Carbon and nitrogen isotopic ratios of urine and faeces as novel nutritional biomarkers of meat and fish intake

Purpose Meat and fish consumption are associated with changes in the risk of chronic diseases. Intake is mainly assessed using self-reporting, as no true quantitative nutritional biomarker is available. The measurement of plasma fatty acids, often used as an alternative, is expensive and time-consuming. As meat and fish differ in their stable isotope ratios, δ13C and δ15N have been proposed as biomarkers. However, they have never been investigated in controlled human dietary intervention studies. Objective In a short-term feeding study, we investigated the suitability of δ13C and δ15N in blood, urine and faeces as biomarkers of meat and fish intake. Methods The dietary intervention study (n = 14) followed a randomised cross-over design with three eight-day dietary periods (meat, fish and half-meat–half-fish). In addition, 4 participants completed a vegetarian control period. At the end of each period, 24-h urine, fasting venous blood and faeces were collected and their δ13C and δ15N analysed. Results There was a significant difference between diets in isotope ratios in faeces and urine samples, but not in blood samples (Kruskal–Wallis test, p < 0.0001). In pairwise comparisons, δ13C and δ15N were significantly higher in urine and faecal samples following a fish diet when compared with all other diets, and significantly lower following a vegetarian diet. There was no significant difference in isotope ratio between meat and half-meat–half-fish diets for blood, urine or faecal samples. Conclusions The results of this study show that urinary and faecal δ13C and δ15N are suitable candidate biomarkers for short-term meat and fish intake.

Effect of processed and red meat on endogenous nitrosation and DNA damage

Haem in red meat (RM) stimulates the endogenous production of mutagenic nitroso compounds (NOC). Processed (nitrite-preserved red) meat additionally contains high concentrations of preformed NOC. In two studies, of a fresh RM versus a vegetarian (VEG) diet (six males and six females) and of a nitrite-preserved red meat (PM) versus a VEG diet (5 males and 11 females), we investigated whether processing of meat might increase colorectal cancer risk by stimulating nitrosation and DNA damage. Meat diets contained 420 g (males) or 366 g (females) meat/per day. Faecal homogenates from day 10 onwards were analysed for haem and NOC and asso- ciated supernatants for genotoxicity. Means are adjusted for differ- ences in male to female ratios between studies. Faecal NOC concentrations on VEG diets were low (2.6 and 3.5 mmol/g) but significantly higher on meat diets (PM 175 ± 19 nmol/g versus RM 185 ± 22 nmol/g; P 5 0.75). The RM diet resulted in a larger pro- portion of nitrosyl iron (RM 78% versus PM 54%; P < 0.0001) and less nitrosothiols (RM 12% versus PM 19%; P < 0.01) and other NOC (RM 10% versus PM 27%; P < 0.0001). There was no statis- tically significant difference in DNA breaks induced by faecal water (FW) following PM and RM diets (P 5 0.80). However, PM re- sulted in higher levels of oxidized pyrimidines (P < 0.05). Surpris- ingly, VEG diets resulted in significantly more FW-induced DNA strand breaks than the meat diets (P < 0.05), which needs to be clarified in further studies. Meats cured with nitrite have the same effect as fresh RM on endogenous nitrosation but show increased FW-induced oxidative DNA damage.

Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N-nitroso compound formation attributed to red meat consumption

Red meat consumption causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC). The genotoxic effects of these ATNCs were investigated using two different Comet assay protocols to determine the genotoxicity of fecal water samples. Fecal water samples were obtained from two studies of a total of 21 individuals fed diets containing different amounts of red meat, protein, heme, and iron. The first protocol incubated the samples with HT-29 cells for 5 min at 4 degrees C, whereas the second protocol used a longer exposure time of 30 min and a higher incubation temperature of 37 degrees C. DNA strand breaks were quantified by the tail moment (DNA in the comet tail multiplied by the comet tail length). The results of the two Comet assay protocols were significantly correlated (r = 0.35, P = 0.003), however, only the second protocol resulted in detectable levels of DNA damage. Inter-individual effects were variable and there was no effect on fecal water genotoxicity by diet (P > 0.20), mean transit time (P = 0.588), or weight (P = 0.705). However, there was a highly significant effect of age (P = 0.019). There was no significant correlation between concentrations of ATNCs in fecal homogenates and fecal water genotoxicity (r = 0.04, P = 0.74). ATNC levels were lower in fecal water samples (272 microg/kg) compared to that of fecal homogenate samples (895 microg/kg) (P < 0.0001). Failure to find dietary effects on fecal water genotoxicity may therefore be attributed to individual variability and low levels of ATNCs in fecal water samples.

The potential to intensify sulforaphane formation in cooked broccoli (Brassica oleracea var. italica) using mustard seeds (Sinapis alba)

Sulforaphane, a naturally occurring cancer chemopreventive, is the hydrolysis product of glucoraphanin, the main glucosinolate in broccoli. The hydrolysis requires myrosinase isoenzyme to be present in sufficient activity; however processing leads to its denaturation and hence reduced hydrolysis. In this study, the effect of adding mustard seeds, which has a more resilient isoform of myrosinase, to processed broccoli was investigated with a view to intensify the formation of sulforaphane. Thermal inactivation of myrosinase from both broccoli and mustard seeds was studied. Thermal degradation of broccoli glucoraphanin was investigated in addition to the effects of thermal processing on the formation of sulforaphane and sulforaphane nitrile. Limited thermal degradation of glucoraphanin (less than 12 %) was observed when broccoli was placed in vacuum sealed bag (sous vide) and cooked in a water bath at 100 ºC for 8 and 12 min. Boiling broccoli in water prevented the formation of any significant levels of sulforaphane due to inactivated myrosinase. However, addition of powdered mustard seeds to the heat processed broccoli significantly increased the formation of sulforaphane.

Was the Australian meat and livestock corporations advertising efficient?

A theory of the allocation of producer levies earmarked for downstream promotion is developed and applied to quarterly series (1970:2–1988:4) on red-meats advertising by the Australian Meat and Live-stock Corporation. Robust inferences about program efficiency are contained in the coefficients of changes in promotion effort regressed against movements in farm price and quantity. Empirical evidence of program efficiency is inconclusive. While the deeper issue of efficient disbursement of funds remains an open question, there is evidence, at least, of efficient taxation.

Heme-induced biomarkers associated with red meat promotion of colon cancer are not modulated by the intake of nitrite

Red and processed meat consumption is associated with the risk of colorectal cancer. Three hypotheses are proposed to explain this association, via heme-induced oxidation of fat, heterocyclic amines, or N-nitroso compounds. Rats have often been used to study these hypotheses, but the lack of enterosalivary cycle of nitrate in rats casts doubt on the relevance of this animal model to predict nitroso- and heme-associated human colon carcinogenesis. The present study was thus designed to clarify whether a nitrite intake that mimics the enterosalivary cycle can modulate hemeinduced nitrosation and fat peroxidation. This study shows that, in contrast with the starting hypothesis, drinking water added with nitrite to mimic the salivary nitrite content did not change the effect of hemoglobin on biochemicalmarkers linked to colon carcinogenesis, notably lipid peroxidation and cytotoxic activity in the colon of rat. However, ingested sodium nitrite increased fecal nitrosocompounds level, but their fecal concentration and their nature (iron-nitrosyl) would probably not be associated with an increased risk of cancer.We thus suggest that the rat model could be relevant for study the effect of red meat on colon carcinogenesis, in spite of the lack of nitrite in the saliva of rats.

Preference and consumption of a taste enhanced meat meal by older hospital patients: a pilot study

BACKGROUND: Older hospital patients are considered to be at risk of malnutrition due to insufficient dietary intake. OBJECTIVE: To determine whether taste enhancement, using ingredients naturally high in umami compounds, increases preference and consumption of a meal by older hospital patients. METHODS: 31 patients (65–92 years) on elderly carewards in aUKNHS Trust hospital took part in a single-blinded preference and consumption study. They tasted two meats (control and enhanced, presented in balanced order) and stated their preference. At lunch, control and enhanced cottage pie and gravy were served concurrently; patients were asked to consume ad libitum and intake was measured. RESULTS: Taste enhanced meat was significantly preferred (P = 0.001). Although mean consumption was higher for the enhanced compared to control meal (137 g versus 119 g), with higher levels of energy (103 kcal versus 82 kcal) and protein (4.6 g versus 3.4 g) consumed; differences were not significant. CONCLUSIONS: Natural ingredients rich in umami taste compounds can successfully be used to increase preference of meat based meals by older hospital patients. Larger trials are needed to determine whether such increases in preference can significantly increase consumption.

Array based detection of antibiotic resistance genes in Gram negative bacteria isolated from retail poultry meat in the UK and Ireland

The use of antibiotics in birds and animals intended for human consumption within the European Union (EU) and elsewhere has been subject to regulation prohibiting the use of antimicrobials as growth promoters and the use of last resort antibiotics in an attempt to reduce the spread of multi-resistant Gram negative bacteria. Given the inexorable spread of antibiotic resistance there is an increasing need for improved monitoring of our food. Using selective media, Gram negative bacteria were isolated from retail chicken of UK-Intensively reared (n = 27), Irish-Intensively reared (n = 19) and UK-Free range (n = 30) origin and subjected to an oligonucleotide based array system for the detection of 47 clinically relevant antibiotic resistance genes (ARGs) and two integrase genes. High incidences of β-lactamase genes were noted in all sample types, acc (67%), cmy (80%), fox (55%) and tem (40%) while chloramphenicol resistant determinants were detected in bacteria from the UK poultry portions and were absent in bacteria from the Irish samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to qualitatively analyse the Gram negative population in the samples and showed the expected diversity based on band stabbing and DNA sequencing. The array system proved to be a quick method for the detection of antibiotic resistance gene (ARG) burden within a mixed Gram negative bacterial population.

O6-carboxymethylguanine DNA adduct formation and lipid peroxidation upon in vitro gastrointestinal digestion of haem-rich meat

Scope Epidemiological and clinical studies have demonstrated that the consumption of red haem-rich meat may contribute to the risk of colorectal cancer. Two hypotheses have been put forward to explain this causal relationship, i.e. N-nitroso compound (NOC) formation and lipid peroxidation (LPO). Methods and Results In this study, the NOC-derived DNA adduct O6-carboxymethylguanine (O6-CMG) and the LPO product malondialdehyde (MDA) were measured in individual in vitro gastrointestinal digestions of meat types varying in haem content (beef, pork, chicken). While MDA formation peaked during the in vitro small intestinal digestion, alkylation and concomitant DNA adduct formation was observed in seven (out of 15) individual colonic digestions using separate faecal inocula. From those, two haem-rich meat digestions demonstrated a significantly higher O6-CMG formation (p < 0.05). MDA concentrations proved to be positively correlated (p < 0.0004) with haem content of digested meat. The addition of myoglobin, a haem-containing protein, to the digestive simulation showed a dose–response association with O6-CMG (p = 0.004) and MDA (p = 0.008) formation. Conclusion The results suggest the haem-iron involvement for both the LPO and NOC pathway during meat digestion. Moreover, results unambiguously demonstrate that DNA adduct formation is very prone to inter-individual variation, suggesting a person-dependent susceptibility to colorectal cancer development following haem-rich meat consumption.

A central role for heme iron in colon carcinogenesis associated with red meat intake

Epidemiology shows that red and processed meat intake is associated with an increased risk of colorectal cancer. Heme iron, heterocyclic amines and endogenous N-nitroso compounds (NOC) are proposed to explain this effect, but their relative contribution is unknown. Our study aimed at determining, at nutritional doses, which is the main factor involved and proposing a mechanism of cancer promotion by red meat. The relative part of heme iron (1% in diet), heterocyclic amines (PhIP+MeIQx, 50+25 μg/kg in diet) and NOC (induced by NaNO2+NaNO3 0.17+0.23 g/l of drinking water) was determined by a factorial design and preneoplastic endpoints in chemically-induced rats and validated on tumors in Min mice. The molecular mechanisms (genotoxicity, cytotoxicity) were analyzed in vitro in normal and Apc- deficient cell lines and confirmed on colon mucosa. Heme iron increased the number of preneoplastic lesions but dietary heterocyclic amines and NOC had no effect on carcinogenesis in rats. Dietary hemoglobin increased tumor load in Min mice (control diet: 67±39 mm2; 2,5% hemoglobin diet: 114±47 mm2, p=0.004). In vitro, fecal water from rats given hemoglobin was rich in aldehydes and was cytotoxic to normal cells, but not to premalignant cells. The aldehydes 4-hydroxynonenal and 4-hydroxyhexenal were more toxic to normal versus mutated cells and were only genotoxic to normal cells. Genotoxicity was also observed in colon mucosa of mice given hemoglobin. These results highlight the role of heme iron in the promotion of colon cancer by red meat and suggest that heme iron could initiate carcinogenesis through lipid peroxidation.

Consumer acceptability and sensory profile of cooked broccoli with mustard seeds added to improve chemoprotective properties

Broccoli, a rich source of glucosinolates, is a commonly consumed vegetable of the Brassica family. Hydrolysis products of glucosinolates, isothiocyanates, have been associated with health benefits and contribute to the flavour of Brassica. However, boiling broccoli causes the myrosinase enzyme needed for hydrolysis to denature. In order to ensure hydrolysis, broccoli must either be mildly cooked or active sources of myrosinase, such as mustard seed powder, can be added post-cooking. In this study, samples of broccoli were prepared in six different ways; standard boiling with and without mustard seeds, sous-vide cooking at low temperature (70 °C) and sous-vide cooking at higher temperature (100 ºC) without mustard and with mustard at two different concentrations. The majority of consumers disliked the mildly cooked broccoli samples (70 ºC, 12 min, sous-vide) which had a hard and stringy texture. The highest mean consumer liking was for standard boiled samples (100 ºC, 7 min). Addition of 1% mustard seed powder developed sensory attributes such as pungency, burning sensation, mustard odour and flavour. One cluster of consumers (32%) found mustard seeds to be a good complement to cooked broccoli, however, the majority disliked the mustard-derived sensory attributes. Where the mustard seeds were partially processed, doubling the addition to 2% led to only the same level of mustard flavour and pungency as 1% unprocessed seeds, and mean consumer liking remained unaltered. This suggests that optimisation of the addition level of partially processed mustard seeds may be a route to enhance bioactivity of cooked broccoli without compromising consumer acceptability.

The effects of tannin-containing ground pine bark diet upon nutrient digestion, nitrogen balance, and mineral retention in meat goats

Background Pine bark is a rich source of phytochemical compounds including tannins, phenolic acids, anthocyanins, and fatty acids. These phytochemicals have potential to significantly impact on animal health and animal production. The goal of this work is to measure the effects of tannins in ground pine bark as a partial feed replacement on feed intake, dietary apparent digestibility, nitrogen balance, and mineral retention in meat goats. Results Eighteen Kiko cross goats (initial BW = 31.8 ± 1.49 kg) were randomly assigned to three treatment groups (n = 6). Dietary treatments were tested: control (0 % pine bark powder (PB) and 30 % wheat straw (WS)); 15 % PB and 15 % WS, and 30 % PB and 0 % WS. Although dry matter (DM) intake and digestibility were not affected (P > 0.10) by feeding PB, neutral detergent fiber (linear; P = 0.01), acid detergent fiber (linear; P = 0.001) and lignin digestibility (linear; P = 0.01) decreased, and crude protein (CP) digestibility tended to decrease (P = 0.09) as PB increased in the diet, apparent retention of Ca (P = 0.09), P (P = 0.03), Mg (P = 0.01), Mn (P = 0.01), Zn (P = 0.01) and Fe (P = 0.09) also increased linearly. Nitrogen intake and fecal N excretion were not affected (P > 0.05) by addition of PB in the diet, but N balance in the body was quadratically increased (P < 0.01) in the 15 % PB diet compared to other diets. This may be due to more rumen escape protein and less excreted N in the urine with the 15 % PB diet. The study showed that a moderate level of tannin-containing pine bark supplementation could improve gastrointestinal nitrogen balance with the aim of improving animal performance. Conclusion These results suggest that tannin-containing PB has negative impact on fiber, lignin, and protein digestibility, but positively impacted on N-balance.