Fungistatic activity of bicyclo[4.3.0]-γ-lactones

Five optically active and sixteen racemic lactones (nine of them new) of bicyclo[4.3.0]nonane structure were synthesized. IC(50) values for the following phytopathogens were determined: Aspergillus ochraceus AM 456, Fusarium culmorum AM 282, Fusarium oxysporum AM 13, Fusarium tricinctum AM 16. Effect of compound structures, especially stereogenic centers, on fungistatic activity has been discussed. The highest fungistatic activity was observed for trans-7,8-dibromo-cis-3-oxabicyclo[4.3.0]nonan-2-one (3c), IC(50) = 30.1 μg/mL (0.10 μM/mL), and cis-7,8-epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (3b), IC(50) = 72.2 μg/mL (0.47 μM/mL), toward F. oxysporum AM 13.     

Fungal biotransformation of (+/-)-linalool

The biotransformation of (+/-)-linalool was investigated by screening 19 fungi. Product accumulation was enhanced by substrate feeding and, for the first time, lilac aldehydes and lilac alcohols were identified as fungal biotransformation byproduct using SPME-GC-MS headspace analysis. Aspergillus niger DSM 821, Botrytis cinerea 5901/02, and B. cinerea 02/FBII/2.1 produced different isomers of lilac aldehyde and lilac alcohol from linalool via 8-hydroxylinalool as postulated intermediate. Linalool oxides and 8-hydroxylinalool were the major products of fungal (+/-)-linalool biotransformations. Furanoid trans-(2 R,5 R)- and cis-(2 S,5 R)-linalool oxide as well as pyranoid trans-(2 R,5 S)- and cis-(2 S, 5 S)-linalool oxide were identified as the main stereoisomers with (3 S,6 S)-6,7-epoxylinalool and (3 R,6 S)-6,7-epoxylinalool as postulated key intermediates of fungal (+/-)-linalool oxyfunctionalization, respectively. With a conversion yield close to 100% and a productivity of 120 mg/L.day linalool oxides, Corynespora cassiicola DSM 62485 was identified as a novel highly stereoselective linalool transforming biocatalyst showing the highest productivity reported so far.     

Synthesis and herbicidal activity of 2alpha, 4alpha-dimethyl-8-oxabicyclo[3.2.1]oct-6-en-3-one derivatives.

The catalytic oxidation of 2alpha,4alpha-dimethyl-8-oxabicyclo[3.2. 1]oct-6-en-3-one with osmium tetraoxide and excess hydrogen peroxide resulted in the formation of 2alpha,4alpha-dimethyl-6, 7-exo-isopropylidenedioxy-8-oxabicyclo[3.2.1]octan-3-one (2), with 91% yield. Addition of aryllithium reagents to this compound resulted in the formation of the aromatic alcohols (6a-h) with 48-76% yield. These alcohols were treated with thionyl chloride in pyridine, and the corresponding alkenes (7a-h) were obtained with 46-80% yield. The effect of compounds 6a-h and 7a-h on the root growth of Sorghum bicolor was evaluated at a concentration of 6.6 microg g(-)(1). The alcohols 6a-h caused an inhibitory effect (8-100%) on the S. bicolor radicle growth. The three most active compounds were 6e (aryl = p-methylphenyl), 6g (aryl = p-chlorophenyl), and 6h (aryl = p-fluorophenyl) and caused 100% inhibition. The effect of alkenes 7a-h was less pronounced and varied from 15% to 46% inhibition. Another experiment was carried out in a greenhouse to evaluate the effect of alcohols 6e, 6g, and 6h, at a 6.6 microg g(-)(1) dose, against Cucumis sativus, S. bicolor and the weeds Bidens pilosa, Desmodiumtortuosum, and Pennisetum setosum. All three compounds showed an inhibitory effect on the development of the aerial parts (26-73%) and roots (13-79%) of the weeds and crops.     

Metabolites of conjugated isomers of alpha-linolenic acid (CLnA) in the rat

Rumelenic (cis-9,trans-11,cis-15 18:3) acid is a naturally occurring conjugated isomer of alpha-linolenic acid (CLnA) in milk fat. Metabolism in rats was studied using a synthetic CLnA mixture, composed mainly by equimolar quantities of cis-9,trans-11,cis-15 and cis-9,trans-13,cis-15 CLnA isomers. Their metabolisms were studied by feeding high quantities of CLnA (150 mg/day) for 4 days to rats that had been reared on a fatfree diet for 2 weeks. After this period, animals were sacrificed and liver and epididymal adipose tissue lipids extracted. Six metabolites of the cis-9,trans-11,cis-15 18:3 CLnA isomers were identified as being cis-7,trans-9,cis-13 16:3, cis-11,trans-13,cis-17 20:3, cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids. Two metabolites of cis-9,trans-13,cis-15 18:3 CLnA isomer were also identified by GC-MS as being cis-7,trans-11,cis-13 16:3 and cis-5,cis-8,cis-11,trans-15,cis-17 20:5.     

Abscisic acid related compounds and lignans in prunes (Prunus domestica L.) and their oxygen radical absorbance capacity (ORAC)

Four new abscisic acid related compounds (1-4), together with (+)-abscisic acid (5), (+)-beta-D-glucopyranosyl abscisate (6), (6S,9R)-roseoside (7), and two lignan glucosides ((+)-pinoresinol mono-beta-D-glucopyranoside (8) and 3-(beta-D-glucopyranosyloxymethyl)-2- (4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran (9)) were isolated from the antioxidative ethanol extract of prunes (Prunus domestica L.). The structures of 1-4 were elucidated on the basis of NMR and MS spectrometric data to be rel-5-(3S,8S-dihydroxy-1R,5S-dimethyl-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (1), rel-5-(3S,8S-dihydroxy-1R,5S-dimethyl-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid 3'-O-beta-d-glucopyranoside (2), rel-5-(1R,5S-dimethyl-3R,4R,8S-trihydroxy-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (3), and rel-5-(1R,5S-dimethyl-3R,4R,8S-trihydroxy-7-oxabicyclo[3,2,1]- oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (4). The antioxidant activities of these isolated compounds were evaluated on the basis of oxygen radical absorbance capacity (ORAC). The ORAC values of abscisic acid related compounds (1-7) were very low. Two lignans (8 and 9) were more effective antioxidants whose ORAC values were 1.09 and 2.33 micromol of Trolox equiv/micromol, respectively.     

Characterization of the aroma-active compounds in pink guava (Psidium guajava, L.) by application of the aroma extract dilution analysis

The volatiles present in fresh, pink-fleshed Colombian guavas ( Psidium guajava, L.), variety regional rojo, were carefully isolated by solvent extraction followed by solvent-assisted flavor evaporation, and the aroma-active areas in the gas chromatogram were screened by application of the aroma extract dilution analysis. The results of the identification experiments in combination with the FD factors revealed 4-methoxy-2,5-dimethyl-3(2 H)-furanone, 4-hydroxy-2,5-dimethyl-3(2 H)-furanone, 3-sulfanylhexyl acetate, and 3-sulfanyl-1-hexanol followed by 3-hydroxy-4,5-dimethyl-2(5 H)-furanone, ( Z)-3-hexenal, trans-4,5-epoxy-( E)-2-decenal, cinnamyl alcohol, ethyl butanoate, hexanal, methional, and cinnamyl acetate as important aroma contributors. Enantioselective gas chromatography revealed an enantiomeric distribution close to the racemate in 3-sulfanylhexyl acetate as well as in 3-sulfanyl-1-hexanol. In addition, two fruity smelling diastereomeric methyl 2-hydroxy-3-methylpentanoates were identified as the ( R,S)- and the ( S,S)-isomers, whereas the ( S,R)- and ( R,R)-isomers were absent. Seven odorants were identified for the first time in guavas, among them 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, 3-hydroxy-4,5-dimethyl-2(5 H)-furanone, trans-4,5-epoxy-( E)-2-decenal, and methional were the most odor-active.     

Anti-inflammatory activities of new succinic and maleic derivatives from the fruiting body of Antrodia camphorata

Six new compounds, trans-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione (1), trans-1-hydroxy-3-(4-hydroxyphenyl)-4-isobutylpyrrolidine-2,5-dione (2), cis-3-(4-hydroxyphenyl)-4-isobutyldihydrofuran-2,5-dione (3), 3-(4-hydroxyphenyl)-4-isobutyl-1H-pyrrole-2,5-dione (4), 3-(4-hydroxyphenyl)-4-isobutylfuran-2,5-dione (5), and dimethyl 2-(4-hydroxyphenyl)-3-isobutylmaleate (6), together with one known compound, 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione (7), were isolated from the fruiting bodies of Antrodia camphorata. The structures of the compounds were elucidated by analysis of their spectroscopic data. To investigate the immunomodulatory potential of the compounds, RAW264.7 macrophage cells were treated with the compounds. Compound 1 significantly increased spontaneous TNF-alpha secretion from unstimulated RAW264.7 cells but suppressed IL-6 production [50% inhibition concentration value (IC50) = 10 microg/mL] in LPS-stimulated cells. Compounds 3, 4, and 6 also suppressed IL-6 production with IC50 values of 17, 18, and 25 microg/mL, respectively, suggesting that these four compounds may have an anti-inflammatory effect on macrophage-mediated responses. Of the six compounds, compound 1 was the most effective, exerting both immunostimulatory and anti-inflammatory effects.     

Studies on the key aroma compounds in soy milk made from three different soybean cultivars

An investigation by aroma extract dilution analysis (AEDA) of the aroma concentrate of soy milk made from a major Japanese soybean cultivar, Fukuyutaka (FK), revealed 20 key aroma compounds having flavor dilution (FD) factors of not less than 64. Among them, 2-isopropyl-3-methoxypyrazine, cis-4,5-epoxy-(E)-2-decenal, trans-4,5-epoxy-(E)-2-decenal, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, and 2'-aminoacetophenone were identified as the key aroma compounds in soy milk for the first time. (E,E)-2,4-Decadienal exhibiting a fatty note and trans-4,5-epoxy-(E)-2-decenal exhibiting a metallic/sweet note were detected as having the highest FD factors of 4096, followed by hexanal (green), (E)-2-nonenal (fatty), and (E,E)-2,4-nonadienal (fatty) having FD factors of 1024. Although all of these compounds might be generated from lipids, various aroma components, which were thought to be generated from amino acids, sugars, and ferulic acid, were detected having FD factors of 64-256. Investigation by comparative AEDA experiments of the soy milk aroma concentrates of two cultivars for soybean curd and soy milk, FK and Vinton81 (VT), and one cultivar for boiled beans, Miyagishirome (MY), revealed that most of the key aroma compounds were common to all of them, but 2-isopropyl-3-methoxypyrazine, exhibiting a pea-like/earthy note, was detected only in FK and VT. In addition, a sensory experiment revealed that the pea-like/earthy notes in FK and VT were significantly stronger than that in MY. These results demonstrated that a pea-like/earthy note contributed by 2-isopropyl-3-methoxypyrazine might be one of the essential characteristics to describe soy milk aromas.     

Fatty acid and carotenoid composition of gac (Momordica cochinchinensis Spreng) fruit

In this study, we analyzed fatty acid and carotenoid composition of fruit tissues, including seed (which are surrounded by a bright red, oily aril) of Momordica cochinchinensis Spreng, known as gac in Vietnam. Carotenoid content was analyzed by reversed-phase HPLC, using a C(30) column and a method separating cis- and trans-isomers of the major carotenoids in this fruit. Mean values obtained in aril tissues were 1342 microg trans-, 204 microg cis-, and 2227 microg total lycopene; 597 microg trans-, 39 microg cis-, and 718 microg total beta-carotene; and 107 microg alpha-carotene/g FW. Mesocarp contained 11 microg trans-, 5 microg cis-beta-carotene/g FW, trace amounts of alpha-carotene, and no lycopene. Gac aril contained 22% fatty acids by weight, composed of 32% oleic, 29% palmitic, and 28% linoleic acids. Seeds contained primarily stearic acid (60.5%), smaller amounts of linoleic (20%), oleic (9%), and palmitic (5-6%) acids, and trace amounts of arachidic, cis-vaccenic, linolenic, and palmitoleic, eicosa-11-enoic acids, and eicosa-13-enoic (in one fruit only) acids.     

Hapten and antibody production for a sensitive immunoassay determining a human urinary metabolite of the pyrethroid insecticide permethrin

Permethrin is the most popular synthetic pyrethroid insecticide in agriculture and public health. For the development of the enzyme-linked immunosorbent assay (ELISA) to evaluate human exposure to permethrin, the glycine conjugate (DCCA-glycine) of a major metabolite, cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was established as the target analyte. Four different types of the cis- and trans-isomers of immunizing haptens were synthesized as follows: N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine (hapten 3), N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)-4-amino-l-phenylalanine (hapten 5), N-(N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine)amino-6-(2,4-dinitrophenyl)aminohexanoic acid (hapten 9), and N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine-4-oxobutanoic acid (hapten 24). Sixteen polyclonal antibodies produced against each cis- or trans-hapten-thyroglobulin conjugate as immunogens were screened against numerous hapten-bovine serum albumin conjugates as coating antigens. Six ELISAs with both a heterologous hapten structure and a heterologous hapten configuration (cis/trans or trans/cis) between antibody and coating antigen showed a high sensitivity for the target analyte. The IC50 was 1.3, 2.1, and 2.2 microg/L for the trans-target analyte and 0.4, 2.3, and 2.8 microg/L for the cis-target analyte. The immunizing haptens, except for hapten 5, provided the target specific antibodies. Molecular modeling of the haptens supported the selection of reasonable immunizing haptens that best mimicked the target analyte. Hapten 5 was suitable as a coating antigen rather than as an immunogen since it had a different geometry. Very low cross-reactivities were measured to permethrin, its free metabolite (DCCA), PBA-glycine conjugate, and glycine. The ELISA will be optimized for the detection of total cis/trans-DCCA-glycine in human urine samples.     

Identification of natural oak lactone precursors in extracts of american and French oak woods by liquid chromatography-tandem mass spectrometry

A method for the screening of potential natural oak lactone precursors in oak wood extracts using LC-MS/MS combined with information-dependent acquisition was developed. The method was applied to extracts of American and French oak woods. As a result, cis-3-methyl-4-galloyloxyoctanoic acid (ring-opened cis-oak lactone gallate), (3S,4S)- and (3S,4R)-3-methyl-4-O-beta-D-glucopyranosyloctanoic acid (ring-opened cis- and trans-oak lactone glucoside), and (3S,4S)-3-methyl-4-O-(6'-O-galloyl)-beta-D-glucopyranosyloctanoic acid (ring-opened cis-oak lactone galloylglucoside) were identified as natural oak lactone precursors in the extracts by comparison with the respective synthetic reference compounds. In addition, the ring-opened oak lactone rutinoside was tentatively identified in the extracts. Three apparent isomers of the ring-opened cis-oak lactone galloylglucoside were also observed.     

Conjugated linoleic acid, cis-9,trans-11, is a substrate for pulmonary 15-lipoxygenase-1 in rat

The objective of this study was to determine whether two of the major conjugated linoleic acid (CLA) isomers, cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12), are possible substrates for pulmonary 15-lipoxygenase-1 (15-LOX-1) and, therefore, they are also involved in the production of 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE] in biological systems. 13(S)-HODE, a major bioactive metabolite of linoleic acid, is an important intracellular signal agent and is involved in cell proliferation and differentiation in various biological systems. Nordihydroguaiaretic acid (NDGA), a known LOX inhibitor, was used as a control for measuring 15-LOX-1 enzyme activity. It was found that c9,t11-CLA was 25% as active as linoleic acid as a substrate for 15-LOX-1; however, t10,c12-CLA was not a substrate for 15-LOX-1 as measured by 13(S)-HODE production. The authenticity of the production of 13(S)-HODE from c9,t11 as a substrate was established by isolation and cochromatography with pure standard on HPLC using non-radioactive and [14C]-c9,t11-CLA.     

Kinetics of photoirradiation-induced synthesis of soy oil-conjugated linoleic acid isomers

Photoirradiation of soy oil with UV/visible light has been shown to produce significant amounts of trans,trans conjugated linoleic acid (CLA) isomers through conversion of various synthesized intermediate cis,trans isomers. The objective of this study was to determine the kinetics of CLA isomers synthesis to better understand the production of various isomers. Soy oil was irradiated with UV/visible light for 144 h in the presence of an iodine catalyst and CLA isomers analyzed by gas chromatography (GC). Arrhenius plots were developed for the conversion of soy oil linoleic acid (A) to form cis-, trans/trans-, cis-CLA (B), conversion of cis-, trans/trans-, cis-CLA to form trans,trans-CLA (C) with respect to B, and formation of trans,trans-CLA isomers with respect to C. The kinetics of consumption of linoleic acid (LA) to form cis-, trans/trans-, cis-CLA was found to be of second-order with a rate constant of 9.01 x 10-7 L/mol s. The rate of formation of cis-, trans/trans-, cis-CLA isomers depends on the rate of formation from LA and its rate of consumption to form trans,trans-CLA isomers. The conversion of cis-, trans/trans-, cis-CLA isomers to trans,trans-CLA isomers was found to be of first-order with a rate constant of 2.75 x 10-6 s-1. However, the formation of thermodynamically stable trans,trans-CLA isomers (C) with respect to C was found to be a zero-order reaction with a rate constant of 10.66 x 10-7 mol/L s. The consumption of LA was found to be the rate-determining step in the CLA isomers formation reaction mechanism. The findings provide a better understanding of the mechanism of CLA isomers synthesis by photoirradiation and the factors controlling the ratio of various isomers.     

Aroma compound analysis of Eruca sativa (Brassicaceae) SPME headspace leaf samples using GC,GC-MS,and olfactometry

The aroma compounds of rocket salad (Eruca sativa) SPME headspace samples of fresh leaves were analyzed using GC, GC-MS, and olfactometry. More than 50 constituents of the Eruca headspace could be identified to be essential volatiles, responsible for the characteristic intense green; herbal; nutty and almond-like; Brassicaceae-like (direction of cabbage, broccoli, and mustard); and horseradish-like aroma of these salad leaves. As aroma impact compounds, especially isothiocyanates, and derivatives of butane, hexane, octane, and nonane were identified. 4-Methylthiobutyl isothiocyanate (14.2%), cis-3-hexen-1-ol (11.0%), cis-3-hexenyl butanoate (10.8%), 5-methylthiopentyl isothiocyanate (9.3%), cis-3-hexenyl 2-methylbutanoate (5.4%), and 5-methylthiopentanenitrile (5.0%) were found in concentrations higher than 5.0% (calculated as % peak area of GC analysis using a nonpolar column).     

Purification of ethoxyquin and its two oxidation products

2,6-Dihydro-2,2,4-trimethyl-6-quinolone (QI) and 1,8'-bis(1, 2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) (DM) are two oxidation products of 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline (ethoxyquin, EQ). This paper describes several methods for the purification of technical grade EQ and for the production of pure QI and DM as standards with the purity required (>99%) for calibration of quantitative determination methods. EQ of high purity was obtained through vacuum distillation followed by a quick column chromatographic purification on silica gel. Preparative scale purity DM could be obtained through recrystallization from methanol, but QI could be purified only by a high-pressure liquid chromatographic method.     

Synthesis and antifungal activities of novel 5-amino-6-arylamino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one derivatives

A novel approach was developed to regioselectively synthesize new 5-amino-6-arylamino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 5 derivatives via a tandem aza-Wittig and annulation reactions of iminophosphorane 2, aromatic isocyanates, and hydrazine in 52-92% isolated yields. The compounds 5 reacted with triethyl orthoformate to give 1,8-2H-pyrazolo[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-4-ones 6 in good yields (62-94%). Their structures were clearly confirmed by spectroscopy data (IR, (1)H NMR, MS), elemental analysis, or X-ray diffraction crystallography. The results of preliminary bioassay indicated that the compounds 5 and 6 possessed high antifungal activity against Botrytis cinerea Pers and Sclerotinia sclerotiorum. Compounds 5 showed much better antifungal activities when R was Me instead of PhCH 2. Especially, compounds 6c, 6g, and 6i inhibited Sclerotinia by 100% at the concentration of 50 mg/L and by 83, 83, and 82% at the dosage of 10 mg/L, respectively.     

Botrytone, a new naphthalenone pentaketide produced by Botrytis fabae, the causal agent of chocolate spot disease on Vicia faba

A strain of Botrytis fabae isolated from faba bean (Vicia faba L.) plants displaying clear chocolate spot disease symptoms produced phytotoxic metabolites in vitro. The phytotoxins isolated from the culture filtrate organic extract were characterized by spectroscopic and optical methods. A new naphthalenone pentaketide, named botrytone, was isolated and characterized as (4R)-3,4-dihydro-4,5,8-trihydroxy-1(2H)-naphthalenone together with other well-known closely related naphthalenones such as regiolone and cis- and trans-3,4-dihydro-2,4,8-trihydroxynaphthalen-1(2H)-ones. When tested on leaves of the host plant, with the cis- and trans-3,4-dihydro-2,4,8-trihydroxynaphthalen-1(2H)-ones assayed in mixture, regiolone demonstrated the highest level of phytotoxicity together with cis- and trans-3,4-dihydro-2,4,8-trihydroxynaphthalen-1(2H)-ones. Botrytone showed moderate phytotoxic activity at 1 mg/mL and was still phytotoxic at 0.5 mg/mL.     

Determination of the conjugated linoleic acids in cow's milk fat by Fourier transform Raman spectroscopy

The collective term "conjugated linoleic acid" or "CLA" generally refers to a mixture of conjugated positional and geometric isomers of linoleic (cis-9,cis-12-octodecadienoic) acid. In nature, these isomers are mainly formed in the rumen by biohydrogenation of polyunsaturated fatty acids. This study concerns a first trial of CLA determination in cow's milk fat by Raman spectroscopy. The spectra of pure cis-9-oleic, cis-9,cis-12-linoleic, cis-9,trans-11-linoleic, and trans-10,cis-12-linoleic acids have been examined in comparison with the spectra of selected milk-fat samples containing between 0 and 3% of CLA. The trial of CLA determination by Raman spectroscopy on cow milk fat has reached its objective with the two following results. First, the examination of the Raman spectra allows to identify three specific Raman signals of the chemical bonds associated to the cis,trans conjugated C=C in the rumenic and trans-10,cis-12-octodecadienoic acids at 1652, 1438, and 3006 cm(-1). Second, the calibration of Raman spectrometer for the CLA determination has indicated that these three specific signals suit very well for the accurate and reliable measurement of CLA concentration in milk fat. To our knowledge, the present study is the first successful attempt to determine the CLA content of milk fat by a spectrophotometric method.