European Journal of Clinical Nutrition (2016) 70, 306–312 OPEN © 2016 Macmillan Publishers Limited All rights reserved 0954-3007/16 www.nature.com/ejcn ORIGINAL ARTICLE Plasma concentrations and intakes of amino acids in male meat-eaters, fish-eaters, vegetarians and vegans: a cross-sectional analysis in the EPIC-Oxford cohort JA Schmidt1, S Rinaldi2, A Scalbert2, P Ferrari2, D Achaintre2, MJ Gunter3, PN Appleby1, TJ Key1 and RC Travis1 BACKGROUND/OBJECTIVES: We aimed to investigate the differences in plasma concentrations and in intakes of amino acids between male meat-eaters, fish-eaters, vegetarians and vegans in the Oxford arm of the European Prospective Investigation into Cancer and Nutrition. SUBJECTS/METHODS: This cross-sectional analysis included 392 men, aged 30–49 years. Plasma amino acid concentrations were measured with a targeted metabolomic approach using mass spectrometry, and dietary intake was assessed using a food frequency questionnaire. Differences between diet groups in mean plasma concentrations and intakes of amino acids were examined using analysis of variance, controlling for potential confounding factors and multiple testing. RESULTS: In plasma, concentrations of 6 out of 21 amino acids varied significantly by diet group, with differences of − 13% to +16% between meat-eaters and vegans. Concentrations of methionine, tryptophan and tyrosine were highest in fish-eaters and vegetarians, followed by meat-eaters, and lowest in vegans. A broadly similar pattern was seen for lysine, whereas alanine concentration was highest in fish-eaters and lowest in meat-eaters. For glycine, vegans had the highest concentration and meat-eaters the lowest. Intakes of all 18 dietary amino acids differed by diet group; for the majority of these, intake was highest in meat-eaters followed by fish-eaters, then vegetarians and lowest in vegans (up to 47% lower than in meat-eaters). CONCLUSIONS: Men belonging to different habitual diet groups have significantly different plasma concentrations of lysine, methionine, tryptophan, alanine, glycine and tyrosine. However, the differences in plasma concentrations were less marked than and did not necessarily mirror those seen for amino acid intakes. European Journal of Clinical Nutrition (2016) 70, 306–312; doi:10.1038/ejcn.2015.144; published online 23 September 2015 INTRODUCTION The aim of this study was therefore to investigate the Amino acids are the building blocks of proteins1 and are differences in plasma concentrations and in dietary intakes of additionally utilised as a source of energy. They are necessary amino acids between male meat-eaters, fish-eaters, vegetarians for the synthesis of a wide variety of compounds, including and vegans in the Oxford arm of the European Prospective neurotransmitters, haem and DNA. Humans need daily supplies of Investigation into Cancer and Nutrition (EPIC-Oxford). protein including adequate amounts of essential amino acids, 2 which cannot be synthesised endogenously. The amino acid SUBJECTS AND METHODS composition of animal protein better resembles the body’s need than that of single sources of plant protein. However, as different Study population plant proteins complement each other in terms of amino acid The EPIC-Oxford cohort comprises 65 000 participants including 14 600 composition, diets without animal products can fulfil the men over the age of 20 years recruited from throughout the United – requirement. Kingdom in 1993 2001. The study has been described in detail elsewhere.11 The primary aim of this cohort is to investigate diet, lifestyle Although circulating concentrations of amino acids are subject and risk of cancer and other chronic diseases in people with a wide range to homoeostatic control,3 they are also affected by diet, 4 of different dietary habits. Therefore, recruitment was aimed at vegetarians metabolism, lifestyle and genetic factors. Individuals who exclude and vegans, as well as the general population. Most participants (89%) some or all animal products from their diet may thus have were recruited by post, whereas the remaining participants were recruited different circulating concentrations compared with those of meat- via general practitioners’ surgeries. All participants gave written informed eaters, because of differences in dietary protein sources, lifestyle consent, and the protocol for EPIC-Oxford was approved by a multicentre and metabolism. Previous studies have found some variations in research ethics committee (MREC/02/0/90). circulating concentrations or intakes of amino acids between The following eligibility criteria for the current analysis were applied to 5–10 limit variation in amino acid concentrations and intakes because of factors vegans, vegetarians and omnivores. However, none of these other than diet group: male sex; age 30–49 years; provision of a blood studies have investigated a wide range of amino acids in terms of sample at recruitment; known smoking status and diet group; response to both concentration and dietary intake, and limited numbers of 80% or more of the relevant questions in the food frequency questionnaire participants were included (no75). (FFQ) and a daily energy intake between 3.3 and 16.7 MJ (800–4000 kcal); 1Cancer Epidemiology Unit, Nuffield Department of Population Health, University of Oxford, Oxford, UK; 2International Agency for Research on Cancer, Lyon, France and 3Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, UK. Correspondence: JA Schmidt, Cancer Epidemiology Unit, Nuffield Department of Population Health, University of Oxford, Richard Doll Building, Oxford OX3 7LF, UK. E-mail: [email protected] Received 10 November 2014; revised 1 July 2015; accepted 21 July 2015; published online 23 September 2015 Amino acid concentrations and intakes by diet group JA Schmidt et al 307 and no prior cancer (excluding non-melanoma skin cancer), cardiovascular significantly by diet group for lysine, phenylalanine or proline (no disease or treatment for any long-term illness or condition at recruitment. comparison was made for the other amino acids because of the low Of the 110 eligible vegans (who do not eat any animal products), we number of affected samples). Participants were excluded only from selected all those aged 30–39 years and randomly selected four out of analyses of amino acids for which they did not have data. every five aged 40–49 years. In addition, eligible meat-eaters (who eat meat), fish-eaters (who do not eat meat but do eat fish) and vegetarians (who do not eat meat or fish but do eat dairy products and/or eggs) were Diet and lifestyle randomly selected in equal numbers within strata of the ages 30–39 and At recruitment, participants completed a validated semi-quantitative 40–49 years. A total of 392 men, 98 in each diet group, were included in FFQ,12,13 with additional questions on smoking status, height and weight. this analysis. There was good agreement between self-reported and measured height and weight from a sub-sample of the cohort (correlation coefficient 4 14 Blood sampling and laboratory analysis (r) 0.9). If available, measured values were used. Categorisation of the four diet groups was based on the answers to the At recruitment, participants had a blood sample taken at their local general questions 'Do you eat any meat; fish; dairy products; and eggs?' practitioner's surgery. Fasting was not required, but time since last food or Participants were further asked how often they consumed 130 or 113 drink was recorded. Whole blood was sent at ambient temperature to a foods and drinks, depending on their diet group. Mean daily food and laboratory in Norfolk, centrifuged and aliquoted into 0.5 ml straws and fi 15 − drink intakes were estimated using speci ed portion sizes, and mean stored in liquid nitrogen ( 196 °C) until 2011 and subsequently in electric daily nutrient intakes were estimated mostly using data from the fifth freezers (−80 °C). edition of ‘McCance and Widdowson’s The Composition of Foods’ and its In 2013, samples were transported on dry ice to the International – supplements.16 25 Agency for Research on Cancer, Lyon, France, for assay. Before the assay To estimate intakes of 18 amino acids, data from the United States for this study, samples had undergone two or three thaw–freeze cycles; Department of Agriculture, National Nutrient Database for Standard samples from each diet group were equally distributed by the number of 26 cycles. The targeted metabolomics assay, BIOCRATES AbsoluteIDQ p180 Kit Reference, release 26, were used, because such data are only available (Biocrates, Innsbruck, Austria), quantified 21 amino acids using liquid for a limited number of foods in the United Kingdom. All UK foods were fi chromatography-tandem mass spectrometry. For quality control, four to matched to the nearest food in the US nutrient database, paying speci c six blinded samples of pooled plasma were included in each batch. The attention to match on the total protein content so that scaling was not laboratory technicians were blinded to the diet group and the quality necessary. The data in the US database are based on high-performance control samples. The median (range) overall coefficient of variation for the liquid chromatography following protein hydrolysis and thus mainly amino acids was 11.8% (5.4–17.3). No samples had concentrations below represent protein amino acids. The US and UK nutrient tables do not the lower limit of quantification, whereas for five amino acids a few values include information