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.     

Liquid chromatography-mass spectrometry (LC-MS) investigation of the thiobarbituric acid reactive substances (TBARS) reaction

The thiobarbituric acid reactive substances (TBARS) assay is a commonly used method for the detection of lipid peroxidation. Malondialdehyde is formed as a result of lipid peroxidation and reacts with thiobarbituric acid to form a pink pigment that has an absorption maximum at 532 nm. Other compounds also react with thiobarbituric acid to form colored species that can interfere with this assay, but little is known about these interfering species. This is the first investigation using LC-MS and MS-MS to study the structures of the pink adduct as well as a common unstable yellow interference compound, which absorbs at 455 nm. Also, the presence of barbituric acid impurities in the thiobarbituric acid reagent was found to produce 1:1:1 thiobarbituric acid/malondialdehyde/barbituric acid and 2:1 barbituric acid/malondialdehyde adducts that absorbed at 513 and 490 nm, respectively, indicating that thiobarbituric acid should be purified before use.     

Characterization by LC-MS(n) of four new classes of p-coumaric acid-containing diacyl chlorogenic acids in green coffee beans

LC-MS4 has been used to detect and characterize in green coffee beans 15 quantitatively minor p-coumaric acid-containing chlorogenic acids not previously reported in nature. These comprise 3,4-di-p-coumaroylquinic acid, 3,5-di-p-coumaroylquinic acid, and 4,5-di-p-coumaroylquinic acid (Mr 484); 3-p-coumaroyl-4-caffeoylquinic acid, 3-p-coumaroyl-5-caffeoylquinic acid, 4-p-coumaroyl-5-caffeoylquinic acid, 3-caffeoyl-4-p-coumaroyl-quinic acid, 3-caffeoyl-5-p-coumaroyl-quinic acid; and 4-caffeoyl-5-p-coumaroyl-quinic acid (Mr 500); 3-p-coumaroyl-4-feruloylquinic acid, 3-p-coumaroyl-5-feruloylquinic acid and 4-p-coumaroyl-5-feruloylquinic acid (Mr 514); and 4-dimethoxycinnamoyl-5-p-coumaroylquinic acid and two isomers (Mr 528) for which identities could not be assigned unequivocally. Structures have been assigned on the basis of LC-MS4 patterns of fragmentation. Forty-five chlorogenic acids have now been characterized in green Robusta coffee beans.     

Methylation methods for the quantitative analysis of conjugated linoleic acid (CLA) isomers in various lipid samples

Precise methylation methods for various chemical forms of conjugated linoleic acid (CLA), which minimize the formation of t,t isomers and allylmethoxy derivatives (AMD) with the completion of methylation, were developed using a 50 mg lipid sample, 3 mL of 1.0 N H(2)SO(4)/methanol, and/or 3 mL of 20% tetramethylguanidine (TMG)/methanol solution(s). Free CLA (FCLA) was methylated with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). CLA esterified in safflower oil (CLA-SO) was methylated with 20% TMG/methanol (100 degrees C, 5 min), whereas CLA esterified in phospholipid (CLA-PL) was methylated with 20% TMG/methanol (100 degrees C, 10 min), followed by an additional reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). Similarly, CLA esterified in egg yolk lipid (CLA-EYL) was methylated by base hydrolysis, followed by reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). These results suggest that for the quantitative analysis of CLA in lipid samples by GC, proper methylation methods should be chosen on the basis of the chemical forms of CLA in samples.     

Discriminating significance of the free amino acid profile in almond seeds

Known statistical techniques have been applied to the free amino acid composition of 107 samples from 10 different almond cultivars (Marcona, Desmayo-Largueta, Guara, Tuono, Ferragnes, Masbovera, Non Pareil, Titan, Texas, and Primorskyi) cultivated in seven different locations and growing conditions. It is concluded that free amino acid composition can constitute a basis for classifying and typifying these cultivars into five groups: (1) Marcona and Texas, (2) Ferragnes and Masbovera (and probably Primorskyi), (3) Tuono and Guara, (4) Non Pareil (and probably Titan), and (5) an isolated cultivar (Desmayo Largueta). As a result, an easy decision tree is proposed to discriminate the cultivar of an almond flour, as used in confectionery, if it consists of a single cultivar.     

Cytogenetic relationships between Avena insularis (2n=28) and both A. strigosa (2n=14) and A. murphyi (2n=28)

The newly discovered tetraploid oat Avena insularis was crossed with the diploid A. strigosa and the tetraploid A. murphyi. Considerably reduced chromosome association at meiosis and a low average number of chiasmata per cell of the A. strigosa × A. insularis hybrids indicated that the diploid A. strigosa did not participate in the creation of A. insularis. From A. murphyi, A. insularis differed by four chromosomal rearrangements and the hybrids between them were sterile. The tetraploids A. magna, A. murphyi and A. insularis share the two to four floret large diaspore, which is adapted to heavy alluvial soil. They all, however, diverge from one another by four chromosomal rearrangements. At this point it is not possible to determine whether they have diverged from a single tetraploid progenitor, or developed from different diploid species.     

Discriminating between village and commercial hunting of apes

Hunting is the major driver of large mammal decline in Central African forests. In slowly reproducing species even low hunting pressure leaves spatial gradients with wildlife density increasing with distance from transport routes and human settlements. Park management can use this pattern formation to identify sources of threats, but also to discriminate between different threat scenarios, such as the impact of subsistence vs. commercial hunting. We conducted an ape survey in the mountainous Moukalaba Doudou National Park, Gabon, to evaluate whether potential population gradients would emanate from the three human population centers in the region or the villages surrounding the park. Using generalized linear modeling we found hill slope as a good predictor of ape nest occurrence probability and the distance to human population centers a better predictor of ape nest density and ape nest group size than distance to villages. In fact ape nest density was three times lower at the park borders close to the human population centers than in the park’s interior. The results indicate that Moukalaba’s ape population is more impacted by commercial than subsistence hunting and suggest that park management should focus conservation efforts on the human population centers. We conclude that in particular for slowly reproducing species geographic information on wildlife population gradients are of additional value for guiding protected area management. The hunting impact on those species might be easily underestimated, if derived only from market surveys or transport route controls, where they are only rarely found.     

Oxidative stability of conjugated linoleic acid isomers

Conjugated linoleic acids (CLAs) have been shown to be a strong anticarcinogen in a number of animal models. Our previous study demonstrated that CLA as a whole was extremely unstable in air. The present study was undertaken further to examine the oxidative stability of individual CLA isomers using the combination of gas-liquid chromatography (GLC) and silver ion high-performance liquid chromatography (Ag-HPLC). It was found that CLA as a whole oxidized rapidly and more than 80% was degraded within 110 h in air at 50 degrees C. Four c,c-CLA isomers were most unstable followed by four c,t-CLA isomers. In contrast, four t,t-CLA isomers were relatively stable under the same experimental conditions. Both the oxygen consumption and the GLC analysis revealed that 200 ppm jasmine green tea catechins (GTCs) exhibited protection to CLA and were even stronger than 200 ppm butylated hydroxytoluene (BHT) when added to either CLA or canola oil containing 10% CLA. The present study emphasized that oxidative unstability of CLA should not be overlooked although CLA has many biological effects.     

Characterization of the geographical origin of Pecorino Sardo cheese by casein stable isotope ((13)c/(12)c and (15)n/(14)n) ratios and free amino acid ratios

The stable isotope ratios ((13)C/(12)C and (15)N/(14)N) of casein measured by isotope ratio mass spectrometry (IRMS) and some free amino acid ratios (His/Pro, Ile/Pro, Met/Pro, and Thr/Pro) determined by HPLC in samples of ewes' milk cheese from Sardinia, Sicily, and Apulia were found to be parameters independent of ripening time. Multivariate data treatments performed by applying both unsupervised (principal component analysis and cluster analysis) and supervised [linear discriminant analysis (LDA)] methods revealed good discrimination possibilities for the cheeses according to place of origin. In this respect, particularly significant were the variables Ile/Pro, Thr/Pro, (13)C/(12)C, and (15) N/(14)N ratios on which basis 100% discrimination and classification of the samples by LDA was obtained.     

Simultaneous determination of orysastrobin and its isomers in rice using HPLC-UV and LC-MS/MS

Orysastrobin is a new strobilurin-type fungicide to control leaf and panicle blast and sheath blight in rice. An analytical method was developed to determine the residues of orysastrobin and its two isomers, the main metabolite F001 and the major impurity F033, in hulled rice by the use of high-performance liquid chromatography with ultraviolet photometry (HPLC-UV) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). All compounds were extracted with acetone from hulled rice samples. The extract was diluted with saline water, and an extraction step using dichloromethane/n-hexane partition was used to recover analytes from the aqueous phase. An n-hexane/acetonitrile partition and Florisil column chromatography were employed to further remove interfering coextractives prior to instrumental analysis. An octadecylsilyl column was successfully applied to identify orysastrobin and its isomers in sample extracts. Net recovery rates of orysastrobin, F001, and F033 from fortified samples ranged from 80.6 to 114.8% using HPLC-UV and LC-MS/MS. Relative standard deviations for the analytical methods were all <20%, and the quantification limits of the method were in the 0.002-0.02 mg/kg range. The proposed methods were reproducible and sufficiently accurate to evaluate the terminal residue of orysastrobin and its isomers in rice.     

Effects of conjugated linoleic acid (CLA) isomers on oxygen diffusion-concentration products in liposomes and phospholipid solutions

Conjugated linoleic acids (CLAs) are a group of octadecadienoic acids (18:2) that are naturally present in food products and may have beneficial health effects. Liposomes and ethanol solutions were prepared by mixing synthetic phosphatidylcholines (PCs) with c9,t11-CLA, t10,c12-CLA, and linoleic acid (LA) in the sn-2 position into natural PCs from soybean, egg yolk, rat brain, and rat heart at 5 mol %. The oxygen diffusion-concentration products were measured using electron spin resonance spin-label oximetry methods. Individual synthetic PCs, the phospholipid matrix, and the tested lipid systems all exhibited influence on oxygen diffusion-concentration products during lipid peroxidation. Incorporating 5 mol % PC(c9,t11-CLA) into soy and egg yolk PC increased oxygen consumption in liposome suspensions while it was decreased in rat heart and brain PCs. On the other hand, PC(t10,c12-CLA) increased oxygen consumption in mixtures with egg yolk and rat heart PC but decreased it in soybean and rat brain PC. By comparison, PC(LA) decreased oxygen consumption in every case. In ethanol solutions, all of the synthetic PCs suppressed the capacity to generate peroxide radicals in the order of LA > c9,t11-CLA > t10,c12-CLA. In addition, PCs containing individual CLA isomers and LA differed in their capacities to react with and quench DPPH radicals in both ethanol solution and liposome, suggesting differences between CLA isomers and LA in DPPH radical-fatty acid interactions. Incorporation of CLA isomers and LA into dimyristyl-PC reduced the phase transition temperature from 23.6 to 23.1 and 23.3 degrees C, respectively. The results of this study provide evidence that the behavior of CLA isomers differs in the microenvironment of membranes possibly due to structural differences that affect the permeability of membranes to oxygen and lipid peroxidation.     

Quantitation of the hydroxycinnamic acid derivatives and the glycosides of flavonols and flavones by UV absorbance after identification by LC-MS

A general approach was developed to quantify hydroxycinnamic acid derivatives and the glycosides of flavonols and flavones using UV molar relative response factors (MRRFs). More than 90 standards were analyzed by LC-MS and divided into five groups based on the λ(max) of their band I absorbance profiles. For each group, a commercially available standard was chosen as the group reference standard. Response factors were determined for each standard in each group as purchased (MRRF) and, when possible, after vacuum drying (MRRF(D)). The MRRF(D) values for 17 compounds whose λ(max) values fell within ±2 nm of the group reference standard were 1.01 ± 0.03. MRRF values for compounds whose λ(max) values fell within ±10 nm of the group reference standard were 0.96 ± 0.13. Group reference standards were used to quantify 44 compounds in Chinese lettuce, red onion, and white tea. This approach allows quantitation of numerous compounds for which there are no standards.     

Preparation of a t,t conjugated linoleic acid methylester (CLA-Me) isomers mixture from synthetic CLA by methylation with BF3/methanol

Mixtures of t,t conjugated linoleic acid methylester (t,t CLA-Me) isomers were prepared from synthetic CLA, consisting of 47.8% t10,c12 CLA; 45.5% c9,t11 CLA; 2.0% t,t CLA; and 4.7% others, by methylation with BF(3)/methanol (designated TT-TC/CT) in conjunction with purification at -68 degrees C for 24 h. The amount or composition of the TT-TC/CT was greatly affected by the concentration of BF(3) in methanol and the duration of methylation. The methylation of 50 mg of synthetic CLA for 30 min with 1 mL of 7.0% BF(3)/methanol produced a TT-TC/CT (21.54 mg) with the composition of 1.3% t12,t14; 5.9% t11,t13; 42.7% t10,t12; 44.0% t9,t11; 5.0% t8,t10; and 1.1% t 7,t9 CLA, whereas the methylation for 60 min with 14.0% BF(3)/methanol produced a TT-TC/CT (28.62 mg) with the composition of t,t CLA isomers different from that of TT-TC/CT by methylation for 30 min with 7.0% BF(3)/methanol. A large quantity of TT-TC/CT (14.15 g) with the composition similar to that of TT-TC/CT prepared from 50 mg of synthetic CLA was also prepared from 25 g of synthetic CLA. The purity of TT-TC/CT samples was greater than 98%. These results suggest that TT-TC/CT with a purity greater than 98% was easily prepared from synthetic CLA by BF(3)-catalyzed methylation, and the amount and composition of t,t CLA isomers of TT-TC/CT samples could be controlled by methylation conditions.     

Identification, quantitative determination, and antioxidative activities of chlorogenic acid isomers in prune (Prunus domestica L. )

Neochlorogenic acid (3-CQA) and cryptochlorogenic acid (4-CQA), isolated from prune (Prunus domestica L.), were identified by NMR and MS analyses. In addition, the quantity of chlorogenic acid isomers in prune were measured by HPLC. These isomers, 3-CQA, 4-CQA, and chlorogenic acid (5-CQA), were contained in the ratio 78.7:18. 4:3.9, respectively. 4-CQA was identified and quantified in prune for the first time, and relatively high amounts of this isomer were characteristic. Antioxidative activities of the chlorogenic acid isomers, such as scavenging activity on superoxide anion radicals and inhibitory effect against oxidation of methyl linoleate, were also evaluated. Each isomer showed antioxidative activities which were almost the same.     

Identification and characterization of conjugated linolenic acid isomers by Ag+-HPLC and NMR

Interest in the biological activity of conjugated linolenic acids (CLnA) is growing. Technically, previous methods could not fully characterize and identify CLnA isomers. The present study is the first report on application of silver ion impregnated high-performance liquid chromatography (Ag+-HPLC) into separation of CLnA methyl ester (CLnAMe) mixture. Compared with the GC and reversed-phase HPLC techniques, Ag+-HPLC could resolve and separate CLnAMe isomers into three clusters of peaks namely ttt, ctt and ctc geometrical groups. Each positional isomer belonging to the same geometrical group could be further separated by Ag+-HPLC. Quantitative 13C NMR properties of CLnAMe mixture were subsequently studied. Olefinic signals in the CLnAMe mixture were assigned and used to quantify each isomer in combination with Ag+-HPLC. The present study provided a detailed analysis of a complex CLnA mixture and may have an important application in future studies dealing with synthesis, oxidative stability, and bioactivity of individual CLnA isomers.     

Characterization by LC-MS(n) of four new classes of chlorogenic acids in green coffee beans: dimethoxycinnamoylquinic acids, diferuloylquinic acids, caffeoyl-dimethoxycinnamoylquinic acids, and feruloyl-dimethoxycinnamoylquinic acids

LC-MS4 has been used to detect and characterize in green coffee beans 12 chlorogenic acids not previously reported in nature. These comprise three isomeric dimethoxycinnamoylquinic acids (7-9) (Mr 382), three caffeoyl-dimethoxycinnamoylquinic acids (22, 24, and 26) (Mr 544), three diferuloylquinic acids (13-15) (Mr 544), and three feruloyl-dimethoxycinnamoylquinic acids (28, 30, and 32) (Mr 558). Structures have been assigned on the basis of LC-MS4 patterns of fragmentation and relative hydrophobicity and, in the case of the dimethoxycinnamoylquinic acids, by comparison with authentic standards. Several new structure-diagnostic fragmentations have been identified for use with diacyl-chlorogenic acids, for example, m/z 299 and 255 for C4 caffeoyl, m/z 313 and 269 for C4 feruloyl, nearly equal elimination of both cinnamoyl residues for vic-3,4-diacyl, and an increasing ratio of "dehydrated" ions to "non-dehydrated" ions at MS2 with increasing methylation of those cinnamoyl residues. Possible mechanisms have been proposed to account for the fragmentations observed. The mass spectrometric resolution of six isomeric chlorogenic acids (Mr 544) in a crude plant extract by fragment-targeted LC-MS2 and LC-MS3 experiments illustrates the analytical power and advantage of ion trap mass spectroscopy.     

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.     

Determination of advanced glycation endproducts by LC-MS/MS in raw and roasted almonds (Prunus dulcis)

A sensitive and reliable LC-(ESI)MS/MS method was developed and validated for the simultaneous analysis of five common advanced glycation endproducts (AGEs) after enzymatic digestion in raw and roasted almonds. AGEs included carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), pyralline (Pyr), argpyrimidine (Arg-p), and pentosidine (Pento-s). This method allows accurate quantitation of free and AGE-protein adducts of target AGEs. Results indicate that CML and CEL are found in both raw and roasted almonds. Pyr was identified for the first time in roasted almonds and accounted for 64.4% of free plus bound measured AGEs. Arg-p and Pento-s were below the limit of detection in all almond samples tested. Free AGEs accounted for 1.3-26.8% of free plus bound measured AGEs, indicating that protein-bound forms predominate. The roasting process significantly increased CML, CEL, and Pyr formation, but no significant correlation was observed between these AGEs and roasting temperature.     

Determination of angiotensin-converting enzyme inhibitory peptide Leu-Lys-Pro-Asn-Met (LKPNM) in bonito muscle hydrolysates by LC-MS/MS

Proteolytic digestion of dried bonito muscle with thermolysin produces a hydrolysate with strong angiotensin-converting enzyme (ACE) inhibitory activity and is the basis of a dietary supplement with antihypertensive activity. A major portion of the ACE activity was shown previously to arise from the peptide Leu-Lys-Pro-Asn-Met (LKPNM). A straightforward method to quantify this peptide was developed using one-step C18 solid-phase extraction (SPE) followed by LC-MS/MS quantification. The SPE step resulted in a hydrolysate that was still crude, as illustrated by combined size-exclusion chromatography/multi-angle laser light scattering detection that showed that a major fraction of oligopeptides were in the 2-20 kDa range. This fraction has a weight-average molecular weight (M(w)) of approximately 5.0 kDa. Method validation for specificity, linearity, accuracy, precision, and reproducibility showed that standard additions of synthetic LKPNM to bonito extract with SPE enrichment followed by LC-MS/MS is a suitably robust procedure for the determination of LKPNM content. The method was also successful for encapsulated powders in which the excipients used are insoluble in water and could be removed by centrifugation.     

Discriminating between organic matter in soil from grass and forest by near‐infrared spectroscopy

The absorbance of near‐infrared (NIR) radiation by plants depends on the overtones of vibration, bending and stretching of the chemical bonds within their organic components. The wide variety of these bonds gives rise to characteristic NIR spectra, or ‘fingerprints’, of plant materials. Do these fingerprints remain in the soil when the plants decompose and allow discrimination between soils that have supported different types of vegetation? We have compared the NIR spectra of grassland soil with those of soil under forest in the Vosges mountains in France. Near‐infrared reflectances, R, from 370 samples of soil were recorded as A = log₁₀(1/R) at wavelengths between 1100 and 2500 nm and averaged over 10‐nm intervals to give spectra each with 140 values. A canonical variate analysis (CVA) of the raw spectra discriminated well, but not perfectly, between grassland and forest soil, with Mahalanobis distance, D, of 4.87. Standardization along the spectra to remove effects of varied mineral composition achieved complete separation between the two sources with D = 9.81. Canonical variate analysis of first and second derivatives of the spectra distinguished the two groups even better, with D = 12.27 and D = 16.65, respectively. The results show much promise for inferring past vegetative cover from NIR spectra deriving from the organic matter in the soil. The next step will be to extend these studies to see how well we can distinguish other types of vegetation from such spectra.