Hyperhomocysteinaemia, coagulation pathway activation and thrombophilia in patients with inflammatory bowel disease.

BACKGROUND: The 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism encoding the thermolabile variant is, when present as homozygote type (TT variant), a known genetic cause of mild hyperhomocysteinaemia (HHCY). This polymorphism has been observed in increased numbers in patients with inflammatory bowel disease (IBD). Coagulation and fibrinolysis are activated in patients with active IBD, but it is not known whether raised plasma homocysteine (HCY) found in patients with IBD significantly contributes to this activation. The aim of this study was to investigate if HHCY or presence of the TT variant significantly induces a hypercoagulable state in IBD patients receiving anti-inflammatory therapy during active disease, and to study if genetic determinants for thromboembolic disease are more frequent in these patients. METHODS: The study was designed as a cross-sectional study in an outpatient clinic comprising 106 IBD patients receiving anti-inflammatory therapy. Markers of coagulation were measured in order to elucidate whether patients with HHCY or the MTHFR TT variant were hypercoagulant compared with patients with no impairment of HCY metabolism. In addition, markers of inflammation and acute-phase reactants were measured in order to compare activity during active disease and during remission. Genetic determinants of thromboembolic disease in patients with IBD and in relevant controls were investigated in the expectation of a more frequent occurrence of these markers of thrombophilia if hypercoagulability could be a primary or contributory factor in IBD. RESULTS: No significant difference could be found in coagulation activity, acute-phase reactants or inflammatory markers in IBD patients with the TT variant of the 677C-->4T polymorphism or high (>15 micromol/L) plasma HCY levels, compared with IBD patients with no impairment of HCY metabolism. In patients with IBD, the coagulation activity was significantly increased during active disease compared with a state of remission. As expected, a significant difference regarding interleukin 6, C-reactive protein and erythrocyte sedimentation rate was present in IBD, comparing active disease with a state of remission. No significant complement activation was present in either of the groups or during active disease. Neither of the allele frequencies of genetic determinants for thrombophilia (coagulation factor V 1691G-->A (factor V Leiden) and factor II 20210G-->A polymorphisms) in the background population differed significantly from that in IBD patients. CONCLUSIONS: This study found no correlation between the MTHFR TT variant or HHCY and a hypercoagulable state in IBD patients receiving anti-inflammatory treatment. This coagulation activity is high during exacerbations of disease, but a considerable reduction is seen in patients on anti-inflammatory therapy compared with non-treated patients. Coagulation activation in IBD is probably a consequence of the inflammatory nature of the disease. That thrombophilia could be a contributory or primary factor in the development of IBD is not supported by the present study, as the frequencies for the genetic determinants for thrombophilia are similar in IBD patients and controls.

Influence of frying fat on the formation of heterocyclic amines in fried beefburgers and pan residues.

The influence of six frying fats (butter, margarine, margarine fat phase, liquid margarine, rapeseed oil and sunflower seed oil) on the formation of mutagenic/carcinogenic heterocyclic amines (HAs) during the frying of beefburgers was investigated. Frying was performed at 165 and 200 degrees C (i.e. under conditions that represented normal household cooking practices). The fried beefburgers and their corresponding pan residues were purified using solid-phase extraction and analysed for HAs using HPLC with photodiode array UV and fluorescence detection. The HAs 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 9H-pyrido[3,4-b]indole (norharman) and 1-methyl-9H-pyridol[3,4-b]indole (harman) were recovered. The amount increased with the temperature, and the content of HAs in the pan residue was much higher than in the corresponding beefburger. The amounts of MeIQx ranged from 0.2 to 1.6 ng/g in the beefburgers and from 0.8 to 4.3 ng/g in the pan residues. DiMeIQx ranged from undetectable to 0.4 ng/g in the beefburgers and from 0.4 to 1.3 ng/g in the residues. PhIP ranged from 0.08 to 1.5 ng/g in the meat and from 0.4 to 13.3 ng/g in the residues. The total amount of HAs in meat and pan residue combined was significantly lower after frying in sunflower seed oil or margarine than after frying with the other fats. The observed differences in MeIQx and DiMeIQx formation could be explained in terms of oxidation status (peroxide and anisidine value) and antioxidant content (vitamin A, vitamin E and tocopherols/tocotrienols) using partial least squares analysis.

Mutagenicity of pan residues and gravy from fried meat.

Lean pork meat was fried with or without the addition of frying-fat at 200 or 250 degrees C. The pan residues were collected by washing the hot pan with boiling water. When producing thickened gravy the water was substituted by a mixture of water and flour, milk and flour or cream and flour. The basic extracts were tested for mutagenicity in Ames' Salmonella test on strain TA98 with the addition of S9 mix. High amounts of mutagenicity were found in all samples. The amounts of mutagenicity in the pan residues were at a comparable level of the amounts found in the meat crusts. Thickening of the gravy caused only small changes in the mutagenicity.

Influence of frying fat on mutagenic activity in lean pork meat.

Mutagenic activity in lean pork meat fried at two different pan temperatures, 200 degrees C and 250 degrees C, with or without the addition of fat, was measured in Ames' Salmonella test on strain TA98. 9 different fats with varying chemical composition were tested. All fried meat samples were shown to be mutagenic. At the frying temperature of 200 degrees C differences between meat samples fried in different fats or without fat, respectively, were small. All meat samples fried at 250 degrees C were considerably more mutagenic than the samples fried at 200 degrees C. At 250 degrees C, the addition of fat caused a significant rise in mutagenic activity. We believe this is mainly an effect of more efficient heat transfer from the bottom of the frying-pan to the meat samples, although other factors may also contribute.

High mutagenic activity formed in pan-broiled pork.

Lean pork was pan-broiled at various temperatures between 100 and 290 degrees C. Cooking was performed in an open frying pan common for domestic use in Sweden. No fat was added. Cooking procedures are clearly defined in order to facilitate inter-laboratory comparisons. The crust was extracted with organic solvents of varying polarity. The mutagenic activity was assayed with Ames' Salmonella mutagenicity test. Large amounts of mutagenic activity were detected in samples pan-broiled at 200-290 degrees C. The mutagenic activity recovered was about 10 times higher than that reported by previous investigators to be found during cooking of meat under similar conditions. This discrepancy could be due to differences in the composition of Swedish pork as compared to the meat samples used by other investigators or to different methodology in cooking and extraction procedures.