Arthur W. Bull

Determination of malondialdehyde by ion-pairing high-performance liquid chromatography☆

A method for the analysis of malondialdehyde (MDA) by ion-pairing HPLC is described. The method is direct, no derivitization is required, and sample preparation is minimal. After removal of particulates, the samples are injected directly onto an octadecylsilane column which is eluted with 14% (v/v) acetonitrile in 50 mM myristyltrimethylammonium bromide. 1 mM phosphate, pH 6.8. Detection is accomplished by monitoring absorbance at 254 nm or for greater sensitivity at 267 nm. The lower limit for reliable quantitation is 5 pmol MDA and the dynamic range extends to at least 4 nmol MDA. The method has been applied to the quantitation of MDA production during microsomal lipid peroxidation and to an assessment of the stability of MDA in microsomal and urine samples.

Distribution and oxidation of malondialdehyde in mice

The in vivo metabolism of malondialdehyde (MDA) by male and female Swiss mice was investigated. Distribution of an i.p. dose of MDA is rapid and uniform throughout the body. Conversion of 14C-labeled MDA to CO2 is complete 4 hours after an i.p. dose of 5 mumol to 200 mumol with no signs of short term toxicity. The yields of CO2 from [1-14C]-beta-alanine, [3-14C]-beta-alanine, [1-14C]-sodium acetate, and [2-14C]-sodium acetate were also determined. Comparison of the yields of CO2 from this series of compounds suggests the intermediacy of malonic semialdehyde in the metabolism of MDA. High doses (600 mumol) of beta-alanine or acetate given prior to 14C-MDA reduced the yield of 14CO2. Ethanol and disulfiram were both inhibitors of MDA metabolism, indicating the involvement of aldehyde dehydrogenase in the oxidation of MDA. These data demonstrate the ability of animal tissues to rapidly remove exogenously administered MDA. They also have implications with respect to the possible pathological consequences of in vivo MDA generation.

Modulation of human colonic lamina propria lymphocyte proliferation. Effect of bile acids and oxidized fatty acids.

Bile acids were been implicated in several pathologic processes, such as secretory diarrhea, carcinogenesis, and immunomodulation of human peripheral blood lymphocytes. Nevertheless, their effect on the human gut immune system is not known. In this study we investigate the effect of several bile acids (cholate, deoxycholate, chenodeoxycholate) and 13-hydroperoxylinoleic acid (conc. 0.1–1000, μM) on human colonic lamina propria lymphocyte (LPL) DNA synthesis and cell proliferation. In addition, the effect of these bile acids on LPL ornithine decarboxylase activity was also determined. Significant dose-dependent inhibition of [3H]thymidine incorporation in Con A-stimulated LPL was observed. Parallel inhibition was seen on LPL cell proliferation. Furthermore, bile acids inhibited ornithine decarboxylase activity in Con A-stimulated LPL. These effects on cell proliferation were not due to the LPL cytolysis as viability and cell membrane integrity were not altered. Our results suggest that bile acid has an immunoregulatory function on the human mucosal immune system and may have a role during pathological states.

The essential fatty acid requirement for azoxymethane-induced intestinal carcinogenesis in rats

The essential fatty acid requirement for the development of intestinal carcinogenesis was determined and compared to the overall essential fatty acid status of the animals as measured by the triene/tetraene ratio in the plasma, liver and colon. To induce tumors, male Sprague-Dawley rats were given two weekly injections (20 mg/kg body wt) of azoxymethane. Two weeks after the last injection, the rats were divided into groups of 25 and given one of six diets containing various levels of essential fatty acids (as linoleate). The diets contained 5% total fat and were prepared by mixing safflower oil (high essential fatty acids, beef fat (low essential fatty acids), and medium chain triglyceride oil (no essential fatty acids). One group of rats was fed a 20% beef fat diet. The range of essential fatty acids was from <0.03% to 1.28% (w/w). Twenty-six weeks after the first azoxymethane injection, the animals were killed and intestinal tumor incidence and multiplicty were determined. Samples of plasma, liver and colon were also taken for measurement of the triene/tetraene ratio by gas chromatography.Large bowel tumor incidence showed a dependence on the essential fatty acid content of the diet. The results were as follows: (percent essential fatty acids: percent tumor incidence) Group A (1.28∶ 72.4), Group B (0.60∶ 73.3), Group C (0.11∶ 55.2), Group D (0.08∶ 39.3), Group E (<0.03∶ 37.9) and Group F, which was fed 20% beef fat, (0.34∶ 88.5). These data suggest the essential fatty acid requirement for colon tumorigenesis is much lower than values previously reported for tumorigenesis in the breast and pancreas. The plasma and liver triene/tetraene ratios showed clear-cut essential fatty acid deficiency (ratio >0.4) in Groups D and E, although no clinical symptoms were evident. In all dietary groups, the triene/tetraene ratio in the colon was lower than 0.3. In addition in the colon, the percentage of fatty acids present as 20 carbon polyunsaturated fatty acids was lower than in the plasma and liver. These data suggest the colon possesses low levels of the fatty acid desaturase and elongase needed for conversion of linoleate to 20 carbon fatty acids, and therefore, that the colonic requirement for essential fatty acids may be low. Furthermore, in the absence of other clinical symptoms, the reduced tumorigenesis observed in the groups fed low essential fatty acids suggests the essential fatty acid requirement of tumor tissue may be higher than that of normal colon mucosa.

Importance of the duration of inhibition on intestinal carcinogenesis by difluoromethylornithine in rats

The effect of the duration and sequence of inhibition of intestinal tumor formation in rats was studied to determine whether part time inhibition has any value. Four groups of male Sprague-Dawley rats were given 8 weekly s.c. injections of azoxymethane (AOM) 8 mg/rat. Three groups were given the inhibitor, difluoromethylornithine (DFMO) in the drinking water; one for the entire 26 weeks of the study, one for the first 13 weeks only, and one for the last 13 weeks. A control group was not given the inhibitor. While the continuous treatment group developed the least number of tumors per rat (1.5 vs. 5 for controls), still both groups given the inhibitor for just 13 weeks also developed fewer tumors than controls 5 vs. 3.2 (early treatment) and 5 vs. 2.8 (late treatment). These results show that part time inhibition, including its late application, does reduce intestinal tumor formation in rats.

Failure of rectal ornithine decarboxylase to identify adenomatous polyp status

Rectal mucosal ornithine decarboxylase (ODC) activity has been reported to distinguish between patients with and without adenomatous polyps (AP). In the present investigation, ODC activity has been measured in 28 patients with AP and 34 patients without AP. To assess the intraindividual variation in ODC activity, repeat biopsies were performed on 11 patients. In addition, the effect of postbiopsy sample handling was investigated by storage of samples on either dry or wet ice during transport to the laboratory. The mean rectal mucosal ODC activity in patients with AP was 196.0 +/- 195.5, whereas that in AP negative patients was 182.2 +/- 320.5. The rectal mucosal ODC activity in patients with colorectal cancer was 388.2 +/- 581. Repeat samples in individuals were generally within the same range as the original samples. The method of sample transport did not significantly affect the level of ODC measured in a particular biopsy. Because of high variability in rectal mucosal ODC activity within the population, there was wide overlap in ODC values between those patients with and without AP in an unselected general population. Thus, the measurement of flat rectal mucosal ODC activity is not a good predictor of the presence or absence of AP. Additional studies of the factors affecting mucosal ODC activity are necessary before the potential clinical utility of the method can be realized in the general population.

The impact of dietary fat and fiber on intestinal carcinogenesis

The effect of dietary fiber on intestinal carcinogenesis in animals is controversial. Some find that the addition of wheat bran or cellulose inhibits intestinal cancer in rats, while others report no effect. Such mixed results often are due to differences in the design of experiments. One important aspect in this regard is the amount of fat in the diet. Some fiber supplements inhibit cancer formation when the fat content is normal but not when it is high. However, a recent epidemiological study in Scandinavia showed a lower cancer incidence in a rural population compared with an urban area, in spite of the fact that the dietary fat content was high in both regions. There was a modest difference in the amount of fiber, and this may not have accounted completely for the variation in cancer incidence. Other dietary factors might have added inhibitory response to help overcome the promotional effect of an excessive amount of fat. The interaction among dietary components must be considered when designing animal experiments to assess the effect of fiber on cancer development.

Autoxidation Products and Intestinal Carcinogenesis

Cancer of the large bowel is one of the most prevalent forms of neoplastic disease in North America (1). There has been a vast amount of research on the etiology of the disease but the exact cause has not been determined. Nonetheless, a number of factors which influence the risk and progression of large bowel cancer have been identified. A small percentage of large bowel cancer is genetic in origin such as that arising in individuals afflicted with familial polyposis (2). However, the great majority of cases of large bowel cancer are of unknown origin and strongly influenced by environmental factors. The major environmental factor affecting large bowel carcinogenesis is the diet and the most important dietary factor that enhances cancer appears to be the level of fat. The focus of the present chapter concerns a potential mechanism by which dietary fat influences colon carcinogenesis.

Prospects for the prevention of colorectal cancer

Cancer research has been productive in developing new knowledge on the role of diet in cancer. It is clear from epidemiologic observations that diet is the principal factor in the cause of colorectal cancer in most people. Therefore, a significant reduction in incidence is possible in countries where the disease is common. The ingestion of excessive amounts of fat appears to be the major factor that promotes cancer development. Animal studies confirm this and have recently shown that the sources of fat vary in the degree of their promotional effect. Fiber is generally considered to inhibit cancer but it is now clear that only some types of fiber are effective. These include whole grain cereals, and fruit and vegetables containing large amounts of uronic acid. In addition to fiber, a number of micronutrients, chemicals, and drugs have been found to be effective inhibitors. It is clear that the basic information concerning dietary changes that can reduce colorectal cancer incidence in this country has been uncovered. Additional information is needed about specific details of dietary guidelines. These include identification of the best mixture of sources of fat and how to incorporate such a mixture in the diet. Substances in foods need to be identified that, when included in the diet, help to lower cancer risk. People at high risk may require an additional supplement of inhibitors. New epidemiologic studies and human intervention trials should provide the necessary information to design dietary guidelines that are more specific than current ones.

Copper-containing IUDs are not associated with an increase of malondialdehyde levels in cervical mucus.

The malondialdehyde (MDA) content of cervical mucus from 23 healthy adult females was measured using an ion-pairing HPLC method capable of detecting 10 pmol MDA. Ten of the women were wearing copper IUDs, four were wearing plastic IUDs, and nine controls were not wearing an IUD. Cervical mucus was sampled during the follicular, periovulatory, luteal, and menstrual phases. The study was designed to determine if there is a relationship between MDA formation and the use of a copper IUD. A total of 79 samples were analyzed. Only 16 of the samples had sufficient MDA for reliable quantitation with the level ranging from 0.1 nmol/g to 2.32 nmol/g. In 19 of the samples, trace levels (less than 0.06 nmol/g) were detected but could not be reliably quantitated. In the remaining 44 samples, no MDA was detectable. There was no correlation between the presence of copper- or non-copper-containing IUDs and the level of MDA. These results are contrary to a previously published report that used a less specific method for MDA analysis.