Thomas P. Mawhinney

Analysis of amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry

A gas-liquid chromatographic procedure is described which will separate and quantitate the seventeen amino acids typically found in protein acid hydrolyzates. If present, tryptophan, cysteine and carboxymethylcysteine can also be simultaneously assayed via this method. The amino acids, as their tert.-butyldimethylsilylated derivatives, are readily separable on SE-30 (wall-coated open-tubular) and OV-17 (bonded) capillary columns, as well as on packed gas-liquid chromatographic columns coated with the same liquid phases. Retention times and responses for all amino acids and internal standards are given. Mass spectral analysis of all tert.-butyldimethylsilylated amino acids is presented and displays a characteristic and unique [M- 57] fragment ion for each amino acid which often dominates the mass spectrum. Application of this method is demonstrated using four well characterized proteins.

Products of the Colonic Microbiota Mediate the Effects of Diet on Colon Cancer Risk

It is estimated that most colon cancers can be attributed to dietary causes. We have hypothesized that diet influences the health of the colonic mucosa through interaction with the microbiota and that it is the milieu interior that regulates mucosal proliferation and therefore cancer risk. To validate this further, we compared colonic contents from healthy 50- to 65-y-old people from populations with high and low risk, specifically low risk Native Africans (cancer incidence <1:100,000; n = 17), high risk African Americans (risk 65:100,000; n = 17), and Caucasian Americans (risk 50:100,000; n = 18). Americans typically consume a high-animal protein and -fat diet, whereas Africans consume a staple diet of maize meal, rich in resistant starch and low in animal products. Following overnight fasting, rapid colonic evacuation was performed with 2 L polyethylene glycol. Total colonic evacuants were analyzed for SCFA, vitamins, nitrogen, and minerals. Total SCFA and butyrate were significantly higher in Native Africans than in both American groups. Colonic folate and biotin content, measured by Lactobacillus rhamnoses and Lactobacillus plantarum ATCC 8014 bioassay, respectively, exceeded normal daily dietary intakes. Compared with Africans, calcium and iron contents were significantly higher in Caucasian Americans and zinc content was significantly higher in African Americans, but nitrogen content did not differ among the 3 groups. In conclusion, the results support our hypothesis that the microbiota mediates the effect diet has on colon cancer risk by their generation of butyrate, folate, and biotin, molecules known to play a key role in the regulation of epithelial proliferation.

The rapid, quantitative determination of neutral sugars (as aldononitrile acetates) and amino sugars (as O-methyloxime acetates) in glycoproteins by gas--liquid chromatography.

Abstract O -Methyloximes have been prepared from 2-amino-2-deoxy- d -glucose, - d -mannose, and - d -galactose. The acetates of these derivatives yield stable compounds which are readily separated quantitatively by gas-liquid chromatography on a number of polar phases. The above compounds along with the aldononitrile acetates of neutral sugars can be easily separated from one another on a single column in one chromatographic run. The procedures developed were tested on a number of glycoproteins of known composition as reported by other workers utilizing more classical methodologies, resulting in excellent agreement in terms of sugar composition. An improved method is also described for converting neutral sugars to oximes which can be either converted to the trimethylsilyl derivatives or, upon acetylation, derivatized to aldononitrile acetates.

Sulfated sialyl-oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis.

Thirteen novel oligosaccharides, each possessing both a sulfate ester and a sialic acid residue, were isolated from tracheobronchial mucous glycoproteins from a patient with cystic fibrosis via cleavage by alkaline borohydride treatment, and by employing immobilized Limulus polyphemus lectin affinity chromatography, SynChroprep AX300 anion-exchange chromatography, Bio-Gel P-2 size-exclusion chromatography, and Hypersil 120A APS-2 high-performance liquid chromatography (HPLC). Proposed structures for the resulting purified sulfated sialyl-oligosaccharides were based on carbohydrate/permethylation analyses, periodate oxidation, complete sequential exoglycosidase digestion, analysis of desulfated products and, analysis by positive-ion fast-atom-bombardment mass spectrometry (FABMS). Sulfate esters on these sialyl-oligosaccharides resided on C-6 of a terminal or an internal D-galactose or 2-acetamido-2-deoxy-D-glucose residue or C-4 of a terminal D-galactose residue. The sialic acid residues were found to be either bound (2-->6)-alpha to 2-acetamido-2-deoxy-D-galactitol or (2-->3)-alpha or (2-->6)-alpha to a D-galactose residue occupying a nonreducing terminus. For this group of oligosaccharides, ranging in size from tri- to hepta-saccharides, it was also observed that a sialic acid residue and a sulfate ester did not residue on the same oligosaccharide branch when more than one branch existed. On linear unbranched sulfated sialyl-oligosaccharides, the sialic acid residue was bound to a D-galactose residue occupying a nonreducing terminus with the sulfate ester residing on an internal D-galactose or a 2-acetamido-2-deoxy-D-glucose residue. These results demonstrate that it is possible for sialic acid and a sulfate ester to exist on the same oligosaccharide and that this oligosaccharide can be as small as a trisaccharide.

Chemical composition of cultivated seaweed Ulva clathrata (Roth) C. Agardh

Samples of cultivated Ulva clathrata were collected from a medium scale system (MSS, 1.5×1.5m tank), or from a large scale system (LSS, 0.8ha earthen pond). MSS samples were dried directly while the LSS sample was washed in freshwater and pressed before drying. Crude protein content ranged 20-26%, essential amino acids accounting for 32-36% of crude protein. The main analysed monosaccharides were rhamnose (36-40%), uronic acids (27-29%), xylose (10-13%) and glucose (10-16%). Some notable variations between MSS and LSS samples were observed for total dietary fibre (26% vs 41%), saturated fatty acids (31% vs 51%), PUFAS (33% vs 13%), carotenoids (358 vs 169mgkg-1dw) and for Ca (9 vs 19gkg-1), Fe (0.6 vs 4.2gkg-1), Cu (44 vs 14mgkg-1), Zn (93 vs 17mgkg-1) and As (2 vs 9mgkg-1). The chemical composition of U. clathrata indicates that it has a good potential for its use in human and animal food.

A redox‐active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis

We report a detailed functional characterization of an Arabidopsis isopropylmalate dehydrogenase (AtIPMDH1) that is involved in both glucosinolate biosynthesis and leucine biosynthesis. AtIPMDH1 shares high homology with enzymes from bacteria and yeast that are known to function in leucine biosynthesis. In plants, AtIPMDH1 is co-expressed with nearly all the genes known to be involved in aliphatic glucosinolate biosynthesis. Mutation of AtIPMDH1 leads to a significant reduction in the levels of free leucine and of glucosinolates with side chains of four or more carbons. Complementation of the mutant phenotype by ectopic expression of AtIPMDH1, together with the enzymes substrate specificity, implicates AtIPMDH1 in both glucosinolate and leucine biosynthesis. This functional assignment is substantiated by subcellular localization of the protein in the chloroplast stroma, and the gene expression patterns in various tissues. Interestingly, AtIPMDH1 activity is regulated by a thiol-based redox modification. This work characterized an enzyme in plants that catalyzes the oxidative decarboxylation step in both leucine biosynthesis (primary metabolism) and methionine chain elongation of glucosinolates (specialized metabolism). It provides evidence for the hypothesis that the two pathways share a common origin, and suggests a role for redox regulation of glucosinolate and leucine synthesis in plants.

Gas-liquid chromatography-mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers.

tert.-Butyldimethylsilyl ethers of secondary hydroxy fatty acid methyl esters (tBDMS-O-FAMEs) produce stable derivatives amenable to gas-liquid chromatography (GLC) and mass spectrometry (MS). Derivatives produce prominent molecular mass minus 57 [M-57]+ fragment ions and unique marker fragment ions indicating the location of the secondary hydroxyl groups along the aliphatic chain from the omega-2 carbon to carbon numbers 5 from the carboxylic terminus, in addition to yielding information regarding carbon chain length, and degree of unsaturation. The tBDMS-derivatives of C-2, C-3 hydroxy fatty acids and the unique GLC-MS data of gamma- and delta-lactones are also presented. Though several tBDMS-O-FAMEs with centrally located hydroxyl groups were not chromatographically resolved, the combination of GLC retention times and monitoring of key diagnostic fragment ions of each tBDMS-derivative, when applied to mixtures containing all hydroxy isomers of palmitic through arachidic acid methyl esters, and to several monounsaturated, monohydroxylated fatty acid methyl esters, allowed for their unambiguous identification. Coupled with derivative stability, permitting their purification and concentration, this method was applied to the identification of trace lipids isolated from bovine skim milk which contained a complex mixture of hydroxy fatty acids of which 19 were newly identified.

Microwave digestion and ultrasonic nebulization for determination of boron in animal tissues by inductively coupled plasma atomic emission spectrometry with internal standardization and addition of mannitol

Determinations of boron in animal tissues were performed by inductively coupled plasma atomic emission spectrometry (ICP-AES) with an ultrasonic nebulizer. The method is based on the microwave digestion of tissues with HNO 3 –H 2 O 2 and the use of mannitol as a modifier. It was found that mannitol can significantly enhance the analytical sensitivity, improve the precision and minimize the memory effect for the determination of boron. Acid effect and matrix effects were controlled by using the Be I 234.861 nm line as the internal standard. Six animal tissue samples, including two NIST standard reference materials (Oyster Tissue and Bovine Liver), were analyzed to test the reliability of the method. A limit of detection (3 σ ) of 0.7 µg l -1 and recoveries of added boron from selected matrices between 95.0 and 100.2% were obtained.

1-Amino-1-deoxy-d-fructose (“Fructosamine”) and its Derivatives

Fructosamine has long been considered as a key intermediate of the Maillard reaction, which to a large extent is responsible for specific aroma, taste, and color formation in thermally processed or dehydrated foods. Since the 1980s, however, as a product of the Amadori rearrangement reaction between glucose and biologically significant amines such as proteins, fructosamine has experienced a boom in biomedical research, mainly due to its relevance to pathologies in diabetes and aging. In this chapter, we assess the scope of the knowledge on and applications of fructosamine-related molecules in chemistry, food, and health sciences, as reflected mostly in publications within the past decade. Methods of fructosamine synthesis and analysis, its chemical, and biological properties, and degradation reactions, together with fructosamine-modifying and -recognizing proteins are surveyed.

Separation and analysis of sulfate, phosphate and other oxyanions as their tert.-Butyldimethylsilyl derivatives by gas—liquid chromatography and mass spectrometry

The tert.-butyldimethylsilyl derivatives of the oxyanions carbonate, sulfite, sulfate, selenite, selenate, molybdenate, borate, phosphite, phosphate, vanadate, arsenite, arsenate and pyrophosphate were prepared by reacting the free acid and the ammonium salt form of the oxyanions with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide in N,N-dimethylformamide. All derivatives were amenable to gas chromatographic analysis and, except for molybdenate, were stable for over a week at room temperature and over six months at 4°C. Each tert.-butyldimethylsilylated oxyanion produced an easily interpretable mass spectrum dominated by a unique M — 57 fragment ion.