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)

Skin test for diagnosis of penicillin allergy--current status.

The skin test for evaluating allergy to penicillin is reviewed. The reagents, penicillin, penicilloylpolylysine, benzyl penicilloate, and benzyl penilloate provide a safe and effective skin test for screening out the likelihood of severe allergic reactions to penicillin. The skin test gives much more accurate information than the patient's history and will enable many patients to receive penicillin despite a past history of allergy to the drug. Recent developments in the standardization and stabilization of the minor determinant mixture should result in making this reagent more available.

A device for positioning the breast during angiography.

A patient positioning device to be used during angiography of the breast that can easily be mounted and removed from an existing table top is described. The device facilitates patient positioning, provides good compression of the breast, and permits the placement of a water bolus during the studies.

A liquid chromatographic study of stability of the minor determinants of penicillin allergy: a stable minor determinant mixture skin test preparation.

Various skin test reagents supplying minor determinants for detecting penicillin hypersensitivity have been examined by high-performance liquid chromatography (HPLC) for composition and stability. HPLC systems capable of separating and determining the four diastereoisomers of benzyl-D-penicilloic acid and the two benzyl-D-penicilloic acids were developed for this purpose. The "simple skin test reagent," consisting of an aged partial alkaline hydrolysate of penicillin, is possibly an adequate source of (5R,6R)-benzyl-D-penicilloate whereas the "simple skin test reagent," consisting of aged aqueous solution of penicillin, is a questionable source of this compound. A modified Levine, Voss, Redmond, and Zolov minor determinant mixture (MDM) reagent and the components (5R,6R)-benzyl-D-penicilloate and (5R)-benzyl-D-penilloate have been found to be highly labile in aqueous solution, giving rise to a mixture of diastereoisomers. The tendency to epimerize at C-5 was a prominent feature of (5R,6S)- and (5S,6R)- as well as (5R,6R)-benzyl-D-penicilloic acids. The MDM reagent has been prepared in single-dose ampules as a dried, lyophilized powder that can be stored without change and used as needed. Lyophilized MDM has served as a satisfactory substitute for freshly prepared MDM in several individuals with MDM-positive history and, in a recent clinical study, evaluating the question of penicillin skin test sensitization. This convenient, stable, single-dose form of the MDM reagent should facilitate skin testing for penicillin sensitivity.

Routine elective penicillin allergy skin testing in children and adolescents: study of sensitization.

This study aims at evaluating the possibility in children and adolescents of (re)sensitization to penicillin that could result from skin test and challenge. Patients (240) with a history of a reaction to penicillin or one of its analogs were skin-tested with penicillin G, commerical benzlpenicilloyl polylysine, and a minor determinant mixture consisting of sodium benzylpenicilloate and sodium benzylpenilloate. The patients were tested when well, in no immediate need for penicillin, and during a routine office visit. Twenty-one (8.75%) patients had one or more positive skin tests. Three (14%) of the positive reactors reacted only to the MDM mixture, with one reacting only to the benzylpenilloate component. Of the patients with negative skin tests, 219 were given a 10-day course of oral penicillin. Three (1.4%) of the patients developed a-mild skin exanthem 7 to 10 days after starting the penicillin. All skin test-negative patients were retested 4 wk or more after completion of the oral challenge. Only two patients (less than 1%) who tolerated an oral challenge of penicillin had a positive skin test upon retesting. We believe that the described penicillin allergy testing procedure in children and adolescents with a history of allergy to penicillin or certain analogs is a safe, highly predictive, nonsensitizing office procedure in the hands of physicians experienced with skin testing. It should be considered for all such individuals labeled as allergic to penicillin when they are well and not in immediate need of penicillin.

[3-(1,4-Cyclohexadienyl)-L-alanine,8-lysine]vasopressin: synthesis and some pharmacological properties.

[3-(1,4-Cyclohexadienyl)-L-alanine,8-lysine]vasopressin, otherwise known as [3-(2,5-dihydrophenylalanine),8-lysine]vasopressin or [DiHPhe3]lysine-vasopressin, has been synthesized in an attempt to utilize 2,5-dihydrophenylalanine (DiHPhe) to evaluate the contribution of aromaticity in position 3 to biological activity. The analogue has the same primary structure as lysine-vasopressin, except that two additional hydrogen atoms are present on the ring moiety of the phenylalanine residue in position 3. The key intermediate was the protected nonapeptide N-carbobenzoxy-S-benzyl-L-cysteinyl-L-tyrosyldihydrophenyl-L-alanyl-L-glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N epsilon-tosyl-L-lysylglycinamide that was synthesized stepwise by the solid-phase technique. Deprotection with sodium in liquid ammonia was followed by sulfhydryl oxidation with I2 to give the hormone analogue. [DiHPhe3]lysine-vasopressin exhibited 125--130 units/mg of antidiuretic, 129--132 units/mg of rat pressor, and 6 units/mg of rat uterus contracting activity. To confirm the presence of DiHPhe in the analogue, an enzymatic procedure employing Aspergillus oryzae was developed that liberates in high yield the amino acid residue in position 3 of the posterior pituitary hormone structure. This study should be applicable to other biologically active peptides.

Phenylalanine ammonia-lyase: enzymic conversion of 3-(1,4-cyclohexadienyl)-L-alanine to trans-3-(1,4-cyclohexadienyl)acrylic acid.

The phenylalanine analogue 3-(1,4-cyclohexadienyl)-L-alanine is converted to the hitherto unknown cinnamate analogue trans-3-(1,4-cyclohexadienyl)acrylic acid by L-phenylalanine ammonia-lyase (EC 4.3.1.5) from maize, potato, or Rhodotorula glutinis. The structure assigned to the product is confirmed by its 1H nuclear magnetic resonance spectrum and by the chemical synthesis to be described in a subsequent paper. On comparing the above substrate analogue with L-phenylalanine, the Km was lowered only slightly but kcat was reduced 14--40-fold depending on the source of the enzyme. Because the compounds closely resemble each other in size and hydrophobic properties, this lowering of kcat may be attributed to the electronic effect of replacing the pi electrons of the aromatic system by those of a double bond. Correct alignment at the active site appears to depend upon the space-filling properties of the ring system; open chain analogues that retain the gamma, beta double bond were found to be inhibitors, not substrates.

Aminonitriles and aminothioamides related to natural amino acids.

N-p-Methoxybenzyloxycarbonyl and N-tert.-butyloxycarbonyl amino acid amides related to a series of natural amino acids were dehydrated to the corresponding Meoz- and Boc-alpha-aminonitriles. Deprotection of the latter derivatives afforded alpha-aminonitriles related to alanine, tyrosine, phenylalanine, dihydrophenylalanine, histidine, Dopa, ornithine, asparagine and glutamine. Thioamidation with H2S/NH3 or H2S/NEt3 in general converted the protected amino nitriles to Meoz- and Boc-alpha-aminothioamides. When deprotected these furnished the alpha-aminothioamides corresponding to alanine, tyrosine, phenylalanine, dihydrophenylalanine and histidine. For dehydration and thioamidation of histidine and Dopa, N alpha-Boc-im trityl-histidine and N-Boc-O, O'-diacetyldihydroxyphenylalanine were useful. Dopa was obtained as the free and Boc-thiohydrazide. Also prepared were N alpha,omega-diMeoz-ornithine DCHA, Meoz-2,5-dihydrophenylalanine DCHA and N,O-diMeoz-tyrosine as starting materials and N,O-dicarbobenzyloxycarbonyltyrosinamide, N,O-diZ-tyrosine nitrile and Z-beta-cyano-beta-alaninamide as model compounds. During deprotection of Meoz-alanine thioamide, transfer of an anisyl group from the N-Meoz protecting group to sulfur took place as a side reaction that yielded alanine p-methoxybenzyl beta-imidothiolic ester. This study provides two new series of amino acid analogs with potential antimetabolite activity. Also suitable for incorporation into peptide analogs, these afford approaches to relating structure and conformation to activity in biologically active peptides.

Enzymatic dtermination of pyridoxal 5'-phosphate with gamma-cyanoaminobutyric acid aposynthase.

A rapid alternative method is presented for the determination of pyridoxal 5'-phosphate (pyridoxal-P). The method involves the colorimetric analysis of thiocyanate liberated from S-cyanohomocysteine (Hcy (CN)) in the presence of cyanide when catalyzed by the pyridoxal-P dependent enzyme, gamma-cyano-alpha-aminobutyric acid (gamma-CNabu)-synthase (Hcy (CN) thiocyano-lyase [adding CN]). The rate of formation of thiocyanate is determined by the increase in absorbance at 470 nm on treatment of the enzymatic reaction mixture with FeCl3.