1 Medical Research Council, Dunn Human Nutrition Unit, Welcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY and
2 Pollock and Pool Ltd, Ladbroke Close, Reading RG5 4DX, UK
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Abstract |
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Abbreviations: ATNC, apparent total N-nitroso compounds; MTT, mean transit time; NOC, N-nitroso compounds; PABA, p-aminobenzoic acid.
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Introduction |
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Large intestinal N-nitrosation has previously received little attention due to analytical difficulties. The development of a group-selective method for total NOC detection, however, has allowed NOC detection in several biological fluids, including faeces (9). NOC detected by this method are referred to as apparent total N-nitroso compounds (ATNC) because the method may be susceptible to false positives from S-nitrosothiols and nitrolic acids (10). Meat is a source of nitrogenous residues entering the large intestine which are available for large intestinal N-nitrosation. We have therefore investigated whether increasing doses of red meat result in increased large intestinal N-nitrosation in human volunteers. A demonstration of such an effect would support epidemiological associations between red meat intake and colon cancer (11,12). Red meat was chosen because an initial study indicated that red, rather than white, meat initiated large intestinal N-nitrosation (13). We also show that levels of large intestinal N-nitrosation remain high when red meat is given over a 40 day period of time and that longer residence times within the colon are associated with higher levels.
Permission for the studies was given by the Dunn Nutrition Unit Ethics Committee and each volunteer signed a consent form after receiving a detailed explanation of the study protocol and aims. The two studies were carried out in a metabolic suite at separate times where all food and drink was provided and all specimens collected. Only foods and drinks which were provided by the diet technicians, weighed to the nearest gram from a specifically designed diet, were consumed. Subjects remained within the suite for breakfast and dinner and body weights were monitored to ensure a constant weight throughout. Lunches were pre-packed. In order to keep nitrate intake constant, deionized water was given throughout for drinking and used in cooking and low nitrate vegetables were used. All food was bought from the same batch and stored for later use throughout the study in order to minimize day-to-day variation.
To determine the effect of different doses of meat, eight healthy male volunteers (aged 3649 years) were studied over four 10 day dietary periods. Four doses of meat were studied, 0, 60, 240 and 420 g/day [given as 0100 g roast beef at lunch and 0320 g beef (lasagne or steak) or pork (sweet and sour pork) at dinner]. The protein contents of the diets were 42, 58, 100 and 167 g/day or 8, 11, 19 and 28% total energy, respectively (14). A glucose polymer drink and cream were substituted for meat in the low meat diets to equalize the energy content. All diets were constant in fat (28% total energy) and non-starch polysaccharide (13 g/day) and adjusted for the energy needs of each subject with extra bread, low fat margarine and marmalade (14).
For each study diets were randomized using a crossover design and each subject acted as their own control. Faecal samples were collected daily and were weighed, X-rayed and stored at 20°C. Recovery of radio-opaque faecal markers was noted and used to monitor compliance and to calculate mean transit time (MTT) (15). Of the total faecal markers administered 96% was recovered. Mean faecal weights were determined during the final 4 days of each diet and were corrected for faecal marker output by multiplication of mean daily weight by the ratio of marker output to marker input. Faecal samples collected on days 810 were processed within 20 min of excretion. Samples were diluted 4-fold with ultrapure deionized water, homogenized in a stomacher (Colworth 3500; Seward) and centrifuged at 4500 r.p.m. for 10 min. Each supernatant was filtered and stored at 20°C before being analysed for NOC and nitrite by the release of nitric oxide (NO) following chemical denitrosation of each compound via thermal energy analysis (16). The sample was then treated with sulphamic acid to remove nitrite and reinjected into the refluxing solvent to determine NO released from NOC only. Nitrite was calculated by the difference between the two results. During each analysis 160 ng N-nitrosodipropylamine was injected into the system as an internal standard to check recovery.
Each subject completed a 24 h urine collection on day 10 of each dietary period. Compliance with the 24 h collection was checked using the p-aminobenzoic acid (PABA) test (17). With the exception of one sample, all 24 h collections were complete, as PABA recoveries exceeded 85%. Urinary nitrogen was determined in complete collections, using a semi-automated Kjeldahl technique.
Results were log transformed (to the base 10) and analysed using Microsoft DATA DESK 4.0 for the Macintosh. Dietary effects were determined using two-way ANOVA with diet and subject as factors and probability results less than the 0.05 level were regarded as significant. Fischer's least significant difference tests were used to compare individual means when significant F values were found. Possible relationships between variables were calculated using Pearson product correlation coefficients. From repeat analyses of subjects on the high (420 g) meat diet, the within person standard deviation was 56 µg/day and setting = 0.05 and ß as 0.2, the study had sufficient power to detect an 80 µg difference in ATNC between the 60 and 420 g diets with eight subjects.
Faecal ATNC and nitrite and urinary N excretion increased significantly with higher intakes of meat (Table I) and each of these correlated positively with dietary protein (r = 0.75, P < 0.0001, r = 0.41, P = 0.02 and r = 0.95, P < 0.0001). There were no significant differences in faecal ATNC and nitrite and urinary N excretion when meat intake increased from 0 to 60 g/day. ATNC excretion on the 240 and 420 g diets was significantly greater than on the low meat diets, i.e. 0 and 60 g (P < 0.0001 for each comparison) (Figure 1
). There was a consistent increase in ATNC with increased dose of meat for each subject. Inter-individual variation accounted for 15 (P = 0.03) and 35% (P < 0.0001) of the total variability calculated for daily ATNC excretion and ATNC concentration results with an individual range on the high meat diet of 93427 µg/day (7715103 ng/g). Mean faecal NO2 excretion increased at meat intakes >60 g/day (P = 0.023 and P = 0.046 for NO2 concentration and daily excretion, respectively). Daily NO2 and ATNC excretion were positively associated (r = 0.403, P = 0.022). Diet accounted for 91% (P < 0.0001) of the total variability in daily urinary N excretion.
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Taking account of two previous studies from our laboratory, the influence of red meat on faecal ATNC excretion has now been shown in 24 healthy male volunteers, all of whom were studied in a metabolic suite where diet could be carefully controlled (13,18). The present study shows that this association is dose responsive. At the higher levels of meat consumption concentrations of ATNC were found to be of the same order of magnitude as the concentration of tobacco-specific NOC in cigarette smoke (19). Levels of ATNC in foods are low and a diet containing 600 g red meat/day contained only 13 µg ATNC/day (18). Faecal ATNC levels exceeded this value by as much as 30-fold for some subjects, showing that faecal ATNC excretion during the study was due to endogenous formation.
Endogenous formation appears to be the most potent source of human exposure to NOC following the reaction between nitrosating agents and nitrogenous substrates (19). N-nitrosation can occur under acid, neutral or inflammatory conditions and hence NOC are formed at a number of sites in the body (20). In the large intestine endogenous nitrosation is brought about by bacteria present in the large intestine, as has been shown by studies with germ-free animals (6). Even with the no meat diet endogenous nitrosation occurs, because even at low protein intakes there are significant dietary and non-dietary protein residues entering the large bowel from sloughed cells, mucin and enzymes which would be available for proteolysis and nitrosation (2). However, with only 60 g meat/day there was no significant elevation in ATNC or nitrite. Protein intakes were similar on the no and 60 g meat diets so that the amounts of protein residues available for nitrosation reaching the intestine would have been similar. The lack of effect of low doses of meat may be important in formulating public health recommendations for the consumption of meat in relation to cancer (11,12). At higher meat intakes arginine from meat may also influence N-nitrosation, as many mammalian cells produce NO by oxidation of the terminal guanido nitrogen of L-arginine to citrulline by NO synthase in the presence of oxygen and NADPH (21). 420 g of meat is calculated to provide ~7.3 g arginine, as compared with 1.04 g from 60 g meat (22).
Despite the highly controlled conditions of this study, there remained extensive individual variation in faecal ATNC excretion which might be attributable to individual variations in gut flora, with high responders harbouring high populations of nitrate- and nitrite-reducing bacteria. However, a major determinant of individual variation in levels was length of residence in the gut, as ATNC concentration correlated positively with MTT (Figure 2). A longer retention in the gut would favour increased ATNC formation due to more efficient bacterial protein degradation leading to increased nitrosatable substrates.
Ongoing work is investigating the genotoxic effects of faecal NOC levels. In preliminary work, seven other volunteers were maintained on the 420 g meat diet and faecal water isolated and genotoxicity assessed by Comet assay using the method of Venturi et al. (23). Samples from six of seven subjects were genotoxic, with mean tail moments of 1624 units/sample (24). This is a comparatively high percentage genotoxicity, compared with the study of Venturi et al., where only 43% of samples were genotoxic (23). A diet high in fat and meat but low in dietary fibre has also been shown to increase the genotoxic potential of faecal water collected from healthy human volunteers (25). Findings from the present study suggest that NOC produced in response to high meat diets may be one factor contributing to the genotoxic effects of these diets.
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