammatory disease. The inflammatory process contributes significantly to the initiation, progression and rupture of atherosclerotic lipid-rich plaques9,10. The cells responsible for the tissue damage associated with inflammation are recruited and activated by a series of mediators, and the platelet activating factor (PAF) is a phospholipid involved in this process. Phospholipids with a pro-inflammatory activity may be generated through oxidative fragmentation of the phosphatidylcholine fatty acid, which are known as PAF-like phospholipids. The biological activity of PAF and PAF-like phospholipids is attenuated by phospholipase A2 enzymes (PLA2)11.

Studies have shown that PLA2 is involved in the inflammatory process and in atherogenesis, and that both the secretory type II PLA2 and the lipoprotein-associated PLA2 (PAF-acetylhydrolase/ PAF-AH) may be related to the risk of coronary heart disease12,13. Theoretically, this enzyme may promote atherogenesis if the products it releases from LDL phospholipids have a deleterious effect on arterial walls14, or it may be protective if, when hydrolyzing PAF, it reduces the inflammatory and thrombotic tendency of the blood15. Plasma PAF-AH is mainly associated with LDL (preferentially subfraction 5); however, a small amount (< 20% of the enzyme activity) is associated with HDL (preferentially subfraction 1)16,17.

The objective of the present study is to evaluate PAF-AH activity and its relationship with clinical and demographic variables, metabolic control, apolipoproteins A and B levels, and the susceptibility of low-density lipoprotein (LDL) to in vitro oxidation in patients with DM 1, to observe the interference of variables classically related to a higher risk of cardiovascular disease in patients with diabetes and this new factor studied.

Methods

Patients - Fifty patients with DM 1 followed regularly in the outpatient clinic of diabetes of the Hospital Universitário Pedro Ernesto (HUPE) – UERJ, and 48 control subjects matched for gender, age, and body mass index (BMI) were evaluated after giving their written consent previously approved by the ethics committee of HUPE. Clinical and demographic characteristics of the two groups at the moment of sample collection for tests are shown in Table 1.

Exclusion criteria for diabetic patients were: smoking, alcohol consumption, systemic infection and/or use of drugs that could affect the results of determination of the susceptibility of LDL to oxidation, such as ACE inhibitors, ferrous sulphate, and vitamin and mineral supplements containing vitamin C and E, Zn, Se, Fe and Cu, and history of previous cardiovascular events, diabetic retinopathy and nephropathy, and clinical diabetic neuropathy. In the control group, these criteria also included the presence of direct family members with diabetes mellitus.

In accordance with the exclusion criteria, eight patients with type 1 diabetes mellitus were excluded from the study because they had incipient diabetic nephropathy and/or diabetic retinopathy.

The participants had their blood drawn after a 12-hour fast and 2 hours after a 400-kcal breakfast standardized in relation to carbohydrate, protein and fat content. The following tests were performed: fast plasma glucose (FG), postprandial plasma glucose (PPG) (glucose oxidase); total cholesterol (TC), HDL-cholesterol, triglycerides (TG) and uric acid (UA) using colorimetric assay measured on a Cobas-Mira analyzer (Roche); glycosylated hemoglobin (HbA1c – HPLC measured on a Merck Hitachi 9100 analyzer – RV 2.6 to 6.2%, and intra and interassay variation coefficient lower than 1%). LDL was calculated using the Friedewald formula18. The TC/HDL and LDL/HDL ratios were calculated.

All patients had three samples of overnight urine collected in a period of three months, with a minimum two-week interval between each. They were instructed to discard urine at 8p.m. and to collect all the urine until 6 a.m. the next day to determine the urinary albumin excretion rate (UAE). The urine volume was aliquoted and stored in glass flasks at 70º C for further analysis. Urinary albumin concentration was determined using radioimmunoassay (Diagnostic, California, United States, 0.3-µg/mL sensitivity) with intra and interassay variation coefficient of 8.7% and 8.3%, respectively. With base on UAE, only the patients with normal urinary albumin (UAE < 20 µg/min in two out of three urine samples)19 were included. The patients with DM 1 underwent dilated fundoscopy which was performed by the same ophthalmologist.

For the analysis of the susceptibility of LDL to in vitro oxidation, 20 ml of blood were collected in EDTA vacuum tubes which were centrifuged at 4ºC for 20 minutes at 800 x g. The plasma was separated and immediately processed for LDL isolation, in accordance with the following phases: density adjustment at 1.3 g/ml by adding potassium bromide (4.5g of KBr for each 9 ml of plasma) with further preparation of ultracentrifugation tubes with 20 ml of 0.9% saline solution and the 9 ml of plasma previously prepared. These tubes were centrifuged at 4ºC for three hours at 150.00 x g. After centrifugation, the lipoprotein band with density between 1.019 and 1.063 g/ml, compatible with that of LDL, was collected20,21. Protein concentration of this material was measured using the Biuret method, and adjusted for 0.2 mg/ml. Copper sulphate (CuSO4) 20 mM was added to the material obtained in the proportion of 1 µl for each 1 ml of LDL and this solution was placed in a water bath at 37ºC for 24 hours.

Oxidized LDL was indirectly assessed using the calculation of its oxidation rate 23,24, in whose formula absorbance at 3 different UV light wavelengths is used, namely: 205 nm, 232 nm and 280 nm. The oxidation rate was calculated using the formula: Abs 205 – Abs 280 / Abs 232 – Abs 280, where Abs 205 = reading of double bonds of polyunsaturated fatty acids of the LDL phospholipids; Abs 232 = reading of conjugated dienes; and Abs 280 = reading of LDL protein fraction.

The readings were taken in a Shimadzu spectrophotometer at the wavelengths mentioned above at the timepoints pre-addition, 1h, 3h, 6h and 24h post-addition of CuSO4 to assess LDL susceptibility to the oxidative process.

A resting 12-lead electrocardiogram (ECG) was performed and classified by two doctors according to the Minnesota protocol25 in a double-blind manner. Based on this coding, the patients were defined in the following categories: without coronary heart disease, possible or definite coronary heart disease, or possible or definite myocardial infaction26.

PAF-AH activity was analyzed using the colorimetric method with a commercial kit according to instructions provided by the manufacturer (Cayman Chemical). Ten microliters of serum samples frozen in a freezer at - 70º C distributed in a 90-well plate were used. In each well, 5 µl of buffer solution, 10 µl of developing substance and 200 µl of substrate (2-thio PAF) were added. The kit material is enough to analyze 60 samples, including the negative and positive controls. Absorbance readings at 405 nm were further taken at timepoints 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 minutes. PAF-AH activity is expressed in µmol/min/ml and was calculated using the following formula:

PAF-AH activity = DA405/min/10 mM-1 * (0.225 ml/0.01 ml) * sample dilution, where DA405/min = [ DA405 (timepoint 2) - DA405 (timepoint 1)] / [timepoint 2 (min) – timepoint 1 (min)].

Statistical analysis - The results of all variables with a normal distribution analyzed were expressed as mean ± standard deviation, and those with a non-Gaussian distribution were expressed as median (minimum – maximum). The non-paired Student’s t test was used for the comparison of the means of variables with a normal distribution between the two groups analyzed, and the Mann-Whitney test was used for those with a non-Gaussian distribution. The Pearson and Spearman correlation tests, according to variable distribution, were used to test the correlations between PAF-AH activity and metabolic control variables and susceptibility of LDL to in vitro oxidation.

Results

The glycemic control of the patients studied was inadequate and no statistically significant difference of total cholesterol, and LDL and HDL levels was observed between the two groups assessed (Tab. 2). Uric acid and triglyceride levels were lower in diabetics than in control subjects [3.65 ± 0.98 mg/dl vs. 4.69 ± 1.34 mg/dl; p < 0.001 and 69.00 (26.00-201.00) vs. 87.50 (20.00-278.00); p = 0.027, respectively].

Patients with DM 1 had a mean baseline LDL oxidation rate similar to that of control subjects. However, 3 hours after CuSO4 addition this rate was lower in the diabetic group (6.98 ± 1.36 vs. 7.91 ± 1.48; p = 0.007) (Fig. 1; Tab. 3).

According to the Minnesota Coding, no patient had possible or definite coronary heart disease, or possible or definite myocardial infarction.

PAF-AH activity analysis showed a higher enzyme activity in patients with DM 1 than in control subjects (0.0150 ± 0.0051 vs. 0.0116 ± 0.0041; p < 0.001 µmol/min/ml) (Fig. 2).

When the correlation between PAF-AH activity and the other variables was analyzed in patients with type 1 diabetes, we observed significant data with: age (r=0.328; p=0.034), HbA1c (r=-0.319; p=0.039), HDL (r=-0.348; p=0.028) and LDL (r=0.324; p=0.041) (Fig. 3). The stepwise multiple regression using PAF-AH activity as a dependent variable and age, gender, BMI, HbA1c, HDL and LDL as independent variables showed HDL and LDL as independent explanatory variables of changes in PAF-AH activity (step 1: r = 0.231, r2 = 0.053, beta = - 0.231, p = 0.039; step 2: r = 0.316, r2 = 0.100, beta = - 0.252, p = 0.017).

Discussion

There is controversy in the literature regarding the importance of the PAF-AH activity and concentration in the atherosclerotic process27-33. Some studies demonstrate that patients with DM 1 have low levels of PAF-AH, unlike patients with DM 227,29. The literature also correlates changes in PAF-AH activity with several inflammatory diseases including atherosclerosis, and shows that these changes may reflect a slight increase or decrease in the enzyme activity. However, it remains unclear whether these changes have an impact on the progression, severity and resolution of the diseases associated with them11.

In regard to patients with DM 1, Cavallo-Perin et al28 did not find any difference in PAF-AH activity among groups of patients with DM 1 without microalbuminuria, with microalbuminuria, and control subjects. Although they did not find any difference in PAF-AH activity among the groups studied, Cavallo-Perin et al28 and Nathan et al27 demonstrated that PAF levels are increased in patients with DM 1, and that this would be basically associated with a higher production of this particle. The disagreement between data found by these authors and our own may reflect the characteristics of the population studied – our sample had a lower age range, no microvascular complications and lower BMI, as well as the sample size – 42 diabetics versus 7 patients with microalbuminuria and 7 without microalbuminuria assessed by Cavallo-Perin et al. The higher PAF-AH activity in patients with DM 1 may represent an attempt at protection against pathophysiological mechanisms induced by increased levels of PAF found in these patients34 or may exert pro-inflammatory and pro-atherogenic effects30,32,33.

The analysis of oxidation rate evidenced a higher LDL oxidation in patients with type 1 diabetes 3 hours after the addition of the oxidative agent than in control subjects. These data are also controversial in the literature35-38. The differences may result from the number of patients studied and from differences in their clinical and demographic characteristics. No correlation between PAF-AH activity and oxidation rate was observed, and apparently no relation between the higher susceptibility of LDL to oxidation in patients with diabetes and PAF-AH activity levels in the sample studied was observed either.

The evaluation of correlations of PAF-AH activity in patients with type 1 diabetes was positive with age and LDL, and negative with HbA1c and HDL. Several studies demonstrated an increase in the activity of this enzyme with age39-42. The cause of this increase is perhaps related to the elevation of LDL levels that accompanies aging43, since a significant correlation between LDL levels and PAF-AH activity occurs42,44-47. Data from the literature corroborate the correlation of PAF-AH activity with HDL and LDL found in our sample16,17.

Our study had limitations regarding the analysis of other variables associated with atherosclerotic disease, such as acute phase proteins, and the use of other methods to assess this disease in patients with DM 1, such as the measurement of the common carotid artery intima-media thickness5.

Considering the multiple factors involved in atherosclerotic disease and the controversy on the real function of PAF-AH, the higher activity of this enzyme in patients with DM 1 could reflect both a protective mechanism and a marker of a higher risk for atherosclerotic disease. Further prospective studies are necessary to define the association of PAF-AH with cardiovascular risk in patients with type 1 diabetes.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

References

Simone Henriques de Castro

Rua Maestro Vila Lobos 01/202

20260-220 – Rio de Janeiro, RJ - Brazil

E-mail:

sh.castro@uol.com.br

Manuscript received December 15, 2005; revised manuscript received March 16, 2006; accepted April 11, 2006.