Indirect measurement of betaine - homocysteine methyltransferase activity using 1H nmr spectroscopy

Abstract

The (re)methylation of homocysteine to methionine is an essential process for continuation of the cycle whereby Sadenosylmethionine acts as the major methyl donor for biosynthesis. There are two enzymes capable of mediating the homocysteine to m e t h i o n i n e c o n v e r s i o n : N 5 methyltetrahydrofolate-homocysteine methyltransferase with the methyl group provided by N5-methyltetrahydrofolate o r betaine-homocysteine methyl transferase (BHMT), the methyl group being donated by betaine leaving N-dimethylglycine (DMG) as product. Attempts have been made to assess the relative contributions of these reactions e.g. by estimating the production of sarcosine in patients with sarcosinaemia [l]. However, the accumulation of DMG (the immediate precursor of sarcosine) was not measured. We have investigated patients with the disorder multiple acyl CoA dehydrogenase deficiency (glutaric aciduria type 11; [2]) . The disorder is caused by a deficiency in either electron transfer flavoprotein (ETF) o r ETFubiquinone reductase (ETF-QO) and has been characterised by the excretion of several short-chain dicarboxylic acids as a result of the block in metabolism of precursor CoA analogues and subsequent 61 and 61-1 oxidation [ 3 1 . The enzymes DMG dehydrogenase and sarcosine dehydrogenase are also ETF(-QO) dependent and whereas sarcosine excretion has been reported in such patients no investigation of DMG metabolism has been carried out. Urine from a neonate with multiple acyl-CoA dehydrogenase deficiency was immediately frozen at -20 prior to nmr analysis. Spectra were recorded at 400 and 500 MHz with a sweep of 12 ppm, a 90 pulse and recycling time of 5 s . 120 scans were generally sufficient to give good signal to noise. The spectrum from the patient shows several abnormal resonances. Signals from adipic, suberic , sebacic, glutaric and isovaleric acids are present. At 2.93 and 3.73 ppm are large signals from DMG and at 2.74 ppm a small signal from sarcosine. The excretion rate of DMG expressed as creatinine equivalents was 1.5. mol/mol. Excretion of DMG in healthy neonates is usually less than 0.3 mol/mol creatinine and sarcosine < 0.1 (Davies and Iles, unpublished). Perhaps surprisingly betaine excretion was within the normal range for a child of this age [41. As the excretion of DMG was so large in comparison to sarcosine it is likely that the inhibition of DMG