Alpha-lipoic acid induces elevated S-adenosylhomocysteine and depletes S-adenosylmethionine

Abstract

Lipoic acid is a disulfhydryl-containing compound used in clinical medicine and in experimental models as an antioxidant. We developed a stable isotope dilution capillary gas chromatography/mass spectrometry assay for lipoic acid. We assayed a panel of the metabolites of transmethylation and transsulfuration 30 min after injecting 100 mg/kg lipoic acid in a rat model. Lipoic acid values rose 1000-fold in serum and 10-fold in liver. A methylated metabolite of lipoic acid was also detected but not quantitated. Lipoic acid injection caused a massive increase in serum S-adenosylhomocysteine and marked depletion of liver S-adenosylmethionine. Serum total cysteine was depleted but liver cysteine and glutathione were maintained. Serum total homocysteine doubled, with increases also in cystathionine, N,N-dimethylglycine, and alpha-aminobutyric acid. In contrast, after injection of 2-mercaptoethane sulfonic acid, serum total cysteine and homocysteine were markedly depleted and there were no effects on serum S-adenosylmethionine or S-adenosylhomocysteine. We conclude that large doses of lipoic acid displace sulfhydryls from binding sites, resulting in depletion of serum cysteine, but also pose a methylation burden with severe depletion of liver S-adenosylmethionine and massive release of S-adenosylhomocysteine. These changes may have previously unrecognized deleterious effects that should be investigated in both human disease and experimental models.

Figure 1a

The structures of alpha-lipoic acid (LA) and the reduced form dihydrolipoic acid (DHLA) are shown. The asterisks demonstrate the site of the stable isotope ¹³C labels in the molecule.

Figure 1b

Two of the major metabolites of lipoic acid are shown; 4, 6-bismethylthiohexanoic acid (BMHA) and 2, 4-bismethylthio-butanoic acid (BMBA). The asterisks demonstrate expected sites of the stable isotope ¹³C labels after metabolism/catabolism of labeled LA.

Figure 2

The metabolic pathways of remethylation, transmethylation and transsulfuration are shown (14). Homocysteine can be methylated by either betaine to produce methionine and N, N-dimethylglycine by betaine homocysteine methyltransferase (BHMT) or by 5-methyltetrahydrofolate (5MTHF) by methionine synthase, a reaction dependent on folate and methylcobalamin (MECbl). Methionine is activated to S-adenosylmethionine (SAM) by methionine adenosyltransferase (MAT). SAM is the methyl donor for many transmethylation reactions that produce S-adenosylhomocysteine (SAH). SAH is hydrolysed by S-adenosylhomocysteine hydrolase (SAHH) to homocysteine and adenosine. When methionine is abundant then excess homocysteine is cleared by condensing with serine by cystathionine beta synthase (CBS), a pyridoxal phosphate (PLP)-dependent reaction to form cystathionine and alpha-ketobutyrate. Cystathionine can be cleared by cystathionine gamma lyase (CGL), also PLP-dependent, to form cysteine. Not shown is further metabolism of alpha-ketoglutyrate to alpha -aminobutyric acid. When SAM is in excess, glycine N-methyltransferase (GNMT) methylates glycine to form N-methylglycine.

Figure 3a

Gas chromatography/mass spectrometry chromatograms of LA are shown for an assay of 400 uL of control rat serum, as described in Methods. The m-57 ions were monitored. The internal standard ion m/z 267 is shown above and m/z 263 below for the endogenous LA. The calculated result was 0.67 umol/L.

Figure 3b

The gas chromatography/mass spectrometry chromatograms are shown for assay of 20 uL of rat serum 30 minutes after LA 100 mg/kg was injected IP and assayed as described in Fig.4a. The calculated value for serum LA was 20 umol/L.