The Journal of Nutrition Vol. 128 No. 8 August 1998, pp. 1349-1354

Increased Serum Cholesterol in Healthy Human Methane Producers Is Confounded by Age^1,2,3

Judlyn Fernandes^*, Thomas M. S. Wolever^{*, , 4}, and A. Venketeshwer Rao^*

^* Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 3E2 and  Clinical Nutrition and Risk Factor Modification Centre and Division of Endocrinology and Metabolism, St. Michael's Hospital, Toronto, Ontario, Canada M5C 2T2

	ABSTRACT

Abstract Introduction Methods Results Discussion References

It has been theorized that colonic production and absorption of short-chain fatty acids (SCFA) is different in methane producers (MP) compared with nonproducers (MNP). Because colonic SCFA may influence systemic lipid metabolism, blood lipids may differ in MP and MNP. To compare serum lipids and SCFA in fasting MP and MNP, we measured breath gases, serum lipids and SCFA in 167 healthy subjects and excluded subjects with abnormal blood lipids. The 66 MP were significantly older than the 63 MNP (49.5 ± 16.0 vs. 39.6 ± 17.0 y, P = 0.0009), and breath methane concentrations were weakly correlated with age in MP (r = 0.268, P = 0.03). Mean serum cholesterol was significantly higher in MP compared with MNP, but the differences were not significant after adjusting for age. No significant differences were observed in serum SCFA between the two groups. This study has shown that breath methane increases with age, which may be due to age-related increases in transit time and carbohydrate malabsorption. These results provide no conclusive link between colonic events and serum lipids in MP because, with age, methane production increased as did serum cholesterol. More research is required before any definite conclusions can be drawn.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The fermentation of carbohydrates in the colon results in the production of short-chain fatty acids (SCFA),⁵ carbon dioxide, hydrogen and methane (Miller and Wolin 1979). Highly anaerobic methanogenic bacteria produce methane from the reduction of carbon dioxide as follows: CO₂ + 4H₂  CH₄ + 2H₂O (Miller and Wolin 1986). Excretion of methane in breath can be used as a simple indicator of methane production in the colon (Bond et al. 1971, Pitt et al. 1980) and is observed only in subjects with >10⁸ Methanobrevibacter smithii/g dry feces (Miller and Wolin 1986).

Fecal bacteria from methane producers (MP) produce less propionate from starch in vitro and a higher acetate:propionate ratio than fecal bacteria from methane nonproducers (MNP) (Weaver et al. 1992). Methane producers may also absorb colonic SCFA more readily than MNP (Flick and Perman 1989). These results are consistent with our previous finding of significantly higher fasting and postprandial serum acetate concentrations in MP compared with MNP (Wolever et al. 1993). Colonic SCFA may influence hepatic lipid metabolism because the use of colonic acetate, a major substrate for hepatic lipid synthesis, is inhibited by propionate (Demigné et al. 1995, Nishina and Freedland 1990, Wolever et al. 1991). Rectal infusion studies show that colonic acetate is incorporated into serum cholesterol and triglycerides and that these effects are blocked by propionate (Wolever et al. 1995b). In addition, chronic feeding of lactulose, which produces mainly acetate during in vitro fermentation (Mortensen et al. 1988) and raises serum acetate (Fernandes et al. 1994), also increases serum total and LDL cholesterol, triglyceride and apolipoprotein B levels (Jenkins et al. 1991). Because previous studies suggest that methane-producing status increases systemic acetate availability, we hypothesized that methane producers will have higher serum cholesterol concentrations than nonproducers. Thus the purpose of this study was to measure concentrations of lipids in fasting MP and MNP.

	SUBJECTS AND METHODS

Abstract Introduction Methods Results Discussion References

We studied 167 healthy subjects with the use of a protocol approved by the Human Subjects Review Committee of the University of Toronto. Subjects with a history of diabetes, a thyroid disorder, liver disease, any gastrointestinal disorders or antibiotic use in the 3 mo before the study were excluded. After a 10- to 12-h overnight fast, 10 mL blood from a forearm vein was collected from the subjects into plain glass tubes (red-top Vacutainer, Becton Dickinson, Rutherford, NJ), left to clot at room temperature for 30-60 min, centrifuged at 600 × g for 10 min and the serum removed for lipid, lipoprotein and SCFA analysis.

Serum total cholesterol, triglycerides and HDL cholesterol of fasting subjects were analyzed by the Core Lipid Laboratory, University of Toronto according to the Lipid Research Clinics protocol (Allain et al. 1974). Serum LDL cholesterol was calculated as follows: (LDL) = (total cholesterol  HDL cholesterol  triglycerides/2.2).

Protein-free serum for SCFA analysis was obtained by ultrafiltration by using a micropartition system with a 30,000 Da molecular weight cutoff (MPS-1, Amicon, Danvers, MA). The protein-free serum was vacuum distilled according to the procedures described by Tollinger et al. (1991), with modifications (Wolever et al. 1989). To a 225-µL aliquot of protein-free serum was added 25 µL of internal standard solution containing 1.1 mmol/L methylbutyric acid and 110 mmol/L [¹³C]formic acid (Sigma Chemical, St. Louis, MO) to prevent ghosting in the injector sleeve and reduce pH in the sample to ~3, thus ensuring complete SCFA recovery. SCFA were measured in the protein-free distillate by using a 5890 series II gas chromatograph (Hewlett Packard, Mississauga, Canada), equipped with a split/splitless inlet, a J&W DB-FFAP fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µm film; Alltech Associates, Deerfield, IL) and a flame ionization detector. An HP 7673 automatic sampler was used to inject a 1-µL aliquot of sample into the injection port, which contained a deactivated direct injection glass liner (1.5 mm i.d., volume 140 µL; Supelco, Bellefonte, PA). The gas chromatograph injector and detector were maintained at 220°C and the purge valve was set off for splitless injection. The oven temperature was 80°C until 0.1 min after injection, after which it was increased by 15°C/min to 165°C at which it was held for 1 min. The carrier gas was pure helium at a flow rate of 1 mL/min. The detector was supplied with helium at 30 mL/min, hydrogen at 30 mL/min and air at 350 mL/min. Serum samples were analyzed in duplicate, with duplicate injections for each sample. Details of the method used have been published (Wolever et al. 1996).

On the same day that the blood was collected, alveolar breath samples were also collected by the subjects in 20-mL syringes before breakfast and at hourly intervals for 4 h or duplicate breath samples were collected before breakfast, 10-15 min apart, using a modified Haldane-Priestly tube (Metz et al. 1976). The collection procedure was demonstrated to the subjects who then collected the samples. Before collection, subjects were asked to breathe normally to prevent recollection hyperventilation. Breath hydrogen and methane were measured against known standards by gas chromatography (Quinton Microlyser, Model DP, Milwaukee, WI) on the same day the sample was collected. Subjects also collected a sample of room air in a 20-mL syringe to measure breath methane and hydrogen levels in room air. Breath methane concentration was determined by subtracting the methane of room air from the average concentration of the samples collected. Subjects whose corrected breath methane concentrations were < 0.045 µmol/L above ambient air were considered MNP. MP were defined as subjects with a corrected breath methane level > 0.045 µmol/L. Of the 167 subjects studied, classification of 88 subjects as MP and MNP was done by using five breath samples collected 1 h apart (method 1); 79 subjects were classified by using two breath samples collected 10-15 min apart (method 2). Two different methods were used to classify subjects because of time constraints. Previous studies in our laboratory have shown that MP consistently have a breath methane concentration above room air throughout the day; there is no evidence that collecting breath samples over a 4-h or a 15-min period affects classification of subjects. The difference between the numbers of MP and MNP detected by the two methods was not significant (² = 0.14).

Subjects also completed questionnaires about their bowel habits and information concerning any family history of coronary vascular disease (CVD). The bowel habit questionnaire had questions about the frequency of bowel movements, consistency of stools and use of laxatives. The CVD questionnaire had questions related to family history of heart attack, stroke or sudden death and hypercholesterolemia or hypertriglyceridemia in immediate family members (parents and siblings only).

Marked elevation of blood lipids is likely due to primary hyperlipidemia, caused by one or more genetic abnormalities. Because it was reasoned that the inclusion of subjects with primary hyperlipidemia would obscure the expected small effect of being a MP on serum lipids, our aim was to study the effect of methane production on serum lipids only in subjects with normal serum lipids. Thus, subjects were excluded prospectively from statistical analysis if their serum total or LDL cholesterol or triglyceride concentration fell at or below the 5th percentile or at or above the 95th percentile for an age- and sex-matched North American population (National Institutes for Health 1980). Statistical analysis was done by linear regression (GraphPad Prism, Version 2, GraphPad Software, San Diego, CA), ² test and one- and two-way ANOVA with the use of a computer spreadsheet (Microsoft Excel Version 5.0a, Microsoft, Redmond, WA). Serum cholesterol was adjusted for age, and statistical analysis done by analysis of covariance using a SAS statistical package (SAS, Release 6.11, SAS Institute, Cary, NC). Differences were considered significant at P  0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

We studied 167 subjects, of whom 129 (60 males and 69 females) had serum cholesterol and triglyceride levels between the 5th and 95th percentiles for an age- and sex-matched North American population. Of the 129 subjects with normal blood lipids, 66 were MP (27 males and 39 females) and 63 were MNP (33 males and 30 females) (Tables 1 and 2). The MP were significantly older than the MNP (P = 0.0009), and the male MP were significantly older than the male MNP (P = 0.001, Tables 1 and 2). There were no significant differences in body mass index (BMI) between the MP and MNP (Table 1). The proportion of MP and MNP in the different age groups varied significantly (² test = 0.02, Fig. 1). Mean breath hydrogen levels were significantly higher (P = 0.005) in MNP compared with MP (Table 1). There was a weak positive correlation between breath methane and age in MP (r = 0.268, P = 0.03, Fig. 2). No significant differences were seen in the number of bowel movements, consistency of stool samples and use of laxatives between the MP and MNP.

View larger version (12K): [in this window] [in a new window]   Fig 1. Number of methane producers (MP) and methane nonproducers (MNP) in the different age groups (2 = 0.02).

View larger version (12K): [in this window] [in a new window]   Fig 2. Relationship between age and breath methane concentrations in methane producers, r = 0.268 (P = 0.03).

The mean serum total cholesterol concentration was significantly higher in MP than in MNP (P = 0.02, Table 3), but the difference was not significant after adjusting cholesterol for age. A positive correlation was observed between serum total cholesterol and age in MP (r = 0.643, P < 0.0001) and MNP (r = 0.773, P < 0.0001, Fig. 3). The female MP had significantly higher total cholesterol concentrations (P = 0.03) than female MNP (Table 2) but the difference was not significant after adjusting cholesterol for age.

View larger version (18K): [in this window] [in a new window]   Fig 3. Relationship between age and cholesterol in methane producers (MP) (top) and methane nonproducers (MNP) (bottom). Correlation coefficients for age vs. cholesterol: MP, r = 0.643 (P < 0.0001); MNP, r = 0.773 (P < 0.0001).

Methane producers reported a significantly greater proportion of immediate family members with incidents of heart attack, stroke or sudden death (² test = 0.003) and with a history of hypercholesterolemia or hypertriglyceridemia (² test = 0.03) than that for family members of methane nonproducers (Fig. 4).

View larger version (9K): [in this window] [in a new window]   Fig 4. Reported incidents of coronary vascular disease (CVD) (2 = 0.003) or hyperlipidemia (2 = 0.03) in all family members of methane producers (MP) and methane nonproducers (MNP).

There were no significant differences in mean serum concentrations of acetate (93 ± 37 vs. 96 ± 36 µmol/L), propionate (3.8 ± 1.7 vs. 3.8 ± 1.6 µmol/L) and butyrate (2.4 ± 1.6 vs. 2.2 ± 1.5 µmol/L) between MP and MNP. Significant differences between the MP and MNP were also not seen in the serum acetate:propionate and acetate:butyrate ratios, and serum concentrations of acetate, propionate and butyrate as a percentage of total SCFA produced.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The prevalence of methane production observed in our adult population was 51%, 56.5% among females and 45% in males. Our subjects were mainly Caucasian, and our results compare well with previously reported prevalences of 48% in Caucasians and 58% in Caucasian females in Canada; however, incidence in males in our study was higher than the reported incidence of 35% (Pitt et al. 1980). This may be because of the unequal number of males and females in our study. In our study, 59% of the MP were female; this preponderance of female over male MP has been seen before in some studies (Pitt et al. 1980) but not in others (Bjorneklett and Jenssen 1982, Bond et al. 1971, McKay et al. 1981).

An interesting finding of this study, not consistently reported in the literature, was that the MP were significantly older than the MNP; we also observed that age was weakly correlated with increased breath methane concentration. Most studies have not observed an effect of age (Bjorneklett and Jenssen 1982, Bond et al. 1971, McKay et al. 1985), but Haines et al. (1984) observed an increase in the proportion of MP with age in 1398 subjects. There are a few factors that may change with age and may also affect methanogenesis, including transit time, carbohydrate malabsorption and lactose malabsorption.

Methanogens are relatively slow-growing bacteria, and a longer transit time may facilitate their growth by decreasing the turnover of digestive contents (Mah et al. 1977). There have been conflicting reports of transit time in MP compared with MNP. Stephen et al. (1986) observed a longer transit time in MP compared with MNP; however, other studies (Cloarec et al. 1990, McKay et al. 1981) found no difference in transit time, and a recent study (El Oufir et al. 1996) showed that MP had a longer mean transit time than MNP. We did not measure transit time in our study and we did not observe differences in bowel habits between the two groups. There are conflicting reports in the literature on the influence of aging on colonic transit time; Nagengast et al. (1988) did not see an age effect, but other studies reported a longer mean colonic transit time in older subjects (Madsen 1992). Transit time may not be the only factor involved because urban and rural black Africans have shorter transit times than Caucasians, but a higher prevalence of methane producers (Segal et al. 1988).

A progressive reduction in carbohydrate absorptive capacity with age was observed by Feibusch and Holt (1982); there is evidence that exogenous substrates can also contribute to methanogenesis (Bjorneklett and Jenssen 1982, McKay et al. 1981, Pitt et al. 1980). It is unclear if lactose malabsorption is involved because in some studies, an increase in breath methane is observed in lactose-intolerant patients (Corazza et al. 1994, Medow et al. 1993) but not in others (O'Keefe et al. 1990, Zuccato et al. 1983). Recent phylogenetic and genetic data suggest a heritable competence for intestinal methanogenesis, which may appear repeatedly in families (Hackstein et al. 1995). Factors that are influenced by aging such as transit time and carbohydrate malabsorption may affect methanogenesis as methane producers age.

The significantly higher total cholesterol we observed in MP was confounded by age. However, this was a statistical confounding, and the correlation between serum cholesterol and age does not prove causality. It is difficult to conclude that the increase in cholesterol in MP was solely age related because the MP females had higher mean total cholesterol than MNP females despite no significant difference in age. We have previously reported that positive methane-producing status is associated with increased serum total and LDL cholesterol concentrations in age-matched subjects with impaired glucose tolerance (Wolever et al. 1995a). No other studies have linked methane production with cholesterol, but there is increasing evidence that events in the colon may influence lipid metabolism. In a recent study, we reported a significant positive relationship between serum acetate:propionate ratio and total and LDL cholesterol concentrations in men, and also between serum acetate:propionate ratio and age in men and women (Wolever et al. 1996). Methane-producing status, acetate:propionate ratio and total cholesterol seem to be related to age; thus, to determine the effect of methane-producing status on blood lipids, future studies will have to include age-matched subjects.

Because methane production is heritable (Bond et al. 1971), we might also expect to find increased methane production among family members of methane producers. It is tempting when interpreting the results from the family history of CVD data to conclude that methane production may be associated with increased CVD and hyperlipidemia; however, our results may provide only indirect evidence and may also be confounded by age. Because the methane producers in this study were older, they were more likely to have family members with CVD and hyperlipidemia. Our observations may not prove a relationship but do raise interesting questions about hypercholesterolemia in methane producers and its possible relation to CVD. Aorto-iliac disease was associated with a high incidence of methane excretion in a study by McKay et al. (1983), which may be due to impaired colonic circulation altering the intestinal environment, lowering oxygen tension and favoring the growth of methanogens.

In this study, no significant differences in fasting peripheral blood SCFA concentrations were observed between the two groups. In humans after a 12-h overnight fast, the source of SCFA in peripheral blood may be largely endogenous from the liver and peripheral tissues and to a lesser extent from gut fermentation (Pomare et al. 1985). The gut-derived SCFA make an additional contribution, largely from the fermentation of endogenous substrates, protein (Sheppach et al. 1991) and mucus secreted by the gastrointestinal tract and the pancreas. The lack of difference observed here is therefore not surprising; in an earlier study, however, Wolever et al. (1993) reported significantly higher blood acetate concentrations in six fasting MP compared with six fasting MNP who were consuming a highly controlled metabolic diet and fed a polysaccharide-free meal the night before blood was drawn from fasting subjects. In this study, we were interested mainly to determine whether differences in colonic production affected concentrations of SCFA in fasting subjects consuming their regular diets. Studies looking at postprandial SCFA in MP and MNP may provide an alternative means to assess differences in colonic production of SCFA in MP and MNP.

We have shown that breath methane is weakly correlated with age, which may be due to age-related increases in transit time and carbohydrate malabsorption. These results are inconclusive in proving a link between colonic events and serum lipids in MP but raise intriguing questions. With age, methane production increases as does serum cholesterol but does the hyperlipidemia in MP play a role in the development of CVD or do the hyperlipidemia and CVD lead to increased methane production? On the other hand, if the increase in cholesterol we observed is solely age related, there might be differences in SCFA metabolism that we did not observe with this protocol. More information is needed on postprandial SCFA and absorption of SCFA in MP and MNP before any firm conclusions are made about SCFA metabolism in MP and MNP.

	FOOTNOTES

Manuscript received 13 November 1997. Initial reviews completed 28 January 1998. Revision accepted 13 April 1998.

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