Geraldine Cuskelly

Optimised extraction of heterocyclic aromatic amines from blood using hollow fibre membrane liquid-phase microextraction and triple quadrupole mass spectrometry.

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of proteinaceous foods, particularly meat. To assist in the ongoing search for biomarkers of HCA exposure in blood, a method is described for the extraction from human plasma of the most abundant HCAs: 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (and its isomer 7,8-DiMeIQx), using hollow fibre membrane liquid-phase microextraction. This technique employs 2.5cm lengths of porous polypropylene fibres impregnated with organic solvent to facilitate simultaneous extraction from an alkaline aqueous sample into a low volume acidic acceptor phase. This low cost protocol is extensively optimised for fibre length, extraction time, sample pH and volume. Detection is by UPLC-MS/MS using positive mode electrospray ionisation with a 3.4min runtime, with optimum peak shape, sensitivity and baseline separation being achieved at pH 9.5. To our knowledge this is the first description of HCA chromatography under alkaline conditions. Application of fixed ion ratio tolerances for confirmation of analyte identity is discussed. Assay precision is between 4.5 and 8.8% while lower limits of detection between 2 and 5pg/mL are below the concentrations postulated for acid-labile HCA-protein adducts in blood.

Validation study to compare effects of processing protocols on measured N (ε)-(carboxymethyl)lysine and N (ε)-(carboxyethyl)lysine in blood

Epidemiological studies show that elevated plasma levels of advanced glycation end products (AGEs) are associated with diabetes, kidney disease, and heart disease. Thus AGEs have been used as disease progression markers. However, the effects of variations in biological sample processing procedures on the level of AGEs in plasma/serum samples have not been investigated. The objective of this investigation was to assess the effect of variations in blood sample collection on measured Nε-(carboxymethyl)lysine (CML), the best characterised AGE, and its homolog, Nε-(carboxyethyl)lysine (CEL). The investigation examined the effect on CML and CEL of different blood collection tubes, inclusion of a stabilising cocktail, effect of freeze thaw cycles, different storage times and temperatures, and effects of delaying centrifugation on a pooled sample from healthy volunteers. CML and CEL were measured in extracted samples by ultra-performance liquid chromatography-tandem mass spectrometry. Median CML and CEL ranged from 0.132 to 0.140 mM/M lys and from 0.053 to 0.060 mM/M lys, respectively. No significant difference was shown CML or CEL in plasma/serum samples. Therefore samples collected as part of epidemiological studies that do not undergo specific sample treatment at collection are suitable for measuring CML and CEL.

Acid-labile protein-adducted heterocyclic aromatic amines in human blood are not viable biomarkers of dietary exposure: A systematic study

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of protein-rich foods. HCA residues adducted to blood proteins have been postulated as biomarkers of HCA exposure. However, the viability of quantifying HCAs following hydrolytic release from adducts in vivo and correlation with dietary intake are unproven. To definitively assess the potential of labile HCA-protein adducts as biomarkers, a highly sensitive UPLC-MS/MS method was validated for four major HCAs: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx). Limits of detection were 1-5 pg/ml plasma and recoveries 91-115%. Efficacy of hydrolysis was demonstrated by HCA-protein adducts synthesised in vitro. Plasma and 7-day food diaries were collected from 122 fasting adults consuming their habitual diets. Estimated HCA intakes ranged from 0 to 2.5 mg/day. An extensive range of hydrolysis conditions was examined for release of adducted HCAs in plasma. HCA was detected in only one sample (PhIP, 9.7 pg/ml), demonstrating conclusively for the first time that acid-labile HCA adducts do not reflect dietary HCA intake and are present at such low concentrations that they are not feasible biomarkers of exposure. Identification of biomarkers remains important. The search should concentrate on stabilised HCA-peptide markers and use of untargeted proteomic and metabolomic approaches.

Untargeted metabolomic analysis of human serum samples associated with different levels of red meat consumption: A possible indicator of type 2 diabetes?

Red meat consumption has been associated with negative health effects. A study to identify biomarkers of meat consumption was undertaken using serum samples collected from combining high resolution mass spectrometry (UPLC-QTof-MS) and chemometrics. Using orthogonal partial last-squares discriminant analysis (OPLS-DA), multivariate models were created for both modes of acquisition (ESI-/ESI+) and red meat intake classes (YES/NO). In the serum samples, a total 3280 and 3225 ions of interest were detected in positive and negative modes, respectively. Of these, 62 were found to be significantly different (p<0.05) between the two groups. Glycerophospholipids as well as other family lipids, such as lysophospholipids or sphingomyelin, were found significantly (p<0.05) different between yes and no red meat intake groups. This study has shown metabolomics fingerprints have the capability to identify potential biomarkers of red meat consumption, as well as possible health risk factors (e.g., key metabolic families related to the risk of development type 2 diabetes).

Meat consumption trends and relationship with body composition measurements in adolescents and young adults: the Northern Ireland Young Hearts Project

S. Watson, S. Brennan, J.V. Woodside, M. Cantwell, C.A. Boreham, C.E. Neville, Y.Y. Gong and G.J. Cuskelly Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, Belfast BT9 5BN, UK, Centre for Public Health, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast BT12 6BA, UK and Institute for Sport and Health, University College Dublin, Dublin, Republic of Ireland

Trends in Dietary Recommendations: Nutrient Intakes, Dietary Guidelines, Food Guides, Food Labels, and Dietary Supplements

The ultimate goal of nutritional research is the provision of diets that will give the best chance of long-term health. This chapter explores the various types of nutrition recommendations. They all share a goal of improving public health.