Vicianin, prunasin, and beta-cyanoalanine in common vetch seed as sources of urinary thiocyanate in the rat.

When young rats were fed a diet containing common vetch seed for 1 month, they excreted in the urine approximately 7 times more thiocyanate than they had ingested. Vicianin, prunasin, and beta-cyanoalanine were identified as principal dietary sources of the excreted thiocyanate. Vicianin was isolated by chromatography and crystallization. Its structure was confirmed by mass spectrometry and by identification of its monosaccharides and aglycon. Prunasin was identified chromatographically by HPLC. The combined seed content of vicianin (0.68 micromol/g) and prunasin (0.16 micromol/g) corresponded to the cyanogen content of the seed (0.91 +/- 0.14 micromol/g; n = 7), determined as cyanide after autolysis. When vicianin was fed, the urinary thiocyanate output was 21% of the ingested amount of vicianin, whereas beta-cyanoalanine yielded a urinary thiocyanate output of < 0.2%. Calculations show that 73% of the thiocyanate can be derived from vicianin and prunasin, with 27% derived from beta-cyanoalanine. High urinary output of thiocyanate has been associated with endocrine and neurological disorders.

N epsilon-cyclosuccinyllysine: synthesis and formation from hemisuccinylated polylysine and its hapten conjugates with sulfamethoxazole during acid hydrolysis.

The novel amino acid N epsilon-cyclosuccinyllysine (4) forms as a side reaction during acid hydrolysis of N epsilon-hemisuccinylated succinylsulfamethoxazole-polylysine conjugates. The presence of 4 in hydrolysates can be obscured in amino acid analysis. Identification of 4 was based on chromatographic and electrophoretic comparisons with authentic N epsilon-cyclosuccinyllysine, which was synthesized in high yield by acid-catalyzed ring closure, under anhydrous conditions, of N epsilon-hemisuccinyl-L-lysine. The latter was prepared by an improved route, starting from N-alpha-(tert-butyloxycarbonyl)-L-lysine. The amount of 4 formed is influenced by the extent of succinylation and the conditions of hydrolysis and has reached 30 mol % of lysine. Both the hemisuccinyllysyl and the haptenized succinyllysyl residues participate in cyclization. This was confirmed by the synthesis and hydrolytic behavior of hemisuccinylated polylysine (n = 16) and N4,N epsilon-succinyl(sulfamethoxazole)(lysine). Formation of 4 can result in error in the estimation of the degree of ligand substitution based on lysine content in synthetic hemisuccinylated skin test antigens and peptide immunogens, especially when the degree of epitope substitution is low. 4 appears to be sufficiently stable for trial as an amino acid surrogate.

Synthesis and characterization of protein and polylysine conjugates of sulfamethoxazole and sulfanilic acid for investigation of sulfonamide drug allergy.

Conjugates of sulfamethoxazole (SMX) with human serum albumin (HSA), transferrin (TR), and poly(L-lysine) (PL, degrees of polymerization 16 and 430) have been prepared. As a model, succinylSMX-glycine methyl ester was synthesized by carbodiimide and active ester routes. The proteins and PL were acylated with succinylSMX succinimido ester, affording conjugates (succinylSMX)2-21-HSA, (succinylSMX)17,27-TR, (succinylSMX)11-Lys16, and (succinylSMX)71-Lys430 in which SMX was linked by a spacer chain of four carbons. This represents substitution of up to 35, 46, 65, and 17% of the amino groups of HSA, TR, PL16, and PL430, respectively. HSA was also acylated with the succinimido esters of succinylSMX-glycine and succinylSMX-epsilon-aminohexanoic acid, affording conjugates (succinylSMX-Gly)53-HSA and (succinylSMX-epsilon-NH2hex)51-HSA. In these conjugates SMX was linked by a spacer chain of 7 and 11 carbons, respectively, and almost all the amino groups of HSA were substituted. Factors apparently influencing the extent of conjugation to HSA were the stability of the active ester and the solubility of the conjugation reaction mixture. A sulfanilic acid (SA) conjugate, containing 12 mol of ligand/mol of HSA, was also prepared. The route of synthesis involved acylation of HSA with sulfanilyl fluoride. N-epsilon-Sulfanilyl-L-lysine dihydrochloride, required for quantitation of bound SA, was synthesized by a new route starting from alpha-Boc-L-lysine. Conjugates (sulfanilyl)12-HSA and (succinylSMX)13-HSA, differing in molecular weight from HSA by only 2.6 and 6.5%, were distinguishable from HSA by gel-filtration HPLC, as were the more highly substituted conjugates from their respective unsubstituted materials.(ABSTRACT TRUNCATED AT 250 WORDS)