Abstract
beta-Carbolines are tricyclic compounds deriving from the condensation of indoleamines or tryptophan with fairly electrophilic carbonylic compounds to form a fused benzene-pyrrole-pyridine ring system (Pictet-Spengler reaction). Previous investigation carried out with model systems proved the facile formation of various tetrahydro-beta-carboline-3-carboxylic acids (beta-CCA) from tryptophan and reactive carbonyls present in wood smoke, under conditions simulating those of foods during their smoking process. The present work has been undertaken to investigate and confirm the actual occurence of such compounds in real smoked foodstuffs of animal origin. Five representative beta-CCA were selected taking into account the high content of their precursor aldehydes in smoke (formaldehyde, acetaldehyde, glycolaldehyde) and/or the facility of their formation during the model system experiments (benzaldehyde and salicylaldehyde derivatives). They were synthesized following methods based on the Pictet-Spengler reaction as they were described in the literature, or by slightly modifying and altering them. The structure of the synthesised products was confirmed using spot tests, melting points, elemental analysis and spectrometric methods such as fluorescence, UV, 1HNMR, MS and 13CNMR spectroscopy. A method was developed for the isolation of these compounds from smoked solid foods. The samples were extracted with an aqueous acidic medium containing semicarbazide to prevent artifactual formation of beta-CCA. The extract was purified on an XAD-4 resin containing, clean-up column. The eluate was concentrated, further purified by extraction with CHaCla and analysed by high performance liquid chromatography on a Spherisorb ODS1 reversed phase column with gradient elution and fluorescence detection. The quantitation was based on calculating the ratio of the peek area of each compound to the peak area of the internal standard (serotonin creatinine sulphate). The mean recovery of the method was about 76% for the more hydrophilic derivatives of formaldehyde, acetaldehyde and glycol aldehyde and 11% for the more hydrophobic derivatives of benzaldehyde and salicylaldehyde. Higher concentrations of analytes in the samples led to decreased recoveries. The detection limit was lower than 25 mug/kg for the more hydrophilic and 2 mg/kg for the more hydrophobic group of compounds. Thirty four samples of miscellaneous smoked food products such as dry salamis, heat processed comminuted meat products, cured meats (heat processed or not), raw or heat processed fish, hard cheese, were analysed. The beta-CCA concentration in them was variable depending on many factors such as pH value, heavy or light smoking and maturation time. Dry, fermented comminuted meat products contained the higher concentrations. Tetrahydro-beta-carboline-3-carboxylic acid prevailed in all samples examined and was detected at a level of 0.86 up to 22.63 mg/kg in dry salamis, whereas in other samples it ranged from 16 to 680 mug/kg. The levels of 1-methyl- and 1- hydroxymethyl-tetrahydro-beta-carboline-3-carboxylic acids were much lower (0-881 mug/kg and 0-444 mug/kg respectively). The occurence of benzaldehyde and salicylaldehyde derivatives was not definitely proved. The synthesised beta-CCA were tried for mutagenicity using the Ames test. None of them was mutagenic to Salmonella typhimurium TA-100, TA-98, TA-97 strains with and without metabolic activation, end without preincubation. At concentrations higher than 100 mug/plate, 1-hydroxymethyl- and unsubstituted tetrahydro-beta-carboline-3-carboxylic acids were toxic to strains TA-98 and TA-100. B-Carbolines are biologically active compounds and their detection in smoked foods might help to prevent potential implications to the consumers' health.