Separation of Lipoproteins for Quantitative Analysis of 14C-Labeled Lipid-Soluble Compounds by Accelerator Mass Spectrometry.

To date, 14C tracer studies using accelerator mass spectrometry (AMS) have not yet resolved lipid-soluble analytes into individual lipoprotein density subclasses. The objective of this work was to develop a reliable method for lipoprotein separation and quantitative recovery for biokinetic modeling purposes. The novel method developed provides the means for use of small volumes (10-200 µL) of frozen plasma as a starting material for continuous isopycnic lipoprotein separation within a carbon- and pH-stable analyte matrix, which, following post-separation fraction clean up, created samples suitable for highly accurate 14C/12C isotope ratio determinations by AMS. Manual aspiration achieved 99.2 ± 0.41% recovery of [5-14CH3]-(2R, 4'R, 8'R)-α-tocopherol contained within 25 µL plasma recovered in triacylglycerol rich lipoproteins (TRL = Chylomicrons + VLDL), LDL, HDL, and infranatant (INF) from each of 10 different sampling times for one male and one female subject, n = 20 total samples. Small sample volumes of previously frozen plasma and high analyte recoveries make this an attractive method for AMS studies using newer, smaller footprint AMS equipment to develop genuine tracer analyses of lipophilic nutrients or compounds in all human age ranges.

Effect of Canning and Freezing on the Nutritional Content of Apricots.

The effect of commercial canning and freezing on the nutritional content of fresh apricots was investigated. Processed samples were analyzed post-processing and after 3 months of storage and compared directly to fresh apricots from the same source. Vitamin C, beta-carotene, total phenols, and antioxidants were quantified. Compared to fresh, canned apricots initially exhibited similar levels of antioxidants, a 17% increase in beta-carotene, and a 48% increase in phenols, while vitamin C was reduced by 37%. After 3 months of storage, antioxidant levels were 47% higher than fresh. Vitamin C did not change significantly following storage and beta-carotene decreased by 15%. The canned apricot fruit packed in light syrup did not have higher total soluble solids (TSS) levels indicating no increase in fruit sugar content. Frozen apricots exhibited large increases in antioxidants (529%), beta-carotene (35%), vitamin C (3,370%), and phenols (406%) compared to fresh. After 3 months of storage, frozen apricots decreased in vitamin C (29%) and phenols (17%), but remained 2,375% and 318% higher than fresh, respectively. Beta-carotene increased during storage, reaching levels 56% higher than fresh while antioxidant activity was unchanged. This study demonstrates that key nutrients in canned and frozen apricots are retained or amplified upon processing, with the exception of vitamin C in canned apricots. The routine addition of citric and ascorbic acid to fruit prior to freezing resulted in significantly higher antioxidants, vitamin C, and phenols. Consumers eating canned or frozen apricots can feel confident of similar or superior nutritional content as compared to fresh apricots. PRACTICAL APPLICATION: The apricot industry is limited by the short shelf life of the fruit and consumer belief that processed produce is not as nutritious as fresh. Assessing the nutritional content of canned and frozen apricots and determining that processed apricots can deliver nearly comparable nutrient levels to fresh apricots provides the evidence needed to dispel these misconceptions and potentially increase demand for processed apricots among consumers.

Phenolic metabolites and substantial microbiome changes in pig feces by ingesting grape seed proanthocyanidins.

Proanthocyanidin (PAC) consumption has been linked to better colonic health, but PACs are poorly absorbed, making them a target for colonic metabolism. The resulting metabolites are low molecular weight and could potentially be absorbed. To understand the effects of dietary PACs it would be important to resolve the metabolic issue and link these changes to microbial population changes in a suitable model for human digestion. Here, six crossbred female pigs were fed a diet containing 1% (w/w) of MegaNatural® Gold grape seed extract (GSE) daily for 6 days. Fecal samples were analyzed by normal phase LC coupled to fluorescence detection and LC-MS/ToF. DNA was extracted from pig fecal samples and the V3/V4 region of the 16S rRNA gene was sequenced using an Illumina MiSeq. Intact parent PACs (dimer-pentamer) were observed in the feces on days 3 and 6 at similar high levels (∼400 mg kg(-1) total) during ingestion of GSE but were absent 48 h post-feeding. The major phenolic metabolites were 4-hydroxyphenylvaleric acid and 3-hydroxybenzoic acid which increased by ∼30 and 3 mg kg(-1) respectively. The GSE diet also caused an ecological shift in the microbiome, dramatically increasing Lachnospiraceae, Clostridales, Lactobacillus and Ruminococcacceae. The relationship between dietary PACs and colon health may be attributable to the altered bacterial populations or phenolic compounds in the colon.

2,3-cis-2R,3R-(-)-epiafzelechin-3-O-p-coumarate, a novel flavan-3-ol isolated from Fallopia convolvulus seed, is an estrogen receptor agonist in human cell lines.

BACKGROUND: The plant genus Fallopia is well-known in Chinese traditional medicine and includes many species that contain bioactive compounds, namely phytoestrogens. Consumption of phytoestrogens may be linked to decreased incidence of breast and prostate cancers therefore discovery of novel phytoestrogens and novel sources of phytoestrogens is of interest. Although phytoestrogen content has been analyzed in the rhizomes of various Fallopia sp., seeds of a Fallopia sp. have never been examined for phytoestrogen presence. METHODS: Analytical chemistry techniques were used with guidance from an in vitro estrogen receptor bioassay (a stably transfected human ovarian carcinoma cell line) to isolate and identify estrogenic components from seeds of Fallopia convolvulus. A transiently transfected human breast carcinoma cell line was used to characterize the biological activity of the isolated compounds on estrogen receptors (ER) α and ß. RESULTS: Two compounds, emodin and the novel flavan-3-ol, (-)-epiafzelechin-3-O-p-coumarate (rhodoeosein), were identified to be responsible for estrogenic activity of F. convolvulus seed extract. Absolute stereochemistry of rhodoeosein was determined by 1 and 2D NMR, optical rotation and circular dichroism. Emodin was identified by HPLC/DAD, LC/MS/MS, and FT/ICR-MS. When characterizing the ER specificity in biological activity of rhodoeosein and emodin, rhodoeosein was able to exhibit a four-fold greater relative estrogenic potency (REP) in breast cells transiently-transfected with ERß as compared to those transfected with ERα, and emodin exhibited a six-fold greater REP in ERß-transfected breast cells. Cell type-specific differences were observed with rhodoeosein but not emodin; rhodoeosein produced superinduction of reporter gene activity in the human ovarian cell line (> 400% of maximum estradiol [E2] induction) but not in the breast cell line. CONCLUSION: This study is the first to characterize the novel flavan-3-ol compound, rhodoeosein, and its ability to induce estrogenic activity in human cell lines. Rhodoeosein and emodin may have potential therapeutic applications as natural products activating ERß, and further characterization of rhodoeosein is necessary to evaluate its selectivity as a cell type-specific ER agonist.

This kinetic, bioavailability, and metabolism study of RRR-α-tocopherol in healthy adults suggests lower intake requirements than previous estimates.

Kinetic models enable nutrient needs and kinetic behaviors to be quantified and provide mechanistic insights into metabolism. Therefore, we modeled and quantified the kinetics, bioavailability, and metabolism of RRR-α-tocopherol in 12 healthy adults. Six men and 6 women, aged 27 ± 6 y, each ingested 1.81 nmol of [5(-14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol; each dose had 3.70 kBq of (14)C. Complete collections of urine and feces were made over the first 21 d from dosing. Serial blood samples were drawn over the first 70 d from dosing. All specimens were analyzed for RRR-α-tocopherol. Specimens were also analyzed for (14)C using accelerator MS. From these data, we modeled and quantified the kinetics of RRR-α-tocopherol in vivo in humans. The model had 11 compartments, 3 delay compartments, and reservoirs for urine and feces. Bioavailability of RRR-α-tocopherol was 81 ± 1%. The model estimated residence time and half-life of the slowest turning-over compartment of α-tocopherol (adipose tissue) at 499 ± 702 d and 184 ± 48 d, respectively. The total body store of RRR-α-tocopherol was 25,900 ± 6=220 µmol (11 ± 3 g) and we calculated the adipose tissue level to be 1.53 µmol/g (657 µg/g). We found that a daily intake of 9.2 µmol (4 mg) of RRR-α-tocopherol maintained plasma RRR-α-tocopherol concentrations at 23 µmol/L. These findings suggest that the dietary requirement for vitamin E may be less than that currently recommended and these results will be important for future updates of intake recommendations.

Gender and single nucleotide polymorphisms in MTHFR, BHMT, SPTLC1, CRBP2, CETP, and SCARB1 are significant predictors of plasma homocysteine normalized by RBC folate in healthy adults.

Using linear regression models, we studied the main and 2-way interaction effects of the predictor variables gender, age, BMI, and 64 folate/vitamin B-12/homocysteine (Hcy)/lipid/cholesterol-related single nucleotide polymorphisms (SNP) on log-transformed plasma Hcy normalized by RBC folate measurements (nHcy) in 373 healthy Caucasian adults (50% women). Variable selection was conducted by stepwise Akaike information criterion or least angle regression and both methods led to the same final model. Significant predictors (where P values were adjusted for false discovery rate) included type of blood sample [whole blood (WB) vs. plasma-depleted WB; P < 0.001] used for folate analysis, gender (P < 0.001), and SNP in genes SPTLC1 (rs11790991; P = 0.040), CRBP2 (rs2118981; P < 0.001), BHMT (rs3733890; P = 0.019), and CETP (rs5882; P = 0.017). Significant 2-way interaction effects included gender × MTHFR (rs1801131; P = 0.012), gender × CRBP2 (rs2118981; P = 0.011), and gender × SCARB1 (rs83882; P = 0.003). The relation of nHcy concentrations with the significant SNP (SPTLC1, BHMT, CETP, CRBP2, MTHFR, and SCARB1) is of interest, especially because we surveyed the main and interaction effects in healthy adults, but it is an important area for future study. As discussed, understanding Hcy and genetic regulation is important, because Hcy may be related to inflammation, obesity, cardiovascular disease, and diabetes mellitus. We conclude that gender and SNP significantly affect nHcy.

Quantitation of [5-14CH3]-(2R, 4'R, 8'R)-α-tocopherol in humans.

Half-lives of α-tocopherol in plasma have been reported as 2-3 d, whereas the Elgin Study required >2 y to deplete α-tocopherol, so gaps exist in our quantitative understanding of human α-tocopherol metabolism. Therefore, 6 men and 6 women aged 27 ± 6 y (mean ± SD) ingested 1.81 nmol, 3.70 kBq of [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol. The levels of (14)C in blood plasma and washed RBC were monitored frequently from 0 to 460 d while the levels of (14)C in urine and feces were monitored from 0 to 21 d. Total fecal elimination (fecal + metabolic fecal) was 23.24 ± 5.81% of the (14)C dose, so feces over urine was the major route of elimination of the ingested [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol, consistent with prior estimates. The half-life of α-tocopherol varied in plasma and RBC according to the duration of study. The minute dose coupled with frequent monitoring over 460 d and 21 d for blood, urine, and feces ensured the [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol (the tracer) had the chance to fully mix with the endogenous [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol (the tracee). The (14)C levels in neither plasma nor RBC had returned to baseline by d 460, indicating that the t(1/2) of [5-CH(3)]-(2R, 4'R, 8'R)-α-tocopherol in human blood was longer than prior estimates.

Aerobic versus Anaerobic Microbial Degradation of Etofenprox in a California rice field soil.

The microbial degradation of etofenprox, an ether pyrethroid, was characterized under anaerobic (flooded) and aerobic (nonflooded) California rice field soil conditions by determination of its half-life (t1/2) and dissipation rate constant (k) and identification and quantification of degradation products at both 22 and 40 °C using LC-MS/MS. The overall anaerobic t1/2 at 22 °C ranged from 49.1 to 100 days (k=-0.0141 to -0.0069 days(-1)) compared to 27.0 days (k=-0.0257 days(-1)) at 40 °C, whereas under aerobic conditions the overall t1/2 was 27.5 days (k=-0.0252 days(-1)) at 22 °C compared to 10.1-26.5 days (k=-0.0686 to -0.0262 days(-1)) at 40 °C. The biphasic dissipation profiles were also fit to a first-order model to determine the t1/2 and k for both the fast and slow kinetic regions of the dissipation curves. Hydroxylation at the 4'-position of the phenoxy phenyl ring was the major metabolic process under anaerobic conditions for both 22 °C (maximum% yield of applied etofenprox mass=1.3±0.7%) and 40 °C (max % yield=1.2±0.8%). Oxidation of the ether moiety to the ester was the major metabolite under aerobic conditions at 22 °C (max% yield=0.5±0.1%), but at 40 °C increased amounts of the hydroxylated form were produced (max% yield=0.7±0.2%, compared to 0.3±0.1% for the ester). The hydrolytic product of the ester, 3-phenoxybenzoic acid (3-PBA), was not detected in any samples. Sterilized control soils showed little etofenprox degradation over the 56-day incubation period. Thus, the microbial population in a flooded soil was able to transform and contribute to the overall dissipation of etofenprox. The simulated summer temperature extreme (40 °C) increased the overall degradation.

Microbial degradation of clomazone under simulated California rice field conditions.

Clomazone (trade names Cerano and Command) is a popular herbicide used on California rice fields to control aquatic weeds. Its physicochemical characteristics indicate that it will persist primarily in the water column, where microbial degradation may drive its environmental fate. The objectives were to determine microbial degradation rates and compare the metabolic products under aerobic and anaerobic conditions similar to those in California rice fields during the summer. Time-series samples were extracted and analyzed by LC/MS/MS. Metabolic profiling revealed the following clomazone-derived transitions: m/z 240 --> 125 (clomazone), m/z 242 --> 125 (ring-open clomazone), m/z 256 --> 125 (5-hydroxyclomazone), m/z 256 --> 141 (aromatic hydroxyclomazone), m/z 268 --> 125 (unknown metabolite), and m/z 272 --> 141 (4'5-dihydroxyclomazone). Results indicate an anaerobic half-life of 7.9 days, with ring-open clomazone reaching 67.4% of application at 38 days. Aerobically, clomazone degraded more slowly (t(1/2) = 47.3 days), forming mostly soil-bound residues. Thus, under summer conditions, clomazone is likely to dissipate rapidly from fields via anaerobic degradation.

High-throughput method for the quantitation of total folate in whole blood using LC-MS/MS.

A high-throughput liquid chromatography tandem mass spectrometry (HT LC-MS/MS) method for red blood cell (RBC) folate analysis was developed from a previously described manual (M) LC-MS/MS method. The HT LC-MS/MS method used 96-well plates in which RBC folates were hydrolyzed with concentrated HCl in the presence of the [13C6]pABA internal standard (IS). The pH of the hydrolysate was adjusted to 5.0 before cleanup using 96-well plate OASIS HLB SPE cartridges. The analyte and IS were eluted with ethyl acetate/hexane (95:5, v/v) and methylated with methanol and trimethylsilyldiazomethane. The methylated analyte and IS were quantified with LC-MS/MS as previously described. The HT LC-MS/MS method was validated by determining the recovery of six different folate vitamers, which were quantitatively recovered (84-105% with CV<9.0%). RBC folate concentrations in whole blood samples correlated between HT and M LC-MS/MS methods (r=0.922, p<0.0001 for n=43 samples) and between the HT LC-MS/MS method and a chemiluminescence assay (r=0.664, p<0.001 for n=325 samples). Comparison of the results between HT LC-MS/MS and chemiluminescence methods with Bland-Altman difference plots and by ROC curve analysis indicates that the chemiluminescence assay underreports RBC folate concentrations. The HT LC-MS/MS method allows for high-throughput sample preparation for the analysis of RBC folate.

Comparison of two dietary folate intake instruments and their validation by RBC folate.

An optimal folate nutritional status is important in minimizing developmental and degenerative disease. Therefore, constant monitoring of folate intake and of biomarkers of folate nutritional status is essential. The objective of this research was to compare two folate intake instruments and validate each one against RBC folate measured by a high-throughput liquid chromatography tandem mass spectrometry (HT LC-MS/MS) method described in the companion paper (Owens, J. E.; Holstege, D. M.; Clifford, A. J. J. Agric. Food Chem. 2007, 55, 3292-3297). A food frequency questionnaire (FFQ) and a folate-targeted semiquantitative Block dietary folate equivalents (DFE) screener were compared and individually validated against an HT LC-MS/MS method. RBC folate was 1178 +/- 259 nmol/L (mean +/- SD) in a population of 337 normal adult subjects. Folate intakes were 556 +/- 265 microg/day by the FFQ and 524 +/- 276 microg/day by the DFE screener. Folate intakes by the DFE screener were approximately 34 microg less than by the FFQ (paired t test, p<0.01), but the intake instruments were highly correlated for total folate intake (r=0.608, p<0.01). Correlations between instruments and RBC folate were low (r<0.35) but strong (p<0.01). ROC curve analysis indicates that the measurement of RBC folate by the HT LC-MS/MS method is a better predictive tool than are intake instruments for the evaluation of marginal folate status.

Quantitation of total folate in whole blood using LC-MS/MS.

An accurate method for measuring whole blood total folate using liquid chromatography with tandem mass spectrometry is described and compared to GC/MS and a chemiluminescence assay. Whole blood from normal adults (n = 15) was fortified with a [(13)C(6)]para-aminobenzoic acid (pABA) internal standard and treated with 12.1 N hydrochloric acid at 110 degrees C for 4 h to hydrolyze all folates to pABA. Contaminants in the hydrolysate were adsorbed onto a C18 SPE cartridge. The eluate containing the folate catabolite pABA was partitioned into ethyl acetate and methylesterified with trimethylsilyldiazomethane. The methyl-pABA derivatives were quantified by positive-ion atmospheric pressure chemical ionization (APCI)LC-MS/MS. An isocratic mobile phase of acetonitrile-water (70:30) (v/v) on a C18 analytical column was used with a postcolumn reagent of 0.025% formic acid. The limit of quantitation for folate was 56.6 nmol/L RBC, and the limit of detection was 22.6 nmol/L RBC. Folate levels as determined by LC-MS/MS correlated well with the chemiluminescence assay and a GC/MS method. This new LC-MS/MS method provides enhanced sample throughput (n = 36 per day) as compared to GC/MS methods. LC-MS/MS will enable accurate measurements of red blood cell (RBC) folate in nutrition surveys and clinical trials.