Combined Markers to Assess Meat Intake-Human Metabolomic Studies of Discovery and Validation.

SCOPE: Biomarkers of red meat may clarify the relationship between meat intake and health. This paper explores the discovery of biomarkers of intake for three types of meat with varying heme iron content. Candidate biomarkers for red and general meat are further evaluated based on defined validation criteria. METHODS AND RESULTS: In a randomized cross-over meal study, healthy volunteers consume a randomized sequence of four test meals: chicken, pork, beef, and a control made of egg white and pea. Fasting and postprandial urine samples are collected to cover 48 h and profiled by untargeted LC-ESI-qTOF-MS metabolomics. The profiles following the meal challenges are explored by univariate and multivariate analyses. Nine red, four white, and eight general meat biomarkers are selected as putative biomarkers, originating from collagen degradation, flavour compounds, and amino acid metabolism. Heme-related metabolites are masked by the chlorophyll content of the control meal. The candidate biomarkers are confirmed in an independent meal study and validated for plausibility, robustness, time-response, and prediction performance. Combinations of biomarkers are more efficient than single markers in predicting meat intake. CONCLUSION: New combinations of partially validated biomarkers are proposed to assess terrestrial meat intake and thus help disentangle the effects of meat consumption on human health.

Development and validation of a sensitive LC-MS/MS assay for the quantification of anserine in human plasma and urine and its application to pharmacokinetic study.

Carnosine (beta-alanyl-L-histidine) and its methylated analogue anserine are present in relevant concentrations in the omnivore human diet. Several studies reported promising therapeutic potential for carnosine in various rodent models of oxidative stress and inflammation-related chronic diseases. Nevertheless, the poor serum stability of carnosine in humans makes the translation of rodent models hard. Even though anserine and carnosine have similar biochemical properties, anserine has better serum stability. Despite this interesting profile, the research on anserine is scarce. The aim of this study was to explore the bioavailability and stability of synthesized anserine by (1) performing in vitro stability experiments in human plasma and molecular modelling studies and by (2) evaluating the plasma and urinary pharmacokinetic profile in healthy volunteers following different doses of anserine (4-10-20 mg/kg body weight). A bio-analytical method for measuring anserine levels was developed and validated using liquid chromatography-electrospray mass spectrometry. Both plasma (CMAX: 0.54-1.10-3.12 µM) and urinary (CMAX: 0.09-0.41-0.72 mg/mg creatinine) anserine increased dose-dependently following ingestion of 4-10-20 anserine mg/kg BW, respectively. The inter-individual variation in plasma anserine was mainly explained by the activity (R2 = 0.75) and content (R2 = 0.77) of the enzyme serum carnosinase-1. Compared to carnosine, a lower interaction energy of anserine with carnosinase-1 was suggested by molecular modelling studies. Conversely, the two dipeptides seems to have similar interaction with the PEPT1 transporter. It can be concluded that nutritionally relevant doses of synthesized anserine are well-absorbed and that its degradation by serum carnosinase-1 is less pronounced compared to carnosine. This makes anserine a good candidate as a more stable carnosine-analogue to attenuate chronic diseases in humans.

Impact of red meat consumption on the metabolome of rats.

SCOPE: The scope of the present study was to investigate the effects of red versus white meat intake on the metabolome of rats. METHODS AND RESULTS: Twenty-four male Sprague-Dawley rats were randomly assigned to 15 days of ad libitum feeding of one of four experimental diets: (i) lean chicken, (ii) chicken with lard, (iii) lean beef, and (iv) beef with lard. Urine, feces, plasma, and colon tissue samples were analyzed using 1 H NMR-based metabolomics and real-time PCR was performed on colon tissue to examine the expression of specific genes. Urinary excretion of acetate and anserine was higher after chicken intake, while carnosine, fumarate, and trimethylamine N-oxide excretion were higher after beef intake. In colon tissue, higher choline levels and lower lipid levels were found after intake of chicken compared to beef. Expression of the apc gene was higher in response to the lean chicken and beef with lard diets. Correlation analysis revealed that intestinal apc gene expression was correlated with fecal lactate content (R2 = 0.65). CONCLUSION: This study is the first to identify specific differences in the metabolome related to the intake of red and white meat. These differences may reflect perturbations in endogenous metabolism that can be linked to the proposed harmful effects associated with intake of red meat.

Analysis of therapeutic peptides in human urine by combination of capillary zone electrophoresis-electrospray mass spectrometry with preparative capillary isotachophoresis sample pretreatment.

The presented study deals with the off-line coupling of preparative isotachophoresis (pITP) with on-line combination of capillary zone electrophoresis with electrospray mass spectrometric detection (CZE-ESI-MS) used for the analysis of therapeutic peptides (anserine, carnosine, and buserelin) in complex matrix (urine). Preparative capillary isotachophoresis, operating in a discontinuous fractionation mode in column-coupling configuration, served as a sample pretreatment technique to separation, and fractionation of mixture of therapeutic peptides present in urine at low concentration level. The fractions isolated by pITP procedure were subsequently analyzed by capillary zone electrophoresis with electrospray mass spectrometric detection. Acetic acid at 200 mmol L(-1) concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. In pITP fractionation procedure, sodium cation (10 mmol L(-1) concentration) as leading ion and beta-alanine as terminating ion (20 mmol L(-1) concentration) were used. While using CZE-ESI-MS, the limits of detection were 0.18 µg mL(-1) for carnosine, 0.17 µg mL(-1) for anserine and 0.64 µg mL(-1) for buserelin in water and 0.19 µg mL(-1) for carnosine, 0.50 µg mL(-1) for anserine and 0.74 µg mL(-1) for buserelin in 10 times diluted urine, respectively. The cleaning power of pITP sample pretreatment was proved as the peptides provided the higher MS signals at lower concentration levels resulting from the minimized matrix effects. The quality of obtained MS/MS spectra was very good so that they can provide information about the structure of analytes, and they were used for verification of the analytes identities. The pITP pretreatment improved the detection limits of the analyzed therapeutic peptides at least 25 times compared to the CZE-ESI-MS itself.

Profiling histidine dipeptides in plasma and urine after ingesting beef, chicken or chicken broth in humans.

The in vitro metabolic stability of histidine-dipeptides (HD), carnosine (CAR) and anserine (ANS), in human serum, and their absorption kinetics after ingesting pure carnosine or HD rich foods in humans have been investigated. Healthy women (n = 4) went through four phases of taking one dose of either 450 mg of pure carnosine, 150 g beef (B), 150 g chicken (C), or chicken broth (CB) from 150 g chicken with a >2-week washout period between each phase. Blood samples were collected at 0, 30, 60, 100, 180, 240, and 300 min, and urine samples before and after (up to 7 h) ingesting pure carnosine or food. Both plasma and urine samples were analyzed for HD concentrations using a sensitive and selective LC-ESI-MS/MS method. CAR was undetectable in plasma after ingesting pure carnosine, B, C or CB. By contrast, plasma ANS concentration was significantly increased (P < 0.05) after ingesting C or CB, respectively. Urinary concentrations of both CAR and ANS were 13- to 14-fold increased after ingesting B, and 14.8- and 243-fold after CB ingestion, respectively. Thus, dietary HD, which are rapidly hydrolyzed by carnosinase in plasma, and excreted in urine, may act as reactive carbonyl species sequestering agents.

A sensitive and specific high performance liquid chromatographic assay for imidazole dipeptides and 3-methylhistidine in human muscle biopsies, serum and urine.

A sensitive and specific high performance liquid chromatographic method is described for measuring imidazole dipeptides and 3-methylhistidine in human muscle biopsies, serum and urine. Muscle extract, serum or urine was reacted with o-phthaldialdehyde and the derivatives were separated by reversed phase chromatography with column switching and fluorescence detection.

Determination of ophidine in human urine.

Ophidine (beta-alanyl-3-methylhistidine) was first detected in the urine of two patients and later in two members of the laboratory staff loaded with whale meat, by column chromatography, high-voltage paper electrophoresis and two-dimensional paper chromatography. The ophidine peak was detected between homocarnosine and dimethylarginine using a lithium buffer gradient in column chromatography. In paper chromatography the ophidine spot was detected at a position close to anserine and homocarnosine. The ophidine in the urine from the patients was of dietary origin since it was absent in the urine a few weeks later.