Determination of Betaines by Fast Atom Bombardment Mass Spectrometry

Abstract

ABSTRACIA rapid, sensitive, and selective method for the determination of betaines is described and discussed. The method entails derivatizing the quaternary ammonium compounds to increas their sensitivity to detection by fast atom bombardment mass spectrometry. Sensitivity of detection increases markedly as the length of the carbon chain of the alcohol used to esterify the betaine carboxylic acid group is inreased (C4 > C3 > C2 > Cl > Ca). The lower limit of detection of glycine betaine as the i-propyl ester is 0.05 nanomole per microliter of glycerol. Betaine aldehyde can be readily derivatized to the di-n-butyl or di-n-propyl acetal derivatives which exhibit lower limits of detection of about 5 picomoles and 10 picomoles per microliter of glycerol respectively. Accurate quantification of these compounds is accomplished by the use of deuterium labeled internal stanards or quaternary ammonium compound homologs of distinct mass. Methods for the synthesis of these internal standards are reported. Some applications of these methods are iflustrated with stable isotope tracer studies on the kinetics of metabolism of choline to betaine aldehyde and glycine betaine in spinach leaf discs, and the identification of several Zea mays genotypes which appear deficient in glycine betaine. Tracer studies with deuterium labeled betaine aldehyde suggest that the deficiency of glycine betaine in one sweet corn hybrid is probably not due to a deficiency in the capacity to oxidize betaine aldehyde.Quaternary ammonium compounds occur widely in nature and may serve important roles as compatible osmotic solutes or osmoprotectants (13,17,22,(28)(29)(30)34). Glycine betaine (N,N, N-trimethyl glycine) is thus of considerable interest in terms of the osmotic stress tolerance of the Chenopodiaceae and Gramineae (7, 15-18, 20, 32, 33). But a key test of the adaptive significance of glycine betaine accumulation in osmoregulation in higher plants must ultimately come from the identification and detailed characterization ofglycine betaine deficient mutants of glycine betaine accumulating species (16,17 In considering screening maize germplasm for genotypic differences in glycine betaine levels, and potential methods for characterizing putative betaine null genotypes, we noted a paucity ofconvenient methods which would lend themselves to both accurate quantification of low glycine betaine levels in the presence of other quaternary ammonium compounds (e.g. choline, phosphoryl-choline, and betaine aldehyde) and stable isotope tracer studies on the betaine biosynthetic pathway. Available methods for betaine determination lack either sensitivity, resolution, and/or amenability to stable isotope tracer work (4, 8, 12-14, 19, 20). We therefore sought sensitive mass spectral methods for quantifying glycine betaine and its stable isotope abundance.Mass spectral analysis of quaternary ammonium compounds of synthetic and natural origin has been approached for the most part by desorption methods. FABMS2 (6), 252Cf fission-fragment and laser desorption (...

Preliminary Genetic Studies of the Phenotype of Betaine Deficiency in Zea mays L.

“…Methanol extracts were phase-separated by addition of 5 mL chloroform + 6 mL H20. The upper aqueous phase was removed and concentrated to dryness under a stream of dry air, at which point known amounts of internal standards were added (250 nmol of L-aamino-n-butyrate for amino acid analyses of Tables I and II, and Figure 1, and 1 125 or 500 nmol dg-glycine betaine for the betaine determinations [21]). Samples for betaine analysis spiked with internal standard were then redissolved in 2 mL H20 and applied to 1 cm x 2 cm columns of Dowex-1-OH resin, and betaines were eluted with 6 mL H20.…”

“…Several betaine-deficient inbreds and hybrids of maize have recently been identified (21), and this has prompted preliminary genetic analysis of the phenotype of betaine deficiency in maize. At the outset, we emphasize that nothing is yet known about the relationship between this phenotype of betaine deficiency and stress susceptibility in maize, and indeed it is unlikely that such relationships could be definitively established without substantial efforts first devoted to creating isogenic lines or isopopulations differing solely for this metabolic character (8,9).…”

Biosynthesis of 3-Dimethylsulfoniopropionate in Wollastonia biflora (L.) DC. (Evidence That S-Methylmethionine Is an Intermediate)

“…DMSP was purified and converted to the HCl form with a 10-mL mixed resin column [Dowex-1 (OH-):BioRex-70 (H+), 2: 1 (v/v)] followed by a 2-mL Dowex-50 (H+) column. After washing both columns with water, DMSP was eluted from Dowex-50 with 10 mL of 2.5 M HCl and freeze dried; excess HCl was removed by twice redissolving in water and freeze (Blackwell, 1981 (Paquet et al, 1994) and [2H9](31y betaine (Rhodes et al, 1987) were prepared as described.…”

“…Converting DMSP.HC1 to the neutra1 form by ion exchange caused losses of about 40%. Sensitivity could not be enhanced by converting DMSP to its n-butyl ester, as can be done for betaines (Rhodes et al, 1987). After derivatization, no peaks corresponding to DMSP or its ester were found, probably because the ester spontaneously decomposes by elimination of DMS (Wagner and Daniels, 1965).…”

Development of Two Isogenic Sweet Corn Hybrids Differing for Glycinebetaine Content

“…Bet2 (betaine) accumulation in response to salinity stress is proposed to play an important role in osmotic adjustment in halophytic members of the Chenopodiaceae and Gramineae (7,(18)(19)(20)22), and may function as a compatible osmotic solute of the cytoplasm and/or chloroplast (17,22 salt-tolerant grasses such as Spartina and Distichlis, betaine levels may exceed 40 ,umol/gfw (16,20). Moderately salttolerant grasses such as Hordeum vulgare and Sorghum bicolor accumulate fairly high amounts of betaine (approximately 20 jAmol/gfw) (3,6,9,12), whereas salt-sensitive grass species such as Zea mays exhibit a fairly low capacity for betaine accumulation; the maximum reported values for betaine level in maize are only about 5 ,tmol/gfw (9,16,18), and some maize genotypes lack betaine almost completely (15,16). Recent evidence suggests that in one betaine-deficient inbred of maize, the phenotype of betaine deficiency is caused by a single, homozygous recessive, nuclear encoded gene (15).…”