Temperature and pH effects on biodegradation of hexachlorocyclohexane isomers in water and a soil slurry

This study was conducted to monitor the biodegradation of alpha-, beta-, gamma-, and delta-hexachlorocyclohexane (HCH) isomers in liquid culture by a Pandoraea species and determine the influence of pH and temperature on the biodegradation of alpha- and gamma-HCH in liquid as well as in soil slurry cultures. The Pandoraea species degraded 79.4% delta-HCH and 34.3% gamma-HCH in liquid culture at 4 weeks of incubation. alpha- and beta-HCH exhibited almost identical rates (41.6 and 42.4%, respectively) of degradation. The highest degradation of alpha- and gamma-HCH (67.1 and 60.2%, respectively) was observed at an initial pH of 8.0 in liquid; 58.4 and 51.7% rates of degradation of alpha- and gamma-HCH, respectively, at an initial pH of 9.0 were found in soil slurry cultures. An incubation temperature of 30 degrees C was optimum for effective degradation of alpha- and gamma-HCH isomers (62.5 and 57.7%, respectively) in liquid culture, and 54.3 and 51.9% rates of degradation of alpha- and gamma-HCH isomers, respectively, were found in a soil slurry. Increasing the soil/water ratio decreased the extent of degradation of both HCH isomers. Degradation of HCH isomers occurred concomitant with bacterial growth. Byproducts of growth from Pandoraea species significantly decreased the pH of the liquid and the soil slurry during the growth on HCH isomers. The results of this study suggest that this bacterial strain may effectively be used for remediating polluted sites and water contaminated with different HCH isomers over a range of environmental conditions.     

The persistence and degradation of chlorothalonil and chlorpyrifos in a cranberry bog

The effect of a spray-tank adjuvant on the persistence, distribution, and degradation of two pesticides, chlorothalonil and chlorpyrifos, was studied in a commercial cranberry bog. Pesticides were applied according to label instructions to cranberry plants in paired plot studies. Dislodgeable foliar and whole fruit residues of both pesticides and several degradation products were assessed over a growing season. Residues were also assessed in soil samples collected at fruit harvest. Adjuvant increased both fruit and foliar residues but did not significantly alter the dissipation rate or metabolism of either pesticide. The dissipation of dislodgeable foliar chlorothalonil and chlorpyrifos residues followed first-order kinetics, with estimated half-lives of 12.7 and 3.5 d, respectively. All residue levels on harvested fruit were well below the current U.S. EPA tolerances for fresh cranberries. Chlorothalonil (58%) was the major residue in fruit at harvest (76 d post-chlorothalonil application), with 4-hydroxy-2,5,6-trichloroisophthalonitrile and 1,3-dicarbamoyl-2,4,5,6-tetrachlorobenzene accounting for 26% and 6% of the total residues, respectively. Degradation products accounted for 88% of the total chlorothalonil residues in soil at fruit harvest. The products 1,3-dicarbamoyl-2,4,5,6-tetrachlorobenzene, 1-carbamoyl-3-cyano-4-hydroxy-2,5,6-trichlorobenzene, 2,5,6-trichloro-4-methylthioisophthalonitrile, and 2,4,5-trichloroisophthalonitrile have not been previously identified in cranberry bog environments. Chlorpyrifos was detected in fruit at harvest (62 d post-chlorpyrifos application), but no metabolites were found. Chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol, however, were detected in earlier fruit samples and in foliage and soil samples.     

Hyperproduction and application of alpha-agarase to enzymatic enhancement of antioxidant activity of porphyran

The nucleotide sequence of the gene for the alpha-agarase, AgaA33, from Thalassomonas sp. strain JAMB-A33 was determined. The open reading frame for AgaA33 was revealed to encode 1463 amino acid residues. We succeeded in extracellular production of recombinant -agarase (AgaA33) efficiently using Bacillus subtilis as a host. This is the first report of recombinant production of -agarase. Furthermore, we demonstrated that hydrolysis of alpha-1,3 linkages in porphyran, a sulfated polysaccharide from marine red algae, by alpha-agarase is an important step for improvement of its antioxidant activity with regard to free-radical-scavenging capacity and superoxide radical anion scavenging activity, whereas the hydrolysis of beta-1,4 linkages in porphyran by beta-agarase did not increase on the antioxidant activity markedly.     

In vivo studies on the metabolism of the monoterpenes S-(+)- and R-(-)-carvone in humans using the metabolism of ingestion-correlated amounts (MICA) approach

The major in vivo metabolites of S-(+)- and R-(-)-carvone in a metabolism of ingestion correlated amounts (MICA) experiment were newly identified as alpha,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), alpha-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone) on the basis of mass spectral analysis in combination with syntheses and NMR experiments. Minor metabolites were identified as reduction products of carvone, namely, the alcohols carveol and dihydrocarveol. The previously identified major in vivo metabolite in rabbits, 10-hydroxycarvone, could not be detected, indicating either concentration effects or interspecies differences. Metabolic pathways for carvone in humans including oxidation of the double bond in the side chain and, to a minor extent 1,2- and 1,4 + 1,2-reduction of carvone, are discussed. No differences in metabolism between S-(+)- and R-(-)-carvone were detected.     

Amylolytic activity in fruits: comparison of different substrates and methods using banana as model

Several methodologies have been developed for cereal amylolytic activity estimation, but there is lack of information about the application of these methods for fruits. Mature green banana fruit can achieve 20% of starch content which is degraded during the ripening period in a complex process involving alpha- and beta-amylases and alpha-1,4 and alpha-1,6-glucosidases, besides phosphorylases that can compete for the same substrates. Methods used to determine total hydrolytic activity and individual activity of enzymes involved in starch breakdown were compared for banana extracts in several ripening stages. Total hydrolytic activity was measured by DNS and iodometric methods. Endoamylolytic activity on amylose-azure substrate was also evaluated. BPNPG7 and PNPG5 chromogenic substrates were used for alpha- and beta-amylase activities, respectively. The results showed that methods that depend on the use of thermal treatment or on inhibitors to inactivate one of the enzymes were not adequate. The use of p-nitrophenol derivatives seemed to be the most specific, reproducible, and easiest method employed for single alpha- and beta-amylases activities determination in complex tissues. The DNS and iodometric methods can be used only for initial screenings of total hydrolytic activity, because the nonspecific substrate used in these procedures allows the action of more than one enzyme simultaneously.     

Structural features of arabinoxylans from bajra (pearl millet)

Structures of arabinoxylans (AX) from bajra, from a variety known to have characteristic quality in the making of roti, an unleavened flat bread, were elucidated by a combination of methylation analysis, (13)C NMR, FT-IR, etc. Arabinoxylans isolated from barium hydroxide-extracted polysaccharides and hemicellulose B had a backbone of xylose residues with beta-(1,4) linkages and were branched mainly through alpha-(1,3) linkages by arabinofuranosyl residues. Completely branched xylose residues were also present. The AX from bajra characteristically had large numbers of branches, and this may be one of the reasons for the crispiness of the rotis.     

Chemical, molecular, and structural characterization of alkali extractable nonstarch polysaccharides from Job's tears

The polished Job's tears ( Coix lachryma-jobi L.) seeds, dark and white husk types, were sequentially extracted by hot water (75 degrees C) and 0.5 M NaOH solution. Nonstarch polysaccharides were not found in the water extract but were present in the alkali extract. The major components of the alkali extract from both Job's tears were protein, ash, and nonstarch polysaccharides, mainly arabinoxylans. The high arabinose to xylose ratio of 1.25 and 1.24 indicated a highly substituted structure. The average molecular weight (MW) of arabinoxylans of the dark and white husk types were 741,000 Da (Pd 1.5) and 1,449,000 Da (Pd 2.6), respectively, and their average MW reduced after treatment with protease. The alkali extractable arabinoxylans were elucidated to have a (1,4)-linked beta- d-xylan main chain highly substituted with single arabinose units. The results showed that the alpha- l-arabinofuranosyl residues (Ara f) were attached to the main chain mostly at O-3, followed by both O-2 and O-3 of xylopyranosyl residues (Xyl p).     

Interaction of cyclodextrins with aliphatic acetate esters and aroma components of La France pear

The interaction between aliphatic acetate esters, which are the major flavor components of La France pear (Pyrus communis L.), and cyclodextrins (CDs) was studied to obtain fundamental data for preparing powdered pear flavor materials. The complexing abilities of alpha-, beta-, or gamma-CD with six kinds of aliphatic acetate esters were compared. alpha-CD formed the highest numbers of complexes with all of the esters. All of the CDs produced complexes more readily with the more hydrophobic esters. Among our samples of freeze-dried pear juice containing dissolved alpha-, beta-, or gamma-CD, the juice with alpha-CD retained the greatest amount of esters. These data demonstrate that alpha-CD is an effective material with which to prepare powdered pear flavor materials. The nuclear Overhauser effect, measured by (1)H NMR spectra, of an alpha-CD-butyl acetate or alpha-CD-hexyl acetate complex showed that these esters were included in the alpha-CD cavity.     

Kinetic studies of the free radical-scavenging actions of tocopherol metabolites (alpha-, gamma-, and delta-carboxyethyl-6-hydroxychroman) and Trolox in ethanol and micellar solutions

The reaction rates ( k s) of tocopherol metabolites (alpha-, gamma-, and delta-CEHC) and Trolox with aroxyl radical have been measured in ethanol and micellar solution by a stopped-flow spectrophotometer, and the k s values obtained were compared with those reported for tocopherols (alpha-, beta-, gamma-, and delta-tocopherol, TocH) and tocol. The rate constants ( k s) increased in the order of Tocol < delta-CEHC < delta-TocH < gamma-CEHC < Trolox approximately gamma-TocH approximately beta-TocH < alpha-CEHC < alpha-TocH in ethanol. The antioxidants that have lower oxidation potentials ( E p) showed higher reactivities. The k s values of alpha-, beta-, gamma-, and delta-tocopherol and tocol in micelle remained constant between pH 4 and pH 10 and decreased rapidly at pH 11~12 by increasing pH value. On the other hand, the k s values of alpha-CEHC, gamma-CEHC, and Trolox showed notable pH dependence. As a result of the detailed analysis of the pH dependence of the rate constants ( k s), the structure-activity relationship in the free radical-scavenging action of the tocopherol metabolites and Trolox has been clarified.     

Punicalagin and catechins contain polyphenolic substructures that influence cell viability and can be monitored by radical chemosensors sensitive to electron transfer

Plant polyphenols may be free radical scavengers or generators, depending on their nature and concentration. This dual effect, mediated by electron transfer reactions, may contribute to their influence on cell viability. This study used two stable radicals (tris(2,3,5,6-tetrachloro-4-nitrophenyl)methyl (TNPTM) and tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl (HNTTM)) sensitive only to electron transfer reduction reactions to monitor the redox properties of polyphenols (punicalagin and catechins) that contain phenolic hydroxyls with different reducing capacities. The use of the two radicals reveals that punicalagin's substructures consisting of gallate esters linked together by carbon-carbon (C-C) bonds are more reactive than simple gallates and less reactive than the pyrogallol moiety of green tea catechins. The most reactive hydroxyls, detected by TNPTM, are present in the compounds that affect HT-29 cell viability the most. TNPTM reacts with C-C-linked gallates and pyrogallol and provides a convenient way to detect potentially beneficial polyphenols from natural sources.     

Compositional analysis and rheological properties of gum kondagogu (Cochlospermum gossypium): a tree gum from India

Gum kondagogu ( Cochlospermum gossypium) is a tree exudate gum that belongs to the family Bixaceae. Compositional analysis of the gum by HPLC and LC-MS revealed uronic acids to be the major component of the polymer ( approximately 26 mol %). Furthermore, analysis of the gum by GC-MS indicated the presence of sugars such as arabinose (2.52 mol %), mannose (8.30 mol %), alpha- d-glucose (2.48 mol %), beta- d-glucose (2.52 mol %), rhamnose (12.85 mol %), galactose (18.95 mol %), d-glucuronic acid (19.26 mol %), beta- d-galactouronic acid (13.22 mol %), and alpha- d-galacturonic acid (11.22 mol %). Gum kondagogu, being rich in rhamnose, galactose, and uronic acids, can be categorized on the basis of its sugar composition as a rhamnogalacturonan type of gum. The rheological measurements performed on the gum suggest that above 0.6% (w/v) it shows a Newtonian behavior and shear rate thinning behavior as a function of gum concentration. The viscoelastic behavior of gum kondagogu solutions (1 and 2%) in aqueous as well as in 100 mM NaCl solution exhibits a typical gel-like system. The G' (viscous modulus)/ G' (elastic modulus) ratios of native gum kondagogu (1 and 2%) in aqueous solution were found to be 1.89 and 1.85 and those in 100 mM NaCl to be 1.54 and 2.2, respectively, suggesting a weak gel-like property of the polymer. Crossover values of G' and G' were observed to be at frequencies of 0.432 Hz for 1% and 1.2 Hz for 2% for native gum in aqueous condition, indicating a predominantly liquid- to solid-like behavior, whereas crossover values of 2.1 Hz for 1% and 1.68 Hz for 2% gum in 100 mM NaCl solution suggest a larger elastic contribution.     

Improved quantitative analysis of oligosaccharides from lichenase-hydrolyzed water-soluble barley beta-glucans by high-performance anion-exchange chromatography

Cereal beta-glucan is a linear biopolymer linked by beta-(1,3)/(1,4)-glycosidic bonds. More specifically, the beta-(1,4)-linked glucose chain is interrupted with beta-(1,3)-linkages in cereal beta-glucan structure. Elucidation of the exact length and distribution of linear beta-(1,4)-linked portion facilitates the understanding of the fine structure of cereal beta-glucan. A HPAEC assisted by lichenase treatment has been used for the structural and quantitative analysis of cereal beta-glucan. The absence of authentic standard oligosaccharides, putatively 3-O-beta-cellobiosyl-D-glucose (DP3) and 3-O-beta-cellotriosyl-D-glucose (DP4), was a potential problem to the characterization of beta-glucan structure. In this study, two major lichenase-hydrolyzed products were generated from the barley beta-glucan, and putative 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose were separated and highly purified by recycling preparative HPLC technology. Structural analysis of highly purified putative 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose was performed by TLC and LC-MS analysis. Two putative DP3 and DP4 displayed the nonreducing end/(1,4)/(1,3) linkage ratios of 1:0.96:0.90 and 1:2.18:1.16, respectively; the molecular masses (m/z) of their sodium adducts were 527.0 and 689.0, respectively. Using these structurally confirmed oligosaccharides, the exact amounts of beta-glucan lichenase hydrolysates from domestic barley cultivars were quantified. The amount of two major DP3 and DP4 accounted for only 71.4-73.3% of water-extractable beta-glucan fraction, and the (1,4)/(1,3) linkage ratios of the extracted beta-glucans were almost identical in the range of 2.24-2.25 among the barley cultivars tested.     

Improvement of oxidative stability of conjugated linoleic acid (CLA) by microencapsulation in cyclodextrins

Oxidative stability of conjugated linoleic acid (CLA) encapsulated in alpha-, beta-, and gamma-cyclodextrins (designated CLA/CDs microencapsules) was studied by measuring the headspace-oxygen depletion in airtight serum bottles and by measuring the peroxide values (POV). The rate of oxygen depletion was reduced from 41.0 (control) to 21.5, 2.1, 1.2, and 1.1 micromol/L.h(-)(1) by CLA/alpha-CD microencapsules at 1:1, 1:2, 1:4, and 1:6 mole ratios, respectively, indicating that CLA oxidation was completely protected by a 1:4 mole ratio of CLA/alpha-CD. Such a protective effect by CLA/beta-CD or CLA/gamma-CD microencapsules was achieved at a 1:6 mole ratio, but the effect by CLA/beta-CD was slightly greater than that by CLA/gamma-CD. The protective effect of alpha-, beta-, and gamma-CDs for CLA oxidation was confirmed by their POV-reducing abilities in CLA/CDs. These results suggest that alpha-CD was the most effective for the protection of CLA oxidation by microencapsulation, followed by beta-CD and gamma-CD.     

Fractionation and characterization of mushroom dietary fiber (nonstarch polysaccharides) as potential nutraceuticals from sclerotia of Pleurotus tuber-regium (Fries) singer

The nonstarch polysaccharides (NSPs) in the total dietary fiber (TDF) from the sclerotia of Pleurotus tuber-regium (tiger milk mushroom) were fractionated by the sequential use of chemical solvents. About half of the TDF was solubilized and two major alkali-soluble fractions (1 and 4 N sodium hydroxide) that contained 126 and 293 g/kg TDF were obtained. Sugar analysis and infrared spectroscopy indicated that the NSPs in these alkali-soluble fractions were mainly beta-glucans and chitin. These alkali-soluble NSPs were further purified by anion-exchange chromatography followed by gel permeation chromatographic separation. Methylation analysis revealed that these purified glucans were highly branched and contained a mixture of sugar linkages of beta-1,3, beta-1,6, and beta-1,4. The potential use of these sclerotial beta-glucans as nutraceuticals was discussed.     

Preparative fractionation of gliadins by electrophoresis at pH 3.1 (A-PAGE).

Purification of gliadin subclasses has been difficult since they share many biochemical and physicochemical properties. In this report, the optimization of a preparative electrophoretic method to fractionate gliadins is described. Separation was performed in preparative 7% polyacrylamide gels at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis. Preparative gels of different lengths were employed. Using 5-cm preparative gels, several fractions of alpha-, beta-, and gamma-gliadins and fast-mobility and slow-mobility omega-gliadins were collected in 40 h of separation. Resolution was maintained at a protein load of up to 30 mg in each run. The highest efficiency of recovery was achieved using aluminum lactate as the collecting buffer. Fractionation with 10 cm in length gels improved resolution but increased operation times. Gels of 2 cm in length did not separate alpha/beta- and gamma-gliadins efficiently but were useful to separate the two main fractions of omega-gliadins in shorter times. In conclusion, preparative electrophoresis at low pH allowed the separation of alpha-, beta-, gamma-, and omega-gliadin fractions from crude material under nondenaturing conditions.     

Organochlorine pesticide contents of tributaries into Blanca Bay,Argentina

Thirteen 1-L replicate water samples from streams emptying into Blanca Bay, Argentina, were taken to study the environmental quality of the surface waters. Out of tests for eleven chlorinated pesticides, five were detected in all the sampling places as both dissolved and adsorbed species: alpha-HCH, gamma-HCH (lindane), heptachlor, delta-HCH, and aldrin. The concentrations were in the following ranges: alpha-HCH = 3 to 68 ng L^?1 and 9 to 239 ng g^?1, lindane = 2 to 42 ng L^?1 and 10 to 542 ng g^?1, heptachlor = 5 to 32 ng L^?1 and 9 to 390 ng g^?1, delta-HCH = 1 to 10 ng L^?1 and n.d. to 87 ng g^?1, and aldrin = 1 to 7 ng L^?1 and n.d. to 33 ng g^?1 for filtered water and suspended matter samples, respectively. Heptachlor epoxide, dieldrin, o-p′ DDD, and p-p′ DDT were found to be present in solution at only one sampling site whereas p-p′ DDD and o-p′DDT were not detected. The analyses revealed a non-uniform distribution of these compounds over the studied area. The higher concentrations of organochlorines were detected downstream urban areas.     

Binding capacity of a barley beta-D-glucan to the beta-glucan recognition molecule dectin-1

To clarify whether barley beta-glucans exhibit their biological effects via binding to dectin-1, a pivotal receptor for beta-1,3-glucan, the structure of barley beta-glucan E70-S (BBG-70) was unambiguously investigated by NMR spectroscopy and studied for its binding capacity and specificity to dectin-1 by ELISA. NMR spectroscopy confirmed that BBG-70 contains two different linkage glucans, namely, alpha-glucan and beta-glucan, which are not covalently attached to one another. Beta-glucan within BBG-70 is a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose residues but does not contain the continuous 1,3-linkage. Competitive ELISA revealed that highly purified barley beta-glucan E70-S (pBBG-70) inhibits the binding of soluble dectin-1 to sonifilan (SPG), a beta-1,3-glucan, although at a concentration higher than that of SPG and laminarin. It was found that barley beta-glucan can be recognized by dectin-1, implying that barley beta-glucan might, at least in part, exhibit its biological effects via the recognition by dectin-1 of the ligand sugar structure, which may be formed by 1,3-beta- and 1,4-beta-glucosyl linkage.     

Thermal behavior of beta-1 subunits in lignin: pyrolysis of 1,2-diarylpropane-1,3-diol-type lignin model compounds

1,2-Diarylpropane-1,3-diol-type lignin model compounds, 1,2-bis(4-hydroxy-3-methoxyphenyl)propane-1,3-diol (1) and 1-(3,4-diethoxyphenyl)-2-(4-methoxyphenyl)propane-1,3-diol (2), were pyrolyzed at 500 degrees C for 4 s to clarify the thermal behavior of beta-1 subunits in lignin. Products were monitored by gas chromatography/mass spectrometry. The cleavage of the Calpha-Cbeta bond to produce benzaldehydes such as 4-hydroxy-3-methoxybenzaldehyde (9) and phenylethanals as the counterparts such as 4-hydroxy-3-methoxyphenylethanal (10) occurred in pyrolyses of both 1 and 2. In pyrolysis of 1, an oxetane pathway leading to the formation of Z/E-stilbenes without the gamma-CH2OH group such as Z/E-4,4'-dihydroxy-3,3'-dimethoxystilbene (3) was predominant. In pyrolysis of 2, the oxetane pathway was minor, while pathways producing a dimer with a =CgammaH2 group by loss of water and a dimer with an alpha-carbonyl group were predominant. Pyrolysis of Japanese cedar wood provided 3 and 10 in approximately 0.8% and 0.6% yields, respectively, based on the Klason lignin content, while pyrolysis of a guaiacyl bulk dehydrogenation polymer gave them in a very small amount.     

Biochemical characterization of a novel thermostable beta-1,3-1,4-glucanase (lichenase) from Paecilomyces thermophila

The purification and characterization of a novel extracellular beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila J18 were studied. The strain produced the maximum level of extracellular beta-glucanase (135.6 U mL(-1)) when grown in a medium containing corncob (5%, w/v) at 50 degrees C for 4 days. The crude enzyme solution was purified by 122.5-fold with an apparent homogeneity and a recovery yield of 8.9%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 38.6 kDa. The molecular masses were 34.6 kDa and 31692.9 Da when detected by gel filtration and mass spectrometry, respectively, suggesting that it is a monomeric protein. The enzyme was a glycoprotein with a carbohydrate content of 19.0% (w/w). Its N-terminal sequence of 10 amino acid residues was determined as H2N-A(?)GYVSNIVVN. The purified enzyme was optimally active at pH 7.0 and 70 degrees C. It was stable within pH range 4.0-10.0 and up to 65 degrees C, respectively. Substrate specificity studies revealed that the enzyme is a true beta-1,3-1,4-D-glucanase. The K m values determined for barley beta-D-glucan and lichenan were 2.46 and 1.82 mg mL(-1), respectively. The enzyme hydrolyzed barley beta-D-glucan and lichenan to yield bisaccharide, trisaccharide, and tetrasaccharide as the main products. Circular dichroism studies indicated that the protein contains 28% alpha-helix, 24% beta-sheet, and 48% random coil. Circular dichroism spectroscopy is also used to investigate the thermostability of the purified enzyme. This is the first report on the purification and characterization of a beta-1,3-1,4-glucanase from Paecilomyces sp. These properties make the enzyme highly suitable for industrial applications.     

Detection of a "nonaromatic" NIH shift during in vivo metabolism of the monoterpene carvone in humans

High-resolution gas chromatography in combination with mass spectrometry and high-resolution mass spectrometry was used to determine the positions and extent of labeling in the metabolites of carvone, namely in alpha,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), alpha-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone), after human ingestion of 9,9-dideutero- and 9-(13)C-carvone. Carvonic acid was formed by oxidation at the methyl carbon of the isopropenyl group of carvone, whereas dihydrocarvonic acid was formed by oxidation at the methylene position, most probably via carvone epoxide. A "nonaromatic" NIH shift must occur during the subsequent reactions yielding dihydrocarvonic acid. Additionally, dehydrogenation of dihydrocarvonic acid and hydrogenation of carvonic acid were observed, resulting in minor amounts of both acids owning a carboxy group of opposite origin. Uroterpenolone was found to be exclusively formed by oxidation at the methylene carbon of the isopropenyl group of carvone, and thus, most probably by hydrolysis of carvone epoxide.