Recent findings have implicated one-carbon compounds and methylation in the secretory function of the pancreas. Gilliland and Steer (1980) showed that consumption of a diet deficient in choline and containing 0.5% ethionine led to a failure to discharge the zymogen granules but had no effect on enzyme levels in the granules. Balaghi et al. (1993b) and Balaghi and Wagner (1995) showed that folate deficiency perturbs pancreatic one-carbon metabolism and results in an inhibition of secretion of amylase. They also showed that the methylation ratio (the ratio of S-adenosylmethionine to S-adenosylhomocysteine) was lower in the folate-deficient group [this ratio is thought to regulate many physiologically important methylation reactions (Cantoni et al. 1978)]. These considerations suggest that factors that affect folate-dependent one-carbon metabolism may perturb pancreatic enzyme secretion. Because vitamin B-12 deficiency (either dietary or induced by the anesthetic gas nitrous oxide) perturbs folate metabolism, we decided to determine the effects of nitrous oxide on pancreatic folate coenzyme distribution.

Exposure to nitrous oxide results in the symptoms of vitamin B-12 deficiency, megaloblastic anemia and neuropathy, in humans (Chanarin 1980), monkeys (Scott et al. 1981) and pigs (Weir et al. 1988). This is due to the inactivation of methionine synthase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13) by oxidizing the enzyme-B-12 (Co⁺) complex formed during catalysis (Chanarin 1980, Drummond and Matthews 1994). Several investigators have used nitrous oxide as a tool for exploring the folate-vitamin B-12 interrelationships (see Shane and Stokstad 1983 and 1985 for reviews). These studies have shown that nitrous oxide exposure results in an accumulation of folates as the 5-methyltetrahydrofolate derivative at the expense of other folates. This has been called the methyl trap hypothesis (Herbert and Zalusky 1962, Noronha and Silverman 1962), which states that in vitamin B-12 deficiency folates are trapped as 5-methyltetrahydrofolate. This is because the synthesis of 5-methyltetrahydrofolate is not reversible in vivo (Green et al. 1988) and methionine synthase is inactive due to vitamin B-12 deficiency (either dietary or via inactivation by nitrous oxide). This trapping of folates results in decreased amounts of 10-formyl- and 5,10-methylenetetrahydrofolate (which are necessary for purine and thymidine synthesis, respectively), which results in megaloblastosis.

The major pools of folate in the liver are in the cytosol and mitochondria (Cook and Blair 1979, Corrocher and Hoffbrand 1972, Shin et al. 1976). We found (Horne et al. 1989), in rats exposed to nitrous oxide, that hepatic 5-methyltetrahydrofolate was about twice the concentration (from 45 to 84% of total cytosolic folates, control and nitrous oxide, respectively) and this change was at the expense of 5-formyl-, 10-formyl- and tetrahydrofolate. The folate distribution in the mitochondria from these rats was not affected by nitrous oxide.

In the present study, we examined the compartmentation of and the effect of nitrous oxide on folate metabolites in rat pancreas.

MATERIALS AND METHODS

RESULTS

Table 1. Nitrous oxide inactivation of methionine synthase affects the concentration of folate coenzymes in cytosol and mitochondria in rat pancreas1 [View Table]

In mitochondria of control rats, tetrahydrofolate was again the major folate derivative, but, unlike in cytosol, the concentration of 5-formyltetrahydrofolate was higher than either 10-formyl- or 5-methyltetrahydrofolate (Table 1). Unlike results in the cytosol, nitrous oxide treatment led to a 50% lower concentration of folates in mitochondria. The concentration of tetrahydrofolate was 62% lower and and 5-formyltetrahydrofolate was 45% lower in nitrous oxide-exposed rats. The concentrations of 10-formyl- and 5-methyltetrahydrofolate were not significantly different between groups.

The activity of methionine synthase was measured in pancreas from control and nitrous oxide-treated rats. The activity was 4.58 ± 0.29 nmol/(min·mg protein) in controls (n = 5) and 0.68 ± 0.18 nmol/(min·mg protein) (P < 0.001) in nitrous oxide-exposed rats (n = 4).

DISCUSSION

Similar to results in liver, the present study showed that, in pancreas, inactivation of methionine synthase also resulted in trapping of 5-methyltetrahydrofolate in the cytosolic compartment. In control rats, 5-methyltetrahydrofolate was 31% of cytosolic folates of pancreas; in nitrous oxide-exposed rats, this value was 59% (Table 1). The greater concentration of 5-methyltetrahydrofolate was exclusively at the expense of tetrahydrofolate, which was 54% of total folate in controls and 32% in nitrous oxide-exposed rats, respectively. Neither 5- nor 10-formyl-tetrahydrofolate concentrations differed significantly. In contrast, in liver cytosol all reduced folates other than 5-methyltetrahydrofolate declined during nitrous oxide exposure (Horne et al. 1989). The observation that nitrous oxide exposure resulted in accumulation of 5-methyltetrahydrofolate to only 59% of cytosolic folates in pancreas, whereas in liver 5-methyltetrahydrofolate accumulated to 84% of cytosolic folates, could be due to more pronounced inhibition of methionine synthase in some cell types than in others in pancreas. However, our results showed that methionine synthase activity was about 86% lower in the pancreas of rats breathing nitrous oxide than in controls, and this is similar to the 84% inhibition seen in liver (Horne et al. 1989). In any case, our studies show that folates are trapped as the 5-methyl derivative in the cytosol of pancreas, as in the liver, when methionine synthase is inhibited.

The effect of methionine synthase inactivation on the spectrum of folate coenzymes in mitochondria from pancreas is quite different from that seen in liver. In liver, there was no change in the mitochondrial folate coenzyme distribution or in the total amount of folate in the nitrous oxide-exposed group (Horne et al. 1989). However, in the pancreas, the total mitochondrial folate concentration was about 50% lower in the nitrous oxide-exposed group compared with air-breathing controls. The reason for this difference is unknown; however, the data suggest that polyglutamate turnover may be greater in mitochondria than in the cytosol of pancreas. Folate polyglutamates do not readily cross the mitochondrial membrane in liver (Horne et al. 1989, Lin et al. 1993); however, our data suggest that folate polyglutamates may cross the mitochondrial membrane to some extent in pancreas, because the concentration of folate was lower and the distribution of the coenzymes was altered in nitrous oxide-exposed rats. Further experiments, using radiolabeled folate, would be necessary to determine whether there is an increased rate of polyglutamate turnover in pancreatic mitochondria. In any event, total mitochondrial folate was lower and this was due to lower concentrations of 5-formyl- and tetrahydrofolate. The concentrations of 10-formyl- and 5-methyltetrahydrofolate did not differ in pancreatic mitochondria of the nitrous oxide group and controls.

As stated in the introduction, there seems to be a connection between perturbed pancreatic secretion and folate-dependent one-carbon metabolism, which results in a lower methylation ratio (the ratio of S-adenosylmethionine to S-adenosylhomocysteine) in rats fed folate-deficient diets (Balaghi and Wagner 1995, Balaghi et al. 1993b). We reasoned that other factors that alter folate-dependent one-carbon metabolism, e.g., vitamin B-12 deficiency might perturb pancreatic secretion. We used nitrous oxide exposure to inactivate the vitamin B-12-dependent enzyme methionine synthase. This causes trapping of folate coenzymes as the 5-methyl derivative at the expense of other reduced folates. Because methionine synthase is inactivated, the concentration of methionine, and consequently S-adenosylmethionine, should decline. Although S-adenosylmethionine concentration in pancreas was not measured in the present study, in another study (Horne 1989) in which rats were exposed to nitrous oxide for 18 h, there were no significant differences between nitrous oxide-exposed and control rats in S-adenosylmethionine or S-adenosylhomocysteine concentrations in liver, kidney, brain or small intestine. Thus inactivation of methionine synthase, at least for short periods of time, has no apparent effect on the methylation ratio in these tissues. Longer exposure to nitrous oxide (up to 12 d) resulted in significant decreases in S-adenosylmethionine concentration in liver (Lumb et al. 1983). Therefore, we might expect that long-term vitamin B-12 deficiency could affect pancreatic secretion. If the diet contains sufficient methionine, we may not expect to see a significant lowering of the methylation ratio due to short-term nitrous oxide exposure. However, if methionine is limiting in the diet, the failure to regenerate methionine because methionine synthase is inactivated may cause a lowered methylation ratio. Further experiments are needed to clarify these issues.

In conclusion, we have found that nitrous oxide administration to rats results in trapping of folate coenzymes as the 5-methyltetrahydrofolate derivative in the cytosol of rat pancreas as it does in liver. However, unlike effects in liver, folate coenzyme concentration was lower in pancreatic mitochondria in rats exposed to nitrous oxide, and the distribution of mitochondrial folate coenzymes was also altered.