There were massive changes in concentration of some of the metabolites of transmethylation and transsulfuration pathways within 30 minutes, after a commonly used experimental dose of LA was injected into rats. Serum SAH increased 20-fold and serum cysteine decreased by 60%. Accompanying the rise in serum and liver SAH was a massive depletion of liver SAM with sparing of serum SAM. Despite the extreme drop in serum cysteine, liver cysteine was unchanged as was liver methionine, and glutathione. Accompanying the rise in tHcys was an increase in N, N-dimethylglycine concentrations, possibly due to methylation of homocysteine by liver betaine homocysteine methyltransferase (22
). The rise in serum and liver cystathionine and alpha-aminobutyric acid probably reflect an increase in transsulfuration flux which is somewhat surprising given the marked SAM depletion in the liver since SAM is an activator of cystathionine beta-synthase (14
). SAH may also activate CBS thus, there may have been further depletion of liver SAM because of loss of homocysteine to transsulfuration (14
). In contrast, MESNA injection only induced serum cysteine and tHcy depletion.
The metabolic changes summarized above may be due to either shifts in sulfhydryl binding caused by the disulfhydryl LA and/or an induced methylation burden due to the metabolism of LA/DHLA. It is believed that absorbed LA is enzymatically converted to the reduced form, DHLA by mitochondrial dihydrolipoamide dehydrogenase, cytosolic glutathione reductase and thioredoxin reductase (2
). It is likely that when large amounts of DHLA are present then other sulphur containing compounds such as homocysteine and cysteine may be released from sulfhydryl binding to albumin and other proteins and compounds. Two other sulfhydryl containing therapeutics, MESNA and N-acetylcysteine, have been shown to cause decreases in plasma cysteine and homocysteine similar to what occurred in this study (19
). The release of free cysteine may promote its uptake by tissues and in this study despite marked serum depletion of cysteine, there was maintenance of hepatic cysteine and no change in glutathione. Previous investigators have found increased glutathione concentrations after LA treatment of rodents (3
) and in tissue culture (31
), possibly because of the displacement of bound cysteine and its increased tissue availability for synthesis of glutathione. This mobilization of cysteine and increased production of glutathione may be part of the beneficial antioxidant effect that has been reported in many experiments. The need for reduction of lipoic acid by the organism would seem to negate some of the antioxidant effect. However, we found comparable changes in metabolites when we compared injections of LA and DHLA which suggests that using the already reduced compound has little benefit.
Detailed investigations of the pharmacokinetics and metabolism of lipoic acid have been reported (11
). LA/DHLA is metabolized by beta hydroxylation of the carbon side chain, and S-methylation (11
). The major metabolite of LA found in healthy volunteers was BMHA (12
) (see ) along with smaller quantities of other methylated metabolites. We were also able to find serum and liver BMHA after injection of labeled or unlabeled LA in rats. The reported studies in humans showed that approximately 60% of the recovered amount in urine after oral dosing was BMHA and only 1.6% the parent compound (12
). We estimated that hepatic BMHA was about 25% of the simultaneous hepatic LA concentration. Thus, it appears that most administered LA is methylated, which could be the cause of the marked depletion of liver SAM in the rats injected in this investigation.
There is an estimated transmethylation flux of 220-260 umol/kg per day for humans (34
). The dose of lipoic acid we injected in rats was 500 umol/kg, thus likely a major consumer of SAM with resulting massive production of SAH. Since MESNA depleted serum cysteine without significantly raising SAH or homocysteine, the latter effects are likely not due to the sulfhydryl reducing capability of LA. Inhibition of SAHH with adenosine-dialdehyde was reported to decrease the extracellular homocysteine in cell cultures exposed to LA (13
) suggesting that production of homocysteine rather than displacement might be the cause of the elevated levels seen in our rats. Two other commonly used drugs, niacin and L-dopa, which are used in comparably large doses have also been shown to increase total homocysteine in humans (35
). This has been attributed to their requirement for methylation. An inhibitor of L-dopa methylation, tolcapone, decreased plasma SAH and homocysteine concentrations in patients treated with L-dopa (37
Commonly used doses of LA in human disease range from 600-1800 mg/day (1
), which for a 70 kg person will range from roughly 9-26 mg/kg, thus 4-10-fold less than in our rat model. However, at the higher doses it is likely that LA might pose a methylation burden and human subjects should be investigated for changes in the transmethylation and transsulfuration metabolites. A recent report suggests that an improved formulation of R-(+)-LA has significantly higher bioavailability and thus, might be expected to have more effect on methylation status (38
). Placebo controlled trials in diabetic neuropathy have shown clear benefit for LA with few demonstrated side effects (1
). Fortunately, there is not a dose response, with lower doses equally effective, which would likely have less potential to cause hyperhomocysteinemia. In Parkinson's disease treatment with L-Dopa, the hyperhomocysteinemia presumptively attributed to the methylation burden has been correlated with vascular disease (35
). Therefore some concern may be warranted if large doses of LA are used in human medicine. An interesting difference in LA dose toxicity has been described in cats as compared to humans and dogs, which could be investigated for methylation defects (39
). LA is being investigated in cell models as a cancer chemotherapeutic agent since high media concentrations (100 umol/L – 5 mmol/L) have been reported to induce apoptosis in a variety of cell lines (40
). The impact of probable SAM depletion and SAH induced transmethylation inhibition should be investigated in these systems.
In summary, we have demonstrated that a common experimental dose of LA causes massive depletion of liver SAM, elevation of serum SAH and increased tHcy in rats. Similar depletion of serum cysteine with MESNA was not accompanied by changes in SAM and SAH suggesting that the former effects are due to different aspects of LA metabolism/catabolism. Further studies in humans may be interesting.