Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC 9h5qm7RMFhsD8ryGTbhYfTs S0066-782X2023001100305 00305 10.36660/abc.20230078 Artigo Original Os Ácidos Graxos Poli-insaturados Ômega-6 e Ômega-3 Presentes nas Hemácias Exercem uma Influência Distinta sobre o Tamanho das Partículas de LDL e suas Alterações Estruturais 0000-0002-2962-5341 Gonçalinho Gustavo Henrique Ferreira Obtenção de dados Análise e interpretação dos dados Análise estatística Redação do manuscrito e Revisão crítica do manuscrito quanto ao conteúdo intelectual importante 1 Sampaio Geni Rodrigues Obtenção de dados Análise e interpretação dos dados 1 0000-0001-7955-656X Soares-Freitas Rosana Aparecida Manólio Obtenção de dados Análise e interpretação dos dados 1 Damasceno Nágila Raquel Teixeira Concepção e desenho da pesquisa e Obtenção de financiamento Obtenção de dados 1 Departamento de Nutrição Faculdade de Saúde Pública Universidade de São Paulo São Paulo SP Brasil Departamento de Nutrição – Faculdade de Saúde Pública – Universidade de São Paulo , São Paulo , SP – Brasil Correspondência: Gustavo Henrique Ferreira Gonçalinho • Universidade de São Paulo – Av. Dr. Enéas Carvalho de Aguiar, 44. CEP 05403-000, Cerqueira César, São Paulo, SP – Brasil E-mail: ghfg93@gmail.com

Editor responsável pela revisão: Gláucia Maria Moraes de Oliveira

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 20 11 2023 11 2023 120 11 e20230078 07 02 2023 05 07 2023 16 08 2023 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Figura Central

: Os Ácidos Graxos Poli-insaturados Ômega-6 e Ômega-3 Presentes nas Hemácias Exercem uma Influência Distinta sobre o Tamanho das Partículas de LDL e suas Alterações Estruturais

Resumo Fundamento

Embora os ácidos graxos poli-insaturados ômega-3 e ômega-6 (AGPIs n-3 e n-6) tenham efeitos bem conhecidos sobre os fatores de risco de doenças cardiovasculares (DCV), ainda existe um conhecimento limitado sobre como eles afetam os indicadores de qualidade da LDL.

 Objetivo

Avaliar as associações dos AGPIs n-3 e n-6 de hemácias com o tamanho da partícula da LDL, LDL-c pequena e densa (sdLDL-c) e com LDL eletronegativa [LDL(-)] em adultos com fatores de risco para DCV.

 Métodos

Estudo transversal com 335 homens e mulheres de 30 a 74 anos com, pelo menos, um fator de risco cardiovascular. Foram realizadas análises de parâmetros bioquímicos, como glicose, insulina, HbA1c, proteína C reativa (PCR), perfil lipídico, subfrações de lipoproteínas, partícula eletronegativa de LDL [LDL(-)] e seu autoanticorpo, e os AGPIs n-3 e n- 6 de hemácias. Os testes t independente/teste de Mann-Whitney, ANOVA unidirecional/teste de Kruskal-Wallis e regressões lineares múltiplas foram aplicados. Todos os testes foram bilaterais e um valor de p inferior a 0,05 foi considerado estatisticamente significativo.

 Resultados

A relação n-6/n-3 de hemácias foi associada ao aumento dos níveis de LDL(-) (β = 4,064; IC de 95% = 1,381 – 6,748) e sdLDL-c (β = 1,905; IC de 95% = 0,863 – 2,947), e redução do tamanho das partículas de LDL (β = -1,032; IC de 95% = -1,585 − -0,478). Individualmente, os AGPIs n-6 e n-3 apresentaram associações opostas com esses parâmetros, realçando os efeitos protetores do n-3 e evidenciando os possíveis efeitos adversos do n-6 na qualidade das partículas de LDL.

 Conclusão

O AGPI n-6, presente nas hemácias, foi associado ao aumento do risco cardiometabólico e à aterogenicidade das partículas de LDL, enquanto o AGPI n-3 foi associado a melhores parâmetros cardiometabólicos e à qualidade das partículas de LDL.

 Ácidos Graxos Insaturados Receptores de LDL Oxidado Lipoproteínas LDL FAPESP 2016/24531-3 FAPESP 2011/12523-2 CAPES 88882.330835/2019-01 Fontes de financiamento: O presente estudo foi financiado pela FAPESP nº2016/24531-3, FAPESP nº 2011/12523-2 e CAPES nº88882.330835/2019-01. Introdução

As terapias redutoras de colesterol representam a primeira linha de prevenção de complicações ateroscleróticas devido à forte relação do LDL-c com DCVs. 1 Apesar da LDL-c ser um excelente preditor de DCV, esta medida não reflete a qualidade das partículas de LDL. 1 Biomarcadores que refletem a qualidade das partículas de LDL, como LDL-c pequena e densa (sdLDL-c), LDL eletronegativa [LDL(-)], e o tamanho da partícula de LDL, são subfrações mais suscetíveis à oxidação e à progressão da aterosclerose e apresentam melhor desempenho na predição do risco de DCVs quando comparado à sua LDL-c equivalente tradicional. 1 , 2

Os ácidos graxos poli-insaturados ômega-3 (AGPI n-3) demonstraram efeitos benéficos sobre os parâmetros de saúde cardiometabólicos, 3 e outras evidências apontam para a influência do AGPI n-3 na composição lipídica das lipoproteínas e níveis de subclasses. 4 Os efeitos do AGPI n-3 dependerão, no entanto, do status do AGPI n-6 devido ao alto teor da família n-6 nas dietas ocidentais. 3 O ácido linoleico (LA; C18:2 n-6) é o AGPI n-6 mais comum na dieta, sendo convertido em ácido araquidônico (AA; C20:4 n-6), uma importante variedade precursora de metabólitos pró-inflamatórios envolvidos na fisiopatologia das DCVs. 3 , 5 Entretanto, vale ressaltar que nem todos os subprodutos do ácido araquidônico (AA) desencadeiam uma resposta pró-inflamatória, 6 e evidências indicam que os AGPIs n-6 circulantes e presentes na dieta estão correlacionados com um menor risco de ocorrência de doença arterial coronariana (DAC), acidente vascular cerebral isquêmico e mortalidade relacionada a DCVs. 7 - 10 Por outro lado, o LA dietético incorporado em todas as lipoproteínas pode aumentar a sua suscetibilidade à oxidação, associada à gravidade da aterosclerose. 11 - 15

Assim, nosso estudo avaliou a associação dos AGPIs n-3 e n-6 presente nas hemácias com o tamanho da partícula de LDL, LDL-c pequena e densa (sdLDL-c) e LDL eletronegativa [LDL(-)] em adultos com fatores de risco para DCV.

 Métodos Desenho do estudo e participantes

Estudo transversal que utilizou os dados iniciais do estudo CARDIONUTRI (Registro Brasileiro de Ensaios Clínicos - ReBEC: RBR-2vfhfv) que incluiu 335 participantes com os seguintes critérios de elegibilidade: indivíduos de 30 a 74 anos sem histórico de doença cardiovascular. Indivíduos com doenças graves agudas ou crônicas, doenças infecciosas, gestantes e/ou lactantes foram excluídos. Todos os participantes foram selecionados no Hospital Universitário da Universidade de São Paulo (HU-USP), e todos os procedimentos seguiram as normas estabelecidas pelo Comitê de Ética em Pesquisa do HU-USP (CAAE n.º 0063.0.207.198-11).

 Avaliação clínica e nutricional

As condições clínicas atuais e prévias foram investigadas por meio de questionários. O exame físico incluiu avaliação do índice de massa corporal (IMC), circunferência da cintura e pressão arterial. A avaliação da dieta foi obtida por meio de três recordatórios de 24 horas e realizada no software Food Processor (ESHA Research, OR, EUA), com posterior ajuste energético.

 Medições bioquímicas

O sangue foi coletado após jejum de 12 horas; as hemácias foram separadas do plasma por centrifugação (3.000× g por 5 minutos a 4 ºC) e subsequentemente congeladas a -80 ºC. O nível plasmático de colesterol total, HDL-c, triglicerídeos (Labtest Diagnostica S.A., MG, Brasil), apoA-I, apoB (Wako Chemicals USA Inc., Richmond, VA, EUA), glicose (Labtest Diagnostica S.A., MG, Brasil), insulina (Life Technologies, Grand Island, NY, EUA) e proteína C reativa de alta sensibilidade (PCR-us) (Diagnostic System Laboratories, Inc., Webster, Texas, EUA) foram medidos por kits comerciais. O nível de LDL-c foi calculado pela equação de Friedewald. 16

 Análise de subfrações de lipoproteínas

O tamanho das lipoproteínas (HDL e LDL) foi analisado utilizando o LipoprintSystem TM (Quantimetrix, Redondo Beach, CA), que se baseia na separação e quantificação de subfrações de lipoproteínas por gel de poliacrilamida não desnaturante. Sob análise eletroforética, as subfrações foram somadas para determinar a área relativa de cada subunidade de lipoproteína (expressa como porcentagem de cada subfração). Em seguida, essa relação foi multiplicada pelo nível plasmático de colesterol total para determinar a concentração de colesterol em cada subfração de LDL, e pelo colesterol em HDL para quantificar a concentração de colesterol em cada subfração de HDL. Com base nos resultados, sete subfrações de LDL foram identificadas, nas quais as subfrações LDL-1 e LDL-2 foram classificadas como grandes, e LDL-3 a LDL-7 como partículas menores e mais densas (sdLDL-c). Para a HDL, dez subfrações foram identificadas, nas quais as partículas de HDL-1 a HDL-3 foram classificadas como grandes, HDL-4 a HDL-7 como intermediárias e HDL-8 a HDL-10 como pequenas. Um ponto de corte de 25,5 nm foi utilizado para determinação dos fenótipos LDL A e não-A. O tamanho médio do LDL (nm) também foi determinado. Uma relação entre partículas grandes e pequenas de HDL foi calculada a partir da porcentagem de subfrações de lipoproteínas, como segue: (HDL-1 + HDL-2) / (HDL-9 + HDL-10). A relação para partículas grandes e pequenas de LDL foi calculada da seguinte forma: (LDL-1 + LDL-2) / LDL-3 a LDL-7).

 Análise eletronegativa de LDL e autoanticorpos

A detecção de LDL eletronegativa [LDL(-)] e de autoanticorpos anti-LDL(-) foi realizada de acordo com método publicado anteriormente. 17 A LDL(-) eletronegativa foi detectada por meio do método ELISA sanduíche. As placas de microtitulação de poliestireno de fundo plano, com 96 poços (Costar, Corning Inc, NY, EUA), foram revestidas com 0,5 mg/poço de anticorpo monoclonal anti-LDL (-) 1A3H2 em 50 mL/poço de tampão carbonato-bicarbonato 0,05 M, pH 9,6, a 4 ºC, durante a noite. As placas foram lavadas três vezes com PBS, pH 7,4, contendo 0,05% de Tween-20 (200 mL/poço). Posteriormente, os sítios de ligação livres foram bloqueados pela adição de 150 mL/poço de PBS contendo 2% de leite em pó desnatado, previamente inativado por aquecimento (100 ºC), e 0,01% de Tween-20 por 1,5 h a 37 ºC, seguido de lavagem conforme mencionado acima. Padrão ou plasma (1:2000 diluído em PBS contendo 1% de leite desnatado e 0,01% de Tween-20) foi adicionado (50 mL/poço) nas placas e incubado por 1,5 h a 37 ºC. Em seguida, as placas foram lavadas e incubadas com 0,5 mg/poço de anticorpo monoclonal anti-LDL(-) 2C7D5F10 conjugado com biotina por uma hora a 37 ºC. Posteriormente, 50 mL/poço de peroxidase de rábano conjugada com estreptavidina (Invitrogen Corp., Carlsbad, CA, EUA) foram adicionados e incubados por uma hora a 37 ºC. A reatividade da peroxidase foi medida em placas lavadas por incubação com 50 mL de ortofenilenodiamina (OPD) diluída em tampão citrato fosfato, pH 5,3, a 37 ºC por 15 min. A reação foi interrompida pela adição de ácido sulfúrico 2 M, e a absorbância a 492 nm foi medida por espectrofotometria, usando um leitor de microplacas (Spectra Count Microplate Photometer, Packard Instruments Company, Downers Grove, IL, EUA). Uma curva de calibração (de 0,6 a 20 mg/mL) foi construída com LDL(-) humano obtido por FPLC usando uma coluna iônica. Os resultados foram expressos em U/L, com unidades representando 1 g/L de equivalente de apolipoproteína B oxidada.

 Análise de ácidos graxos de hemácias

A análise de ácidos graxos (AG) das membranas eritrocitárias foi realizada com base em método previamente descrito. 18 Após a separação do plasma (3.000× g , 10 min, 4 °C), 300 µL de eritrócitos foram lavados com 5 mL de solução salina tamponada com fosfato (PBS) (pH 7,4) quatro vezes. O precipitado foi transferido para tubos rosqueados, aos quais foram adicionados 1,75 mL de metanol, 50 µL de solução padrão interno contendo 1 mg de ácido tridecanóico (C13:0)/1 mL de hexano e 100 µL de cloreto de acetila. Em seguida, a solução foi agitada em vórtex e aquecida em banho-maria a 90 °C por 1 hora. Na sequência, adicionou-se 1,5 mL de hexano e a solução foi homogeneizada por 1 min. As amostras foram centrifugadas a 1500× g , 4 °C, por 2 min, e 800 µL do sobrenadante foram transferidos para um tubo diferente. Esta etapa foi repetida com a adição de 750 µL de hexano. Os tubos contendo os sobrenadantes coletados foram transferidos para um concentrador centrífugo a 40 °C, por 20 min. Em seguida, os ésteres metílicos de AG foram dissolvidos em 150 µL de hexano e transferidos para um inserto de vidro em um frasco, que posteriormente foi enviado para análise por cromatografia gasosa (Shimadzu CG-2010 equipado com coluna capilar DB-FFAP, Agilent Technologies).

Os resultados foram expressos como percentual do total de AG. As análises foram realizadas considerando os ácidos graxos individualmente, bem como o n-3 total, formado pela soma do ácido α-linolênico (ALA; C18:3 n-3), ácido eicosapentaenoico (EPA; C20:5 n-3) e ácido docosahexaenoico (DHA; C22:6 n-3), e n-6 total, formado pela soma de ácido linoleico (LA; C18:2 n-6), ácido γ-linolênico (GLA; C18:3 n-6 ), ácido eicosadienoico (EDA; C20:2 n-6), ácido di-homo-γ-linolênico (DGLA; C20:3 n-6), ácido araquidônico (AA; C20:4 n-6) e ácido di-homo-linolênico (DLA; C22:2 n-6).

 Análise estatística

O presente estudo utilizou amostragem de conveniência baseada em dados secundários de um ensaio clínico randomizado anterior (ReBEC: RBR-2vfhfv).

As variáveis contínuas foram expressas por meio de média ± desvio padrão (DP) ou mediana e intervalo interquartil (IIQ), conforme distribuição dos dados, e as variáveis categóricas foram expressas por meio de frequência absoluta (n) ou relativa (%). O teste de Kolmogorov-Smirnov foi realizado em variáveis contínuas para avaliar a distribuição. Os dados relativos das características da amostra foram obtidos por meio de teste t independente ou teste U de Mann-Whitney para variáveis contínuas de acordo com a normalidade dos dados e teste qui-quadrado para parâmetros categóricos. Os pacientes foram divididos em tercis de AG de hemácias (n-3 total, n-6 total e razão n-6/n-3) e, em seguida, foram aplicados testes ANOVA unidirecional ou Kruskal-Wallis com base na distribuição da variável, usando Bonferroni como teste posthoc.

Regressões lineares múltiplas foram realizadas para associar AGs com LDL(-), tamanho de LDL e sdLDL-c, sendo esta última a variável dependente. Todos os modelos foram ajustados por idade, sexo, tabagismo e uso de estatinas. Todas as suposições da regressão foram cumpridas (ou seja, sem multicolinearidade, homocedasticidade, erros normalmente distribuídos e independentes, independência das variáveis de resultado e linearidade das variáveis).

Todos os testes foram bilaterais, com p < 0,05 considerado significativo, e realizados no software SPSS versão 20.0.

 Resultados

As características dos participantes estão descritas na Tabela 1 e na Tabela Complementar 1. As mulheres mostraram maior frequência de tratamento com estatinas e apresentaram níveis plasmáticos de colesterol total, HDL-c, LDL-c, apoA-I, HDL-c grande, intermediário e pequeno, LDL(-) e hs-CRP em comparação com os homens. Em contraste, os homens apresentaram triglicerídeos plasmáticos, sdLDL-c e glicose mais elevados. Em comparação com os homens, as mulheres apresentaram maior ingestão de lipídios (valores absolutos, mas não ingestão relativa), DGLA e EPA e menos energia (Tabela Complementar 2). O perfil de AG de hemácias analisado está descrito na Tabela Complementar 3.

– Características da amostra
Variáveis Total (n = 335)
Média/mediana ou n DP/IIQ ou %
Idade (anos) 52,4 10,5
Atividade física (pontos) 7,2 1,4
Pressão arterial sistólica (mmHg) 133 18,3
Pressão arterial diastólica (mmHg) 81,1 10
IMC (kg/m 2 ) 30,9 5,7
Circunferência da cintura (cm) 100,6 13,6
Etnia
Branca 226 67,5
Outras 109 12,5
Tabagismo
Tabagista 64 19,1
Não tabagista 271 80,9
Consumo de álcool
Sim 66 19,7
Não 269 80,3
Escolaridade
Ensino médio completo ou incompleto 191 57
Ensino superior completo 144 43
Doenças não transmissíveis
Diabetes melito 66 19,7
Hipertensão 189 56,4
Hipotireoidismo 43 12,8
Dislipidemia 186 55,5
Medicamentos
Estatinas 95 28,4
Anti-hipertensivos 169 50,4
Anti-hiperglicêmicos 69 20,6
Fibratos 9 2,7
Características bioquímicas
Colesterol total (mg/dL) 204,4 42
HDL-c (mg/dL) 36 30,0-43,0
LDL-c (mg/dL) 137,2 38,7
Triglicerídeos (mg/dL) 130 97,0-190,0
Não-HDL-c (mg/dL) 167,8 41,3
ApoA-I (mg/dL) 132,8 26
ApoB (mg/dL) 103,9 24,8
HDL-c grande (mg/dL) 10 7,0-14,0
HDL-c intermediário (mg/dL) 18 15,0-21,0
HDL-c pequeno (mg/dL) 7,6 3
sdLDL-c (mg/dL) 3 1,0-9,0
lbLDL-c (mg/dL) 52,9 16,4
Tamanho da LDL (Å) 270 266,0-272,0
LDL(-) (U/L) 5,3 1,8-17,9
Anti-LDL(-) (μg/mL) 8,1 5,00-11,5
Glicose (mg/dL) 98 91,0-108,0
Insulina (μUI/mL) 16,1 12,7-22,1
HbA1c (%) 5 4,7-5,3
Adiponectina (μg/mL) 8,3 4,7-13,0
Leptina (ng/mL) 34,6 11,0-65,4
hs-CRP (mg/L) 2,7 2,7-5,8

Os dados são apresentados em médias (desvio padrão) ou medianas (intervalo interquartil) dependendo da distribuição para dados contínuos e valor absoluto (n) e frequência (%) para dados categóricos. ApoA-I: apolipoproteína A-I; ApoB: apolipoproteína B; IMC: índice de massa corporal; hs-CRP: proteína C reativa de alta sensibilidade; lbLDL-c: colesterol LDL grande e flutuante; LDL(-): LDL eletronegativa.

Indivíduos do terceiro tercil do n-3 total apresentaram níveis plasmáticos significativamente mais baixos de lipoproteínas plasmáticas associadas a apoB, colesterol, triglicerídeos e apoB. Além disso, melhores aspectos qualitativos de LDL foram observados neste grupo, como menor sdLDL-c (p = 0,001) e maior tamanho de partícula de LDL (p = 0,003) (Tabela Complementar 4).

As associações encontradas com AGPI n-6 total de hemácias foram comparadas de forma oposta com AGPI n-3 (Tabela Complementar 5). Verificou-se que os indivíduos do terceiro tercil apresentavam níveis plasmáticos mais elevados de colesterol total, LDL-c, não-HDL-c, apoB e apoA-I. Quanto à qualidade das partículas de LDL, o terceiro tercil apresentou maiores níveis plasmáticos de sdLDL-c (p = 0,004), lbLDL-c (p = 0,037) e LDL(-) (p = 0,002). Além disso, o tamanho médio das partículas de LDL foi menor neste tercil (p = 0,026). Associações semelhantes foram observadas com o terceiro tercil da relação n-6/n-3 de hemácias (Tabela Complementar 6).

As associações dos AGPIs n-3 e n-6 com a qualidade das partículas de LDL são mostradas na Tabela 2 . O EPA foi inversamente associado ao LDL(-). O ALA foi significativamente associado à menor sdLDL-c e maior tamanho de partícula de LDL, influenciando a associação do n-3 total com esses parâmetros. Em relação aos AGPIs n-6, o n-6 total teve associações positivas com LDL(-) e sdLDL-c, embora uma associação inversa com o tamanho das partículas de LDL tenha sido observada. Entre as associações com LDL(-), DGLA e AA apresentaram associações positivas significativas, enquanto DLA apresentou associação inversa. Entre as associações de sdLDL-c, LA, GLA, DGLA e AA apresentaram associações positivas. Além disso, o tamanho das partículas de LDL foi inversamente associado apenas com LA e DGLA entre os AGPIs n-6. Por fim, a relação de AGPIs n-6/n-3 de hemácias foi positivamente associada ao LDL(-) e ao sdLDL-c e inversamente associada ao tamanho das partículas de LDL.

– Associações entre ácidos graxos da membrana de hemácias e LDL eletronegativa, LDL pequena e densa e tamanho de partícula de LDL
Variáveis LDL(-)
R 2 β EP Valor de p IC de 95%
C18:2 n-6 (LA) 0,034 0,551 0,779 0,480 -0,981 ₋ 2,083
C18:3 n-6 (GLA) 0,055 -6,071 3,643 0,097 -13,238 ₋ 1,096
C20:2 n-6 (EDA) 0,050 -7,125 7,121 0,318 -21,134 ₋ 6,885
C20:3 n-6 (DGLA) 0,065 10,724 4,253 0,012 2,356 ₋ 19,091
C20:4 n-6 (AA) 0,117 2,999 0,588 < 0,001 1,842 ₋ 4,157
C22:2 n-6 (DLA) 0,068 -12,869 4,760 0,007 -22,233 ₋ -3,506
Total n-6 0,075 1,163 0,367 0,002 0,440 ₋ 1,886
C18:3 n-3 (ALA) 0,054 -1,734 1,126 0,124 -3,949 ₋ 0,480
C20:5 n-3 (EPA) 0,061 -21,517 9,711 0,027 -40,621 ₋ -2,413
C22:6 n-3 (DHA) 0,048 -0,523 1,146 0,649 -2,777 ₋ 1,732
Total n-3 0,055 -1,443 0,850 0,090 -3,115 ₋ 0,229
Relação n-6/n-3 0,072 4,064 1,364 0,003 1,381 ₋ 6,748
sdLDL-c
R 2 β EP Valor de p IC de 95%
C18:2 n-6 (LA) 0,053 0,948 0,301 0,002 0,357 ₋ 1,540
C18:3 n-6 (GLA) 0,039 3,105 1,422 0,030 0,307 ₋ 5,903
C20:2 n-6 (EDA) 0,025 -0,263 2,792 0,925 -5,756 ₋ 5,230
C20:3 n-6 (DGLA) 0,049 4,824 1,654 0,004 1,569 ₋ 8,078
C20:4 n-6 (AA) 0,037 0,491 0,237 0,039 0,025 ₋ 0,958
C22:2 n-6 (DLA) 0,030 -2,405 1,835 0,191 -6,015 ₋ 1,205
Total n-6 0,053 0,453 0,144 0,002 0,170 ₋ 0,736
C18:3 n-3 (ALA) 0,052 -1,340 0,437 0,002 -2,199 ₋ -0,481
C20:5 n-3 (EPA) 0,031 -5,492 3,827 0,152 -13,020 ₋ 2,036
C22:6 n-3 (DHA) 0,026 -0,283 0,447 0,527 -1,163 ₋ 0,597
Total n-3 0,049 -0,958 0,329 0,004 -1,606 ₋ -0,311
Relação n-6/n-3 0,062 1,905 0,530 < 0,001 0,863 ₋ 2,947
Tamanho da partícula de LDL
R 2 β EP Valor de p IC de 95%
C18:2 n-6 (LA) 0,079 -0,504 0,160 0,002 -0,818 ₋ -0,190
C18:3 n-6 (GLA) 0,060 -1,313 0,758 0,084 -2,805 ₋ 0,179
C20:2 n-6 (EDA) 0,051 0,069 1,485 0,963 -2,851 ₋ 2,990
C20:3 n-6 (DGLA) 0,065 -1,955 0,884 0,028 -3,695 ₋ -0,215
C20:4 n-6 (AA) 0,061 -0,232 0,126 0,067 -0,480 ₋ 0,017
C22:2 n-6 (DLA) 0,055 1,007 0,977 0,303 -0,914 ₋ 2,928
Total n-6 0,075 -0,224 0,077 0,004 -0,374 ₋ -0,073
C18:3 n-3 (ALA) 0,077 0,699 0,232 0,003 0,242 ₋ 1,156
C20:5 n-3 (EPA) 0,060 3,465 2,032 0,089 -0,533 ₋ 7,462
C22:6 n-3 (DHA) 0,053 0,157 0,238 0,511 -0,311 ₋ 0,624
Total n-3 0,075 0,510 0,175 0,004 0,165 ₋ 0,854
Relação n-6/n-3 0,089 -1,032 0,281 < 0,001 -1,585 ₋ -0,478

Modelos de regressão linear múltipla ajustados por idade, sexo, tabagismo e uso de estatinas. AA: ácido araquidônico; ALA: ácido α-linolênico; DGLA: ácido dihomo-γ-linolênico; DHA: ácido docosahexaenoico; DLA: ácido di-homo-linolênico; EDA: ácido eicosadienoico; EPA: ácido eicosapentaenoico; GLA: ácido γ-linolênico.

 Discussão

O presente estudo mostrou que os AGPIs n-6 e n-3 de hemácias exerceram influências opostas nos marcadores de saúde cardiometabólicos e nas características das partículas de LDL.

Nossos resultados mostraram que indivíduos com níveis de AGPI n-6 de hemácias e relação n-6/n-3 mais elevados, e níveis de AGPI n-3 mais baixos, apresentaram níveis plasmáticos mais elevados de colesterol (total, LDL-c e não-HDL-c), apoB, apoA-I e triglicerídeos. Estes resultados são consistentes com estudos que mostram que uma elevada relação n-6/n-3 foi associada ao aumento da lipogênese e dos lípidos no sangue. 19 - 24

Estudos clínicos de biomarcadores de AGPI n-6 e DCV na literatura mostram resultados inconclusivos. Em um estudo anterior, um padrão de AG de hemácias, contendo mais AGPI n-6, foi um preditor independente de classificação de risco cardiovascular mais elevada associada a fatores de risco estabelecidos. 18 Por outro lado, AGPIs n-6 teciduais ou sanguíneos não foram associados ao risco cardiovascular. 25 , 26 Além disso, estudos mostraram que altos níveis de AGPI n-6, especialmente LA, eram fatores de proteção contra a síndrome coronariana aguda. 27 - 29 Os autores de um desses estudos afirmaram que o consumo de AGPI n-6 deve ser incentivado, apesar do baixo consumo encontrado em um estudo. 29 Além disso, vale ressaltar que o risco de desfechos cardiovasculares mostrou uma associação em forma de U com AA, 28 e o LA não foi significativamente associado ao risco de infarto do miocárdio em um desses estudos, 29 dificultando a conclusão dos autores sobre o real efeito do AGPI n-6 nas DCVs.

As evidências sugerem que os AGPIs n-6 reduzem o nível plasmático do colesterol, particularmente quando o AGS é substituído, sendo favorável para a redução do risco de DCV, 30 - 32 mas os efeitos sobre os resultados permanecem inconclusivos. 31 Uma metanálise de ensaios clínicos randomizados (ECR) mostrou efeitos protetores do LA nas DCVs usando óleos vegetais, que também são fonte de ALA. 33 Curiosamente, um ECR relatou um risco aumentado de DCV e mortalidade por todas as causas após a suplementação de AGPI n-6. 33 Além disso, os resultados do Lyon Diet Heart Study indicaram que a redução do consumo de AGPI n-6 para menos de 5% da ingestão total de energia, abaixo das recomendações dos EUA de 5-10%, resultou em uma diminuição na mortalidade total e cardiovascular, sugerindo a existência de um possível risco associado ao consumo de AGPI n-6. 34 , 35

Em relação aos AGPIs n-3, as evidências clínicas recentes de DCV são mais consistentes. Estudos observacionais com AGPI n-3 de hemácias 18 , 36 - 40 e ensaios clínicos randomizados 41 - 43 mostraram que o AGPI n-3 reduz o risco de DCV. Nossos resultados corroboram o efeito cardioprotetor dos AGPIs n-3, mas mostram forte influência do ALA devido à baixa ingestão de EPA e DHA (Tabela Complementar 1). Os resultados do nosso estudo estão alinhados com uma revisão sistemática e metanálise dose-resposta, que mostrou que uma maior ingestão de ALA foi significativamente associada a 10%, 8% e 11% de mortalidade por todas as causas, DCV e DAC, respectivamente. 44

Os fatores de risco cardiovasculares tradicionais não refletem toda a complexidade da doença aterosclerótica, tanto que os fatores de risco cardiovasculares emergentes ganharam destaque na literatura, 18 , 45 - 48 e os efeitos dos AGPIs nas DCV estão além desses fatores. 18 Entre os diversos fatores de risco não tradicionais, o sdLDL-c demonstrou ser um melhor preditor de risco de DAC do que o LDL-c. 1 O presente estudo descobriu que os AGPIs n-6 das hemácias e a relação n-6/n-3 estavam positivamente associados ao tamanho médio menor das partículas de LDL e a níveis mais elevados de sdLDL-c. Associações semelhantes foram observadas com LA, GLA, DGLA e AA individualmente. Em contraste com nossos achados, um estudo transversal mostrou que os AGPIs n-6 circulantes estavam modestamente associados ao tamanho do LDL. 49

Por outro lado, os AGPIs n-3 foram associados a maiores tamanhos de partículas de LDL e a níveis mais baixos de sdLDL-c. Tais associações corroboram os resultados de ensaios anteriores, nos quais os AGPIs n-3 diminuíram o sdLDL-c e aumentaram o tamanho das partículas de LDL. 50 - 53 Essas associações podem estar ligadas ao TG. TGs mais elevados aumentam a apoC-III, dificultando a depuração de lipoproteínas que contêm apoE, e resultando em aumento dos níveis circulantes de sdLDL. 1 Além disso, os AGPIs n-3 reduzem os níveis de apoC-III em VLDL, diminuindo os níveis de sdLDL. 54

Observamos também que a relação n-6/n-3 de hemácias, AGPI n-6 total, DGLA e AA foram positivamente associados ao LDL (-), enquanto o EPA e o DLA apresentaram uma associação inversa. O LDL(-), intimamente relacionado ao oxLDL e ao sdLDL, possui propriedades aterogênicas, e níveis aumentados dessa partícula são encontrados em condições de estresse oxidativo e doenças não transmissíveis. 1 , 2 Níveis elevados de n-6/n-3 nos tecidos estão associados ao aumento da peroxidação lipídica, 19 - 21 e os AGPIs n-6 nas partículas de LDL, principalmente LA e AA, são mais facilmente oxidados, resultando no aumento da formação de partículas de LDL modificadas. 12 Outro estudo mostrou que a redução do AGPI n-6 na dieta reduz seus metabólitos gerados pela peroxidação. 55 Curiosamente, descobrimos que o EPA era um fator de proteção. Isto pode ser devido ao mecanismo antiinflamatório e antioxidante do EPA e seus metabólitos, que diminuem a modificação das partículas de LDL. 56

Entre as limitações, não podemos inferir causalidade devido à natureza transversal do estudo. Além disso, os AGPIs de hemácias do nosso estudo diferem dos resultados anteriores. 48 , 55 , 57 Isto pode ser devido à falta de padronização da análise de AG circulante. Os AG das hemácias são expressos em percentagens do AG total, o que significa que os níveis de AG dependem dos AG analisados pelo método. Outro fator que pode explicar esta divergência é que o estresse oxidativo diminui a quantidade de AGPI incorporados aos tecidos. 58 Por fim, nosso estudo não possui uma amostra representativa e, portanto, a AG de hemácias e sua associação com fatores de risco cardiovascular podem diferir de outras populações.

Nossos resultados aumentam o conhecimento disponível sobre ácidos graxos e risco cardiovascular, especialmente em relação aos AGPIs n-6. A literatura propõe que uma maior ingestão de AGPI n-6 pode beneficiar os indivíduos em relação ao risco cardiovascular. 7 - 10 Contudo, os estudos apresentam resultados altamente variáveis devido à avaliação de diversos biomarcadores (como plasma, hemácias, tecido adiposo e dieta) e à ampla variação na alimentação das pessoas entre as diferentes regiões estudadas, incluindo países ocidentais e asiáticos. Com a associação de AGPI n-6 de hemácias e a relação n-6/n-3 com LDL(-) demonstrada, nosso estudo corrobora com experimentos de oxidação de LDL que mostraram que os AG oxidados são da família do AGPI n-6, 11 - 14 sugerindo que o consumo excessivo de AGPI n-6 pode causar efeitos adversos. Isso chama a atenção para o fato de que os benefícios identificados do AL e de outros AGPIs n-6 na literatura provavelmente dependem do estado nutricional de AGPIs n-3, uma vez que mostramos associações metabólicas favoráveis com este último.

Por fim, a presente pesquisa evidencia que estudos futuros sobre o estado nutricional dos AGPIs devem considerar ambas as famílias de ácidos graxos, uma vez que a análise por si só pode ignorar fatores de confusão. Mais estudos são necessários para avaliar a associação de AGPIs de hemácias com fatores de risco e desfechos cardiovasculares, bem como fatores de risco emergentes, como subfrações de LDL.

 Conclusão

Nosso estudo mostrou que os AGPIs n-6 de hemácias estavam associados com fatores de risco cardiometabólico desfavoráveis, bem como níveis mais elevados de sdLDL-c e LDL(-), sugerindo que um alto consumo de n-6 na dieta pode aumentar a susceptibilidade da LDL a modificações oxidativas. Por outro lado, os AGPIs ômega-3 das hemácias, especialmente ALA e EPA, demonstraram associações benéficas para a saúde cardiovascular, manifestando níveis mais baixos de sdLDL-c e LDL(-) e maior tamanho da partícula de LDL.

Consequentemente, a ingestão do AGPI n-6 (LA ou AA) deve ser reduzida, ao passo que a ingestão de AGPIs n-3 deve ser estimulada, uma vez que uma relação mais elevada de n-6 para n-3 nas hemácias e uma maior concentração de AGPI n-6 foram associados a uma menor qualidade das partículas de LDL e a fatores de risco cardiometabólicos desfavoráveis.

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Effects of Coffee on Sirtuin-1, Homocysteine, and Cholesterol of Healthy Adults: does the Coffee Powder Matter? J Clin Med. 2022;11(11):2985. doi: 10.3390/jcm11112985. Gonçalinho GHF Nascimento JRO Mioto BM Amato RV Moretti MA Strunz CMC et al Effects of Coffee on Sirtuin-1, Homocysteine, and Cholesterol of Healthy Adults: does the Coffee Powder Matter? J Clin Med 2022 11 11 2985 10.3390/jcm11112985 47. Leal DP, Gonçalinho GHF, Tavoni TM, Kuwabara KL, Paccanaro AP, Freitas FR, et al. The Interplay of Sirtuin-1, LDL-Cholesterol, and HDL Function: A Randomized Controlled Trial Comparing the Effects of Energy Restriction and Atorvastatin on Women with Premature Coronary Artery Disease. Antioxidants (Basel). 2022;11(12):2363. doi: 10.3390/antiox11122363. Leal DP Gonçalinho GHF Tavoni TM Kuwabara KL Paccanaro AP Freitas FR et al The Interplay of Sirtuin-1, LDL-Cholesterol, and HDL Function: A Randomized Controlled Trial Comparing the Effects of Energy Restriction and Atorvastatin on Women with Premature Coronary Artery Disease Antioxidants Basel 2022 11 12 2363 10.3390/antiox11122363 48. Kemp JA, Esgalhado M, Macedo RA, Regis B, Damasceno NRT, Torres EAFS, et al. A Possible Link Between Polyunsaturated Fatty Acids and Uremic Toxins from the Gut Microbiota in Hemodialysis Patients: a Hypothesis. Hemodial Int. 2019;23(2):189-97. doi: 10.1111/hdi.12725. Kemp JA Esgalhado M Macedo RA Regis B Damasceno NRT Torres EAFS et al A Possible Link Between Polyunsaturated Fatty Acids and Uremic Toxins from the Gut Microbiota in Hemodialysis Patients: a Hypothesis Hemodial Int 2019 23 2 189 197 10.1111/hdi.12725 49. Mäntyselkä P, Niskanen L, Kautiainen H, Saltevo J, Würtz P, Soininen P, et al. Cross-Sectional and Longitudinal Associations of Circulating Omega-3 and Omega-6 Fatty Acids with Lipoprotein Particle Concentrations and Sizes: Population-Based Cohort Study with 6-year Follow-Up. Lipids Health Dis. 2014;13:28. doi: 10.1186/1476-511X-13-28. Mäntyselkä P Niskanen L Kautiainen H Saltevo J Würtz P Soininen P et al Cross-Sectional and Longitudinal Associations of Circulating Omega-3 and Omega-6 Fatty Acids with Lipoprotein Particle Concentrations and Sizes: Population-Based Cohort Study with 6-year Follow-Up Lipids Health Dis 2014 13 28 10.1186/1476-511X-13-28 50. Hartwich J, Malec MM, Partyka L, Pérez-Martinez P, Marin C, López-Miranda J, et al. The Effect of the Plasma n-3/n-6 Polyunsaturated Fatty Acid Ratio on the Dietary LDL Phenotype Transformation - Insights from the LIPGENE Study. Clin Nutr. 2009;28(5):510-5. doi: 10.1016/j.clnu.2009.04.016. Hartwich J Malec MM Partyka L Pérez-Martinez P Marin C López-Miranda J et al The Effect of the Plasma n-3/n-6 Polyunsaturated Fatty Acid Ratio on the Dietary LDL Phenotype Transformation - Insights from the LIPGENE Study Clin Nutr 2009 28 5 510 515 10.1016/j.clnu.2009.04.016 51. Griffin MD, Sanders TA, Davies IG, Morgan LM, Millward DJ, Lewis F, et al. Effects of Altering the Ratio of Dietary n-6 to n-3 Fatty Acids on Insulin Sensitivity, Lipoprotein Size, and Postprandial Lipemia in Men and Postmenopausal Women Aged 45-70 y: the OPTILIP Study. Am J Clin Nutr. 2006;84(6):1290-8. doi: 10.1093/ajcn/84.6.1290. Griffin MD Sanders TA Davies IG Morgan LM Millward DJ Lewis F et al Effects of Altering the Ratio of Dietary n-6 to n-3 Fatty Acids on Insulin Sensitivity, Lipoprotein Size, and Postprandial Lipemia in Men and Postmenopausal Women Aged 45-70 y: the OPTILIP Study Am J Clin Nutr 2006 84 6 1290 1298 10.1093/ajcn/84.6.1290 52. Khan S, Minihane AM, Talmud PJ, Wright JW, Murphy MC, Williams CM, et al. Dietary Long-Chain n-3 PUFAs Increase LPL Gene Expression in Adipose Tissue of Subjects with an Atherogenic Lipoprotein Phenotype. J Lipid Res. 2002;43(6):979-85. Khan S Minihane AM Talmud PJ Wright JW Murphy MC Williams CM et al Dietary Long-Chain n-3 PUFAs Increase LPL Gene Expression in Adipose Tissue of Subjects with an Atherogenic Lipoprotein Phenotype J Lipid Res 2002 43 6 979 985 53. Wilkinson P, Leach C, Ah-Sing EE, Hussain N, Miller GJ, Millward DJ, et al. Influence of Alpha-Linolenic Acid and Fish-Oil on Markers of Cardiovascular Risk in Subjects with an Atherogenic Lipoprotein Phenotype. Atherosclerosis. 2005;181(1):115-24. doi: 10.1016/j.atherosclerosis.2004.12.029. Wilkinson P Leach C Ah-Sing EE Hussain N Miller GJ Millward DJ et al Influence of Alpha-Linolenic Acid and Fish-Oil on Markers of Cardiovascular Risk in Subjects with an Atherogenic Lipoprotein Phenotype Atherosclerosis 2005 181 1 115 124 10.1016/j.atherosclerosis.2004.12.029 54. Oscarsson J, Hurt-Camejo E. Omega-3 Fatty Acids Eicosapentaenoic Acid and Docosahexaenoic Acid and Their Mechanisms of Action on Apolipoprotein B-containing Lipoproteins in Humans: a Review. Lipids Health Dis. 2017;16(1):149. doi: 10.1186/s12944-017-0541-3. Oscarsson J Hurt-Camejo E Omega-3 Fatty Acids Eicosapentaenoic Acid and Docosahexaenoic Acid and Their Mechanisms of Action on Apolipoprotein B-containing Lipoproteins in Humans: a Review Lipids Health Dis 2017 16 1 149 10.1186/s12944-017-0541-3 55. Ramsden CE, Ringel A, Feldstein AE, Taha AY, MacIntosh BA, Hibbeln JR, et al. Lowering Dietary Linoleic Acid Reduces Bioactive Oxidized Linoleic Acid Metabolites in Humans. Prostaglandins Leukot Essent Fatty Acids. 2012;87(4-5):135-41. doi: 10.1016/j.plefa.2012.08.004. Ramsden CE Ringel A Feldstein AE Taha AY MacIntosh BA Hibbeln JR et al Lowering Dietary Linoleic Acid Reduces Bioactive Oxidized Linoleic Acid Metabolites in Humans Prostaglandins Leukot Essent Fatty Acids 2012 87 4-5 135 141 10.1016/j.plefa.2012.08.004 56. Garrido-Sánchez L, García-Fuentes E, Rojo-Martínez G, Cardona F, Soriguer F, Tinahones FJ. Inverse Relation Between Levels of Anti-Oxidized-LDL Antibodies and Eicosapentanoic Acid (EPA). Br J Nutr. 2008;100(3):585-9. doi: 10.1017/S0007114508921723. Garrido-Sánchez L García-Fuentes E Rojo-Martínez G Cardona F Soriguer F Tinahones FJ Inverse Relation Between Levels of Anti-Oxidized-LDL Antibodies and Eicosapentanoic Acid (EPA) Br J Nutr 2008 100 3 585 589 10.1017/S0007114508921723 57. Hodson L, Skeaff CM, Fielding BA. Fatty Acid Composition of Adipose Tissue and Blood in Humans and its use as a Biomarker of Dietary Intake. Prog Lipid Res. 2008;47(5):348-80. doi: 10.1016/j.plipres.2008.03.003. Hodson L Skeaff CM Fielding BA Fatty Acid Composition of Adipose Tissue and Blood in Humans and its use as a Biomarker of Dietary Intake Prog Lipid Res 2008 47 5 348 380 10.1016/j.plipres.2008.03.003 58. Schuster S, Johnson CD, Hennebelle M, Holtmann T, Taha AY, Kirpich IA, et al. Oxidized Linoleic Acid Metabolites Induce Liver Mitochondrial Dysfunction, Apoptosis, and NLRP3 Activation in Mice. J Lipid Res. 2018;59(9):1597-609. doi: 10.1194/jlr.M083741. Schuster S Johnson CD Hennebelle M Holtmann T Taha AY Kirpich IA et al Oxidized Linoleic Acid Metabolites Induce Liver Mitochondrial Dysfunction, Apoptosis, and NLRP3 Activation in Mice J Lipid Res 2018 59 9 1597 1609 10.1194/jlr.M083741

Vinculação acadêmica

Este artigo é parte de dissertação de mestrado de Gustavo Henrique Ferreira Gonçalinho pela Departamento de Nutrição – Faculdade de Saúde Pública – Universidade de São Paulo.

Aprovação ética e consentimento informado

Este estudo foi aprovado pelo Comitê de Ética do Hospital Universitário da Universidade de São Paulo sob o número de protocolo 0063.0.207.198-11. Todos os procedimentos envolvidos nesse estudo estão de acordo com a Declaração de Helsinki de 1975, atualizada em 2013. O consentimento informado foi obtido de todos os participantes incluídos no estudo.

Fontes de financiamento: O presente estudo foi financiado pela FAPESP nº2016/24531-3, FAPESP nº 2011/12523-2 e CAPES nº88882.330835/2019-01.

Para tabelas suplementares, por favor, clique aqui .

 Original Article Red Blood Cells’ Omega-6 and Omega-3 Polyunsaturated Fatty Acids Have a Distinct Influence on LDL Particle Size and its Structural Modifications 0000-0002-2962-5341 Gonçalinho Gustavo Henrique Ferreira Acquisition of data Analysis and interpretation of the data Statistical analysis Writing of the manuscript and Critical revision of the manuscript for important intellectual content 1 Sampaio Geni Rodrigues Acquisition of data Analysis and interpretation of the data 1 0000-0001-7955-656X Soares-Freitas Rosana Aparecida Manólio Acquisition of data Analysis and interpretation of the data 1 Damasceno Nágila Raquel Teixeira Conception and design of the research and Obtaining financing Acquisition of data Analysis and interpretation of the data 1 Brazil Departamento de Nutrição – Faculdade de Saúde Pública – Universidade de São Paulo, São Paulo, SP – Brazil Mailing Address: Gustavo Henrique Ferreira Gonçalinho • Universidade de São Paulo – Av. Dr. Enéas Carvalho de Aguiar, 44. CEP 05403-000, Cerqueira César, São Paulo, SP – Brazil E-mail: ghfg93@gmail.com

Potential conflict of interest

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

Editor responsible for the review: Gláucia Maria Moraes de Oliveira

 Central Illustration

: Red Blood Cells’ Omega-6 and Omega-3 Polyunsaturated Fatty Acids Have a Distinct Influence on LDL Particle Size and its Structural Modifications

Abstract Background

While Omega-3 and omega-6 polyunsaturated fatty acids (n-3 and n-6 PUFAs) have established effects on cardiovascular disease (CVD) risk factors, little is known about their impacts on LDL quality markers.

 Objective

To assess the associations of n-3 and n-6 PUFA within red blood cells (RBC) with LDL particle size, small dense LDL-c (sdLDL-c), and electronegative LDL [LDL(-)] in adults with CVD risk factors.

 Methods

Cross-sectional study involving 335 men and women aged 30 to 74 with at least one cardiovascular risk factor. Analyses were conducted on biochemical parameters, such as glucose, insulin, HbA1c, C-reactive protein (CRP), lipid profile, lipoprotein subfractions, electronegative LDL particle [LDL(-)] and its autoantibody, and RBC n-3 and n-6 PUFAs. Independent t-test/Mann-Whitney test, one-way ANOVA/Kruskal-Wallis test, and multiple linear regressions were applied. All tests were two-sided, and a p-value of less than 0.05 was considered statistically significant.

 Results

The RBC n-6/n-3 ratio was associated with increased LDL(-) (β = 4.064; 95% CI = 1.381 – 6.748) and sdLDL-c (β = 1.905; 95% CI = 0.863 – 2.947) levels, and reduced LDL particle size (β = -1.032; 95% CI = -1.585 − -0.478). Separately, n-6 and n-3 PUFAs had opposing associations with those parameters, reinforcing the protective effects of n-3 and showing the potential negative effects of n-6 on LDL particle quality.

 Conclusion

RBC n-6 PUFA was associated with increased cardiometabolic risk and atherogenicity of LDL particles, while n-3 PUFA was associated with better cardiometabolic parameters and LDL particle quality.

 Fatty Acids, Unsaturated Oxidized LDL Receptors Lipoproteins, LDL Introduction

Cholesterol-lowering therapies are the first-line conducts for preventing atherosclerotic complications due to the strong relationship of LDL-c with CVD. 1 Despite LDL-c being an excellent predictor of CVD, this measure does not reflect the quality of LDL particles. 1 Biomarkers that reflect LDL particle quality, such as small dense LDL-c (sdLDL-c), electronegative LDL [LDL(-)], and LDL particle size, are subfractions that are more susceptible to oxidation and atherosclerosis progression and have better performance in CVD risk prediction when compared to its traditional counterpart LDL-c. 1 , 2

The omega-3 polyunsaturated fatty acids (n-3 PUFA) have demonstrated beneficial effects on cardiometabolic health parameters, 3 and there is further evidence that n-3 PUFA influence lipoprotein lipid composition and subclasses levels. 4 The effects of n-3 PUFA will, however, depend on the n-6 PUFA status due to the high content of the n-6 family in Western diets. 3 Linoleic acid (LA; C18:2 n-6) is the most common dietary n-6 PUFA and is converted to arachidonic acid (AA; C20:4 n-6), an important precursor variety of proinflammatory metabolites involved in the pathophysiology of CVD. 3 , 5 However, not all AA byproducts exert a proinflammatory response, 6 and evidence showed that circulating and dietary n-6 PUFA were associated with lower risks of coronary artery disease (CAD), ischemic stroke, and CVD mortality. 7 - 10 Conversely, dietary LA incorporated into all lipoproteins may increase their susceptibility to oxidation, which is associated with atherosclerosis severity. 11 - 15

Therefore, our study evaluated the association of RBC n-3 and n-6 PUFA with LDL particle size, small dense LDL-c (sdLDL-c), and electronegative LDL [LDL(-)] in adults with CVD risk factors.

 Methods Study design and participants

A cross-sectional study that used the baseline data from the CARDIONUTRI study (Brazilian Clinical Trial Registry - ReBEC: RBR-2vfhfv) that included 335 participants with the following eligibility criteria: individuals aged 30 to 74 without previous history of cardiovascular disease. Those with acute or chronic severe diseases, infectious diseases, pregnant and/or lactating women were excluded. All participants were selected at the University Hospital of the University of São Paulo (HU-USP), and all procedures followed the rules established by the Research Ethics Committee of HU-USP (CAAE No. 0063.0.207.198-11).

 Clinical and nutritional assessment

Current and past clinical conditions were investigated through questionnaires. Physical examination included body mass index (BMI) assessment, waist circumference, and blood pressure. Dietary intake was obtained through three 24-hour recalls and assessed in the Food Processor software (ESHA Research, OR, USA), with subsequent energy adjustment.

 Biochemical measurements

Blood was drawn after fasting for 12 hours; RBCs were separated from plasma by centrifugation (3,000× g for 5 minutes at 4 ºC), then frozen at -80 ºC. Plasma total cholesterol, HDL-c, triglycerides (Labtest Diagnostica S.A., MG, Brazil), apoA-I, apoB (Wako Chemicals USA Inc., Richmond, VA, USA), glucose (Labtest Diagnostica S.A., MG, Brazil), insulin (Life Technologies, Grand Island, NY, USA), and high-sensitivity C-reactive protein (hs-CRP) (Diagnostic System Laboratories, Inc., Webster, Texas, USA) were measured by commercial kits. The LDL-c level was calculated by Friedewald equation. 16

 Lipoprotein subfractions analysis

Lipoprotein size (HDL and LDL) was analyzed using LipoprintSystem TM (Quantimetrix, Redondo Beach, CA), which is based on the separation and quantification of lipoprotein subfractions by non-denaturing polyacrylamide gel. In light of electrophoresis, the subfractions were integrated to determine the relative area of each lipoprotein subunit (percentage of each subfraction), which was then multiplied by the plasma total cholesterol to calculate the cholesterol concentration in each LDL subfraction, and by the cholesterol in HDL to quantify the cholesterol concentration in each HDL subfraction. Based on the results, seven LDL subfractions could be identified, in which LDL-1 and LDL-2 subfractions were classified as large, and LDL-3 to LDL-7 as smaller and denser particles (sdLDL-c). In terms of HDL, ten subfractions were identified, in which HDL-1 to HDL-3 particles were classified as large, HDL-4 to HDL-7 as intermediate, and HDL-8 to HDL-10 as small. A cut-off point of 25.5 nm was used for determining LDL A and non-A phenotypes. The mean LDL size (nm) was also determined. A ratio between large and small HDL particles was calculated from the percentage of lipoprotein subfractions as follows: (HDL-1 + HDL-2) / (HDL-9 + HDL-10). The ratio for large and small LDL particles was calculated as follows: (LDL-1 + LDL-2) / LDL-3 to LDL-7).

 Electronegative LDL and autoantibodies analysis

The electronegative LDL [LDL(-)] and the autoantibody anti-LDL(-) detection were performed according to a previously published method. 17 The electronegative LDL(-) was detected through sandwich ELISA. The 96-well flat-bottom polystyrene microtiter plates (Costar, Corning Inc, NY, USA) were coated with 0.5 mg/well of anti-LDL (–) 1A3H2 monoclonal antibody in 50 mL/well of 0.05 M carbonate-bicarbonate buffer, pH 9.6, at 4 ºC overnight. The plates were washed thrice with PBS, pH 7.4, containing 0.05% Tween-20 (200 mL/well). Afterward, free binding sites were blocked by adding 150 mL/well of PBS containing 2% skimmed dry milk, previously inactivated by heating (100 ºC), and 0.01% Tween-20 for 1.5 h at 37 ºC, followed by washing as mentioned above. Standard or plasma (1:2000 diluted in PBS containing 1% skimmed milk and 0.01% Tween-20) was added (50 mL/well) in the plates and incubated for 1.5 h at 37 ºC. Then, the plates were washed and incubated with 0.5 mg/well of anti-LDL(-) 2C7D5F10 monoclonal antibody conjugated to biotin for one hour at 37 ºC. Afterward, 50 mL/well streptavidin-conjugated horseradish peroxidase (Invitrogen Corp., Carlsbad, CA, USA) was added and incubated for one hour at 37 ºC. The reactivity of peroxidase was measured on washed plates by incubating with 50 mL ortho-phenylenediamine (OPD) diluted in citrate phosphate buffer, pH 5.3, at 37 ºC for 15 min. The reaction was stopped by adding 2 M sulfuric acid, and the absorbance at 492 nm was measured by spectrophotometry using a microplate reader (Spectra Count Microplate Photometer, Packard Instruments Company, Downers Grove, IL, USA). A calibration curve (from 0.6 to 20 mg/mL) was built with human LDL(-) obtained by FPLC using an ionic column. Results were expressed as U/L, with units representing 1 g/L of oxidized apolipoprotein B equivalent.

 Red blood cell fatty acids analysis

The analysis of fatty acids (FA) from erythrocyte membranes was performed based on a previously described method. 18 After plasma separation (3000× g , 10 min, 4 °C), 300 µL of erythrocytes were washed with 5 mL of phosphate-buffered saline (PBS) solution (pH 7.4) four times. The precipitate was transferred to threaded tubes, to which 1.75 mL of methanol, 50 µL of an internal standard solution containing 1 mg tridecanoic acid (C13:0)/1 mL hexane, and 100 µL of acetyl chloride were added. Thereafter, the solution was vortexed and heated in a water bath at 90 °C for 1 hour. After that, 1.5 mL of hexane was added, and the solution was homogenized for 1 min. The samples were centrifuged at 1500× g , 4 °C for 2 min, and 800 µL of the supernatant was transferred to a different tube. This step was repeated with the addition of 750 µL of hexane. The tubes containing the collected supernatants were placed on a centrifugal concentrator at 40 °C for 20 min. Then, the FA methyl esters were dissolved in 150 µL of hexane and transferred to a glass insert in a vial, which subsequently went for gas chromatography analysis (Shimadzu CG-2010 equipped with a capillary column DB-FFAP, Agilent technologies).

The results were expressed as a percentage of the total FA. Analyses were conducted considering the fatty acids individually, as well as the total n-3, formed by the sum of α-linolenic acid (ALA; C18:3 n-3), eicosapentaenoic acid (EPA; C20:5 n-3), and docosahexaenoic acid (DHA; C22:6 n-3), and total n-6, formed by the sum of linoleic acid (LA; C18:2 n-6), γ-linolenic acid (GLA; C18:3 n-6), eicosadienoic acid (EDA; C20:2 n-6), di-homo-γ-linolenic acid (DGLA; C20:3 n-6), arachidonic acid (AA; C20:4 n-6), and di-homo-linoleic acid (DLA; C22:2 n-6).

 Statistical analyses

This study used convenience sampling based on secondary data from a previous randomized clinical trial (ReBEC: RBR-2vfhfv).

Continuous variables were expressed through mean ± standard deviation (SD) or median and interquartile range (IQR), according to data distribution, and categorical variables were expressed through absolute (n) or relative (%) frequency. The Kolmogorov-Smirnov test was performed on continuous variables to assess distribution. Data in sample characteristics were obtained through an independent t-test or Mann-Whitney U test for continuous variables according to data normality and a chi-square test for categorical parameters. Patients were divided into tertiles of RBC FA (total n-3, total n-6, and n-6/n-3 ratio), and then one-way ANOVA or Kruskal-Wallis tests were applied based upon the variable’s distribution, using Bonferroni as posthoc test.

Multiple linear regressions were conducted to associate FA with LDL(-), LDL size, and sdLDL-c, the latter being the dependent variable. All models were adjusted by age, sex, smoking status, and use of statins. All regression assumptions were fulfilled (i.e., no multicollinearity, homoscedasticity, normally distributed and independent errors, independence of the outcome variables, and linearity of the variables).

All tests were two-sided, with p < 0.05 considered significant, and performed on the SPSS software version 20.0.

 Results

Characteristics of participants are described in Table 1 and Supplementary Table 1 . Women reported a higher frequency of statin treatment and had levels of plasma total cholesterol, HDL-c, LDL-c, apoA-I, large, intermediate, and small HDL-c, LDL(-), and hs-CRP than men. In contrast, men had higher plasma triglycerides, sdLDL-c, and glucose. Compared to men, women had a higher intake of lipids (absolute values, but not relative intake), DGLA, and EPA and less energy (Supplementary Table 2 ). The analyzed RBC FA profile is described in Supplementary Table 3.

– Sample characteristics
Variables Total (n = 335)
Mean/median or n SD/IQR or %
Age (years) 52.4 10.5
Physical activity (points) 7.2 1.4
Systolic blood pressure (mmHg) 133 18.3
Diastolic blood pressure (mmHg) 81.1 10
BMI (kg/m 2 ) 30.9 5.7
Waist circumference (cm) 100.6 13.6
Race
White 226 67.5
Non-white 109 12.5
Smoking
Current smoker 64 19.1
Non-smoker 271 80.9
Alcohol consumption
Yes 66 19.7
No 269 80.3
Education
High school or less 191 57
College 144 43
Non-communicable diseases
Diabetes Mellitus 66 19.7
Hypertension 189 56.4
Hypothyroidism 43 12.8
Dyslipidemia 186 55.5
Medication
Statins 95 28.4
Antihypertensives 169 50.4
Antihyperglycemics 69 20.6
Fibrates 9 2.7
Biochemical characteristics
Total cholesterol (mg/dL) 204.4 42
HDL-c (mg/dL) 36 30.0-43.0
LDL-c (mg/dL) 137.2 38.7
Triglycerides (mg/dL) 130 97.0-190.0
Non-HDL-c (mg/dL) 167.8 41.3
ApoA-I (mg/dL) 132.8 26
ApoB (mg/dL) 103.9 24.8
Large HDL-c (mg/dL) 10 7.0-14.0
Intermediate HDL-c (mg/dL) 18 15.0-21.0
Small HDL-c (mg/dL) 7.6 3
sdLDL-c (mg/dL) 3 1.0-9.0
lbLDL-c (mg/dL) 52.9 16.4
LDL size (Å) 270 266.0-272.0
LDL(-) (U/L) 5.3 1.8-17.9
Anti-LDL(-) (μg/mL) 8.1 5.00-11.5
Glucose (mg/dL) 98 91.0-108.0
Insulin (μUI/mL) 16.1 12.7-22.1
HbA1c (%) 5 4.7-5.3
Adiponectin (μg/mL) 8.3 4.7-13.0
Leptin (ng/mL) 34.6 11.0-65.4
hs-CRP (mg/L) 2.7 2.7-5.8

Data is shown in mean (standard deviation) or median (interquartile range) depending on the distribution for continuous data and absolute value (n) and frequency (%) for categorical data. ApoA-I: apolipoprotein A-I; ApoB: apolipoprotein B; BMI: body mass index; hs-CRP: high-sensitivity C-reactive protein; lbLDL-c: large buoyant LDL-cholesterol; LDL(-): electronegative LDL.

– Associations between red blood cell membrane fatty acids and electronegative LDL, small dense LDL-cholesterol, and LDL particle size
Variables LDL(-)
R 2 β SE p-value 95% CI
C18:2 n-6 (LA) 0.034 0.551 0.779 0.480 -0.981 ₋ 2.083
C18:3 n-6 (GLA) 0.055 -6.071 3.643 0.097 -13.238 ₋ 1.096
C20:2 n-6 (EDA) 0.050 -7.125 7.121 0.318 -21.134 ₋ 6.885
C20:3 n-6 (DGLA) 0.065 10.724 4.253 0.012 2.356 ₋ 19.091
C20:4 n-6 (AA) 0.117 2.999 0.588 < 0.001 1.842 ₋ 4.157
C22:2 n-6 (DLA) 0.068 -12.869 4.760 0.007 -22.233 ₋ -3.506
Total n-6 0.075 1.163 0.367 0.002 0.440 ₋ 1.886
C18:3 n-3 (ALA) 0.054 -1.734 1.126 0.124 -3.949 ₋ 0.480
C20:5 n-3 (EPA) 0.061 -21.517 9.711 0.027 -40.621 ₋ -2.413
C22:6 n-3 (DHA) 0.048 -0.523 1.146 0.649 -2.777 ₋ 1.732
Total n-3 0.055 -1.443 0.850 0.090 -3.115 ₋ 0.229
n-6/n-3 ratio 0.072 4.064 1.364 0.003 1.381 ₋ 6.748
sdLDL-c
R 2 β SE p-value 95% CI
C18:2 n-6 (LA) 0.053 0.948 0.301 0.002 0.357 ₋ 1.540
C18:3 n-6 (GLA) 0.039 3.105 1.422 0.030 0.307 ₋ 5.903
C20:2 n-6 (EDA) 0.025 -0.263 2.792 0.925 -5.756 ₋ 5.230
C20:3 n-6 (DGLA) 0.049 4.824 1.654 0.004 1.569 ₋ 8.078
C20:4 n-6 (AA) 0.037 0.491 0.237 0.039 0.025 ₋ 0.958
C22:2 n-6 (DLA) 0.030 -2.405 1.835 0.191 -6.015 ₋ 1.205
Total n-6 0.053 0.453 0.144 0.002 0.170 ₋ 0.736
C18:3 n-3 (ALA) 0.052 -1.340 0.437 0.002 -2.199 ₋ -0.481
C20:5 n-3 (EPA) 0.031 -5.492 3.827 0.152 -13.020 ₋ 2.036
C22:6 n-3 (DHA) 0.026 -0.283 0.447 0.527 -1.163 ₋ 0.597
Total n-3 0.049 -0.958 0.329 0.004 -1.606 ₋ -0.311
n-6/n-3 ratio 0.062 1.905 0.530 < 0.001 0.863 ₋ 2.947
LDL particle size
R 2 β SE p-value 95% CI
C18:2 n-6 (LA) 0.079 -0.504 0.160 0.002 -0.818 ₋ -0.190
C18:3 n-6 (GLA) 0.060 -1.313 0.758 0.084 -2.805 ₋ 0.179
C20:2 n-6 (EDA) 0.051 0.069 1.485 0.963 -2.851 ₋ 2.990
C20:3 n-6 (DGLA) 0.065 -1.955 0.884 0.028 -3.695 ₋ -0.215
C20:4 n-6 (AA) 0.061 -0.232 0.126 0.067 -0.480 ₋ 0.017
C22:2 n-6 (DLA) 0.055 1.007 0.977 0.303 -0.914 ₋ 2.928
Total n-6 0.075 -0.224 0.077 0.004 -0.374 ₋ -0.073
C18:3 n-3 (ALA) 0.077 0.699 0.232 0.003 0.242 ₋ 1.156
C20:5 n-3 (EPA) 0.060 3.465 2.032 0.089 -0.533 ₋ 7.462
C22:6 n-3 (DHA) 0.053 0.157 0.238 0.511 -0.311 ₋ 0.624
Total n-3 0.075 0.510 0.175 0.004 0.165 ₋ 0.854
n-6/n-3 ratio 0,089 -1,032 0,281 < 0,001 -1,585 ₋ -0,478

Multiple linear regressions models adjusted by age, sex, smoking status, and use of statins. AA: arachidonic acid; ALA: α-linolenic acid; DGLA: dihomo-γ-linoleic acid; DHA: docosahexaenoic acid; DLA: di-homo-linoleic acid; EDA: eicosadienoic acid; EPA: eicosapentaenoic acid; GLA: γ-linolenic acid.

Individuals of the third tertile of total n-3 had significantly lower plasma apoB-associated lipoproteins cholesterol, triglycerides, and apoB levels. Also, better qualitative aspects of LDL were observed in this group, such as lower sdLDL-c (p = 0.001) and larger LDL particle size (p = 0.003) (Supplementary Table 4).

Associations found with RBC total n-6 PUFA were oppositely compared to n-3 PUFA (Supplementary Table 5). It was found that individuals within the third tertile had higher plasma total cholesterol, LDL-c, non-HDL-c, apoB, and apoA-I. As regards LDL particle quality, the third tertile had higher plasma levels of sdLDL-c (p = 0.004), lbLDL-c (p = 0.037), and LDL(-) (p = 0.002). Additionally, the mean size of LDL particles was smaller in this tertile (p = 0.026). Similar associations were found with the third tertile of the RBC n-6/n-3 ratio (Supplementary Table 6).

Associations of n-3 and n-6 PUFA with LDL particle quality are shown in Table 2 . EPA was inversely associated with LDL(-). ALA was significantly associated with lower sdLDL-c and larger LDL particle size, influencing the association of total n-3 with these parameters. Regarding n-6 PUFA, total n-6 had positive associations with LDL(-) and sdLDL-c, while it had an inverse association with LDL particle size. Among the associations with LDL(-), DGLA and AA showed significant positive associations, while DLA showed an inverse association. Among sdLDL-c associations, LA, GLA, DGLA, and AA showed positive associations. Furthermore, LDL particle size was inversely associated only with LA and DGLA among n-6 PUFA. Finally, the RBC n-6/n-3 PUFA ratio was positively associated with LDL(-) and sdLDL-c and inversely associated with LDL particle size.

 Discussion

This study showed that RBC n-6 and n-3 PUFA had opposing influences on cardiometabolic health markers and LDL particle characteristics.

Our results showed that individuals with higher levels of RBC n-6 PUFA and n-6/n-3 ratio and lower levels of n-3 PUFA had higher plasma cholesterol (total, LDL-c, and non-HDL-c), apoB, apoA-I, and triglycerides. These results are consistent with studies showing that a high n-6/n-3 ratio was associated with increased lipogenesis and blood lipids. 19 - 24

Clinical studies of n-6 PUFA biomarkers and CVD in the literature show inconclusive results. In a previous study, an RBC FA pattern containing more n-6 PUFA was an independent predictor of higher cardiovascular risk classification associated with established risk factors. 18 Conversely, tissue or blood n-6 PUFA were not associated with cardiovascular risk. 25 , 26 Moreover, studies have found that high n-6 PUFA, especially LA, were protective factors against acute coronary syndrome. 27 - 29 Authors of one of these studies stated that n-6 PUFA consumption should be encouraged despite the low consumption found in one study. 29 Furthermore, it is important to note that the risk for CV outcomes had a U-shaped association with AA, 28 and LA was not significantly associated with myocardial infarction risk in one of these studies, 29 making it difficult to conclude the real effect of n-6 PUFA on CVD.

Evidence suggests that n-6 PUFA reduces plasma cholesterol, particularly when SFA is replaced, which is favorable for CVD risk reduction, 30 - 32 but the effects on outcomes remain inconclusive. 31 A meta-analysis of randomized controlled trials (RCTs) showed protective effects of LA on CVD using vegetable oils, which are also a source of ALA. 33 Interestingly, one RCT reported an increased risk for CVD and all-cause mortality after n-6 PUFA supplementation. 33 Furthermore, results of the Lyon Diet Heart Study showed that the reduction of n-6 PUFA to less than 5% of total energy intake, below the US recommendations of 5-10%, decreased total and cardiovascular mortality, suggesting that there is a risk of the consumption associated with n-6 PUFA. 34 , 35

Concerning n-3 PUFA, the recent clinical evidence for CVD is more consistent. Observational studies with RBC n-3 PUFA 18 , 36 - 40 and randomized clinical trials 41 - 43 showed that n-3 PUFA reduce CVD risk. Our results corroborate with the cardioprotective effect of n-3 PUFA but showed a strong influence of ALA due to the low EPA and DHA intake (Supplementary Table 1 ). Our study’s results align with a systematic review and dose-response meta-analysis, which showed that a higher intake of ALA was significantly associated with 10%, 8%, and 11% of mortality from all causes, CVD, and CAD, respectively. 44

Traditional cardiovascular risk factors do not reflect the full complexity of the atherosclerotic disease, so much so that emerging cardiovascular risk factors have gained emphasis in the literature, 18 , 45 - 48 and the effects of PUFA on CVD are beyond these factors. 18 Among the various non-traditional risk factors, it has been shown that sdLDL-c is a better predictor of CAD risk than LDL-c. 1 The current study found that RBC n-6 PUFA and n-6/n-3 ratio were positively associated with smaller average LDL particle size and higher levels of sdLDL-c. Similar associations were observed with LA, GLA, DGLA, and AA individually. In contrast to our findings, a cross-sectional study showed that circulating n-6 PUFA was modestly associated with LDL size. 49

Conversely, n-3 PUFA were associated with larger LDL particle size and lower levels of sdLDL-c. Such associations corroborate previous trials’ results that n-3 PUFA decreased sdLDL-c and increased LDL particle size. 50 - 53 These associations may be linked to TG. Higher TG increases apoC-III, which hinders clearance of apoE-containing lipoproteins, resulting in increased circulating sdLDL levels, 1 and n-3 PUFA reduce apoC-III levels in VLDL, decreasing sdLDL levels. 54

We also found that RBC n-6/n-3 ratio, total n-6 PUFA, DGLA, and AA were positively associated with LDL(-), whereas EPA and DLA presented an inverse association. LDL(-), closely related to oxLDL and sdLDL, has atherogenic properties, and increased levels of this particle are found in oxidative stress conditions and non-communicable diseases. 1 , 2 High n-6/n-3 tissue levels are associated with increased lipid peroxidation, 19 - 21 and n-6 PUFA in LDL particles, primarily LA and AA, are most readily oxidized, resulting in the increased formation of modified LDL particles. 12 Another study showed that lowering dietary n-6 PUFA reduces its metabolites generated from peroxidation. 55 Interestingly, we found that EPA was a protective factor. This may be due to the anti-inflammatory and antioxidant mechanism of EPA and its metabolites, which decrease the modification of LDL particles. 56

Among limitations, we cannot infer causality because of the study’s cross-sectional nature. In addition, RBC PUFA in our study differs from previous results. 48 , 55 , 57 This may be due to a lack of standardization of circulating FA analysis. RBC FA are expressed in percentages of total FA, meaning that the FA levels depend on FA analyzed by the method. Another factor that may explain this divergence is that oxidative stress decreases the amount of PUFA incorporated into the tissues. 58 Finally, our study does not have a representative sample, so RBC FA and their association with cardiovascular risk factors may differ from other populations.

Our findings add to the knowledge about fatty acids and cardiovascular risk, especially regarding n-6 PUFA. The literature proposes that higher n-6 PUFA intake may benefit individuals concerning cardiovascular risk. 7 - 10 However, the studies show very inconsistent results since different biomarkers (e.g., plasma, RBC, adipose tissue, dietary) are evaluated, and the diet of the individuals may vary greatly among the regions studied (e.g., Western countries vs. Asian countries). With the association of RBC n-6 PUFA and the n-6/n-3 ratio with LDL(-) that was demonstrated, our study corroborates with LDL oxidation experiments that showed that the oxidized FA are from the n-6 PUFA family, 11 - 14 suggesting that excess n-6 PUFA consumption may cause adverse effects. This calls attention to the fact that the benefits found from LA and other n-6 PUFA in the literature probably depend on the nutritional status of n-3 PUFA since we showed favorable metabolic associations with the latter.

This study finally shows that upcoming studies of the nutritional status of PUFA should consider both families of fatty acids since analysis alone may ignore confounding factors. Further studies are required to evaluate the association of RBC PUFA with cardiovascular risk factors and outcomes, as well as emerging risk factors such as LDL subfractions.

 Conclusion

Our study showed RBC n-6 PUFA were associated with worse cardiometabolic risk factors and higher levels of sdLDL-c and LDL(-), suggesting that high dietary n-6 PUFA increases LDL susceptibility to oxidative modifications. In turn, RBC n-3 PUFA, especially ALA and EPA, showed cardioprotective associations, with lower levels of sdLDL-c and LDL(-) and larger LDL particle size.

Finally, n-6 PUFA intake (both LA or AA) should be decreased at the same time as the n-3 PUFA should be stimulated since a higher RBC n-6/n-3 ratio and n-6 PUFA have been associated with worse LDL particle quality and cardiometabolic risk factors.

Study association

This article is part of the thesis of master submitted by Gustavo Henrique Ferreira Gonçalinho, from Departamento de Nutrição – Faculdade de Saúde Pública – Universidade de São Paulo.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Hospital Universitário da Universidade de São Paulo under the protocol number 0063.0.207.198-11. All the procedures in this study were in accordance with the 1975 Helsinki Declaration, updated in 2013. Informed consent was obtained from all participants included in the Informed consent was obtained from all participants included in the study.

Sources of funding: This study was partially funded by FAPESP nº2016/24531-3, FAPESP nº 2011/12523-2 and CAPES nº88882.330835/2019-01

For supplementary tables, please click here .