Arecoline in buccal cells reflects Areca nut dose.

Areca nut (AN) is a carcinogen; its chewing cessation is, therefore, of worldwide interest. However, cessation biomarkers are lacking. We sought to establish arecoline in chewers' buccal cells (BCs) as a biomarker for AN dose. Self-reported AN doses, expressed as the average AN load ("AANL"), the product of AN amount, chewing time, and chewing frequency, were correlated by regression analysis with chewers' BC arecoline, measured by liquid chromatography mass spectrometry. We then determined whether associations differed between Class 1 chewers (who consume AN alone or with slaked lime, leaf, and/or spices) and Class 2 chewers (who consume any combination of the aforementioned ingredients plus tobacco). Among the 103 chewers, 28 Class 1 and 39 Class 2 chewers had detectable arecoline levels, which were used for analyses. A linear regression of cube-root transformed AANL on equally transformed BC arecoline levels provided the best model fit; resulting slopes and corresponding correlation coefficients were 0.86 and 0.40 (p < 0.01) for all; 1.09 and 0.51 (p < 0.01) for Class 1 chewers; 0.35 and 0.17 (p = 0.29) for Class 2 chewers; and 0.94 and 0.45 (p < 0.01), and 0.79 and 0.37 (p = 0.08), respectively, for those who included or excluded lime. Relationships between AANL and BC arecoline levels were similar between chewers who included or excluded lime (p = 0.76), but less between chewing classes (p = 0.14). This provides confidence that BC arecoline can generally act as a reliable biomarker for AN dose, useful for estimating efficacy in AN cessation studies and population-based chewing assessments.

Salivary Areca and tobacco alkaloids for bioverification in the Betel Nut Intervention Trial.

BACKGROUND: The Betel Nut Intervention Trial (BENIT) is the first known randomized controlled intervention trial designed to help minority Pacific Islanders in Guam and Saipan quit chewing the carcinogenic Areca catechu nut (AN). We report the BENIT's saliva bioverification results against the self-reported chewing status ("quitter" or "chewer") at day 22 follow-up. MATERIAL AND METHODS: AN-specific (arecoline, arecaidine, guvacoline, and guvacine) and tobacco-specific (nicotine, cotinine, and hydroxycotinine) alkaloids were analyzed in saliva from 176 BENIT participants by an established and sensitive liquid chromatography mass spectrometry-based assay. RESULTS: The combined four AN alkaloid levels decreased from baseline in quitters (n = 50) and chewers (n = 108) by 32% and 9%, respectively. In quitters, decreases were significant for arecoline (p = 0.044)-the most prominent AN alkaloid, along with arecaidine (p = 0.042) and nicotine (p = 0.011). In chewers, decreases were significant only for hydroxycotinine (p = 0.004). Similar results were obtained when quitters and chewers were stratified by treatment arm. DISCUSSION: Salivary AN alkaloid levels generally agreed with self-reported chewing status, which suggests the former can be used to verify the latter. CONCLUSION: Our results can help to objectively evaluate compliance and program effectiveness in AN cessation programs.

Pilot study on the urinary excretion of the glyphosate metabolite aminomethylphosphonic acid and breast cancer risk: The Multiethnic Cohort study.

Breast cancer is the most commonly diagnosed female cancer and the second leading cause of death in women in the US, including Hawaii. Accumulating evidence suggests that aminomethylphosphonic acid (AMPA), the primary metabolite of the herbicide glyphosate-a probable human carcinogen, may itself be carcinogenic. However, the relationship between urinary AMPA excretion and breast cancer risk in women is unknown. In this pilot study, we investigated the association between pre-diagnostic urinary AMPA excretion and breast cancer risk in a case-control study of 250 predominantly postmenopausal women: 124 cases and 126 healthy controls (individually matched on age, race/ethnicity, urine type, date of urine collection, and fasting status) nested within the Hawaii biospecimen subcohort of the Multiethnic Cohort. AMPA was detected in 90% of cases and 84% of controls. The geometric mean of urinary AMPA excretion was nearly 38% higher among cases vs. controls (0.087 vs 0.063 ng AMPA/mg creatinine) after adjusting for race/ethnicity, age and BMI. A 4.5-fold higher risk of developing breast cancer in the highest vs. lowest quintile of AMPA excretion was observed (ORQ5 vs. Q1: 4.49; 95% CI: 1.46-13.77; ptrend = 0.029). To our knowledge, this is the first study to prospectively examine associations between urinary AMPA excretion and breast cancer risk. Our preliminary findings suggest that AMPA exposure may be associated with increased breast cancer risk; however, these results require confirmation in a larger population to increase study power and permit careful examinations of race/ethnicity differences.

Analysis of glyphosate, aminomethylphosphonic acid, and glufosinate from human urine by HRAM LC-MS.

Aminomethylphosphonic acid (AMPA) is the main metabolite of glyphosate (GLYP) and phosphonic acids in detergents. GLYP is a synthetic herbicide frequently used worldwide alone or together with its analog glufosinate (GLUF). The general public can be exposed to these potentially harmful chemicals; thus, sensitive methods to monitor them in humans are urgently required to evaluate health risks. We attempted to simultaneously detect GLYP, AMPA, and GLUF in human urine by high-resolution accurate-mass liquid chromatography mass spectrometry (HRAM LC-MS) before and after derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) or 1-methylimidazole-sulfonyl chloride (ImS-Cl) with several urine pre-treatment and solid phase extraction (SPE) steps. Fmoc-Cl derivatization achieved the best combination of method sensitivity (limit of detection; LOD) and accuracy for all compounds compared to underivatized urine or ImS-Cl-derivatized urine. Before derivatization, the best steps for GLYP involved 0.4 mM ethylenediaminetetraacetic acid (EDTA) pre-treatment followed by SPE pre-cleanup (LOD 37 pg/mL), for AMPA involved no EDTA pre-treatment and no SPE pre-cleanup (LOD 20 pg/mL) or 0.2-0.4 mM EDTA pre-treatment with no SPE pre-cleanup (LOD 19-21 pg/mL), and for GLUF involved 0.4 mM EDTA pre-treatment and no SPE pre-cleanup (LOD 7 pg/mL). However, for these methods, accuracy was sufficient only for AMPA (101-105%), while being modest for GLYP (61%) and GLUF (63%). Different EDTA and SPE treatments prior to Fmoc-Cl derivatization resulted in high sensitivity for all analytes but satisfactory accuracy only for AMPA. Thus, we conclude that our HRAM LC-MS method is suited for urinary AMPA analysis in cross-sectional studies.

Improved oxytocin analysis from human serum and urine by orbitrap ESI-LC-HRAM-MS.

Native circulating oxytocin (OT) levels in non-pregnant/non-lactating/non-medicated humans are very low (≤ 8 pg/mL). The lower limit of detection (LLOD) of our previous liquid chromatography mass spectrometry (LC-MS) method (10-25 pg/mL) precluded their quantification in serum and urine. Thus, we sought to improve the LC-MS sensitivity of OT measurements in these matrices by hydrophobic tagging and solid phase extraction (SPE). In the former approach, OT was reduced then alkylated with N-alkyl acetamide (C12, C14, C16, and C18) tags or derivatized using sulfonyl chloride-based reagents. In the latter approach, native OT in serum and urine was concentrated by offline SPE using gradient acetonitrile washings after first crashing with acetonitrile. Peak urinary eluate fractions were further concentrated online then analyzed by orbitrap-based LC-MS with electrospray ionization. All hydrophobic OT derivatives had lower sensitivity than native OT. Washing with a water-acetonitrile gradient during SPE improved the LLOD of OT in spiked serum to 2.5 pg/mL, while adding a subsequent online-concentration step improved the LLOD in spiked urine to 1-5 pg/mL and allowed us to detect OT in urine from lactating women. We were unable to improve the sensitivity of OT measurements by hydrophobic tagging or by derivatization using sulfonyl chloride-based reagents. However, we were successful in improving the sensitivity of native OT measurements in serum and urine 2- and 5-fold, respectively, from our previous orbitrap-based LC-MS method. Offline SPE was mandatory for both matrices and a subsequent online-concentration step was required for urine.

Chemical Markers for Short- and Long-Term Areca Nut Exposure.

Background: Areca nut (AN) chewing causes oral cancer. AN cessation programs are the most effective approach to reduce AN chewing induced cancers but require biomarkers to determine program compliance and success. Objectives: To explore chemical markers for short- and long-term AN exposure using non-invasively collected saliva, buccal cells (BCs), and scalp hair of chewers. Methods: Saliva was collected from a male chewer before and up to 2 days after AN chewing. Saliva was separated into supernatant and pellet (BCs) then analyzed by spectrophotometry and liquid chromatography (LC) with UV/VIS detection. Scalp hair was collected from four chewers and analyzed for areca alkaloids using direct analysis in real time-tandem mass spectrometry (DART-MSMS). Results: The red pigmented saliva after chewing showed no valuable signals when either the saliva supernatant or pellet (BCs) were analyzed by spectrophotometry. Saliva analysis by LC-UV/VIS showed diagnostically valuable signals at 488 nm up to 5 and 24 h post chewing in the supernatant and pellet, respectively. DART-MSMS analysis detected two of the four AN specific alkaloids (arecoline and arecaidine) in male but none in female hair. Conclusions/Importance: LC-UV/VIS analysis of the red pigments extracted from saliva and BCs after AN chewing showed distinct signals up to 24 h post chewing while DART-MSMS analysis in BCs and scalp hair showed selective signals of AN alkaloids for several weeks or months after AN exposure. Chemical hair treatment might prevent detection of areca alkaloids in hair. AN cessation trials and other programs now have essential tools for bioverification.

Areca alkaloids measured from buccal cells using DART-MS serve as accurate biomarkers for areca nut chewing.

BACKGROUND: Areca nut (AN) chewing is carcinogenic and biomarkers reflecting it are urgently needed to determine the effectiveness of emergent cessation programs. Buccal cells (BCs) may serve as an ideal matrix to measure such biomarkers; however, their utility for this purpose is unknown. Direct analysis in real time-mass spectrometry (DART-MS) is a sensitive technique that analyzes materials in the open air and requires minimal/no sample preparation. We utilized DART-MS to analyze BCs to test the usefulness of this method in measuring areca alkaloids as biomarkers for AN chewing. METHODS: We applied DART-MS in positive-ion mode to quantitate over time human BCs: (a) exposed ex vivo to betel quid extracts (BQE) consisting of young AN, Piper betle L. leaf, slaked lime, and tobacco; and (b) obtained from seven chewers before and after BQ chewing. Quantification was performed by normalizing DART-MS alkaloid signal intensities to cholesterol intensities. RESULTS: Signals for areca alkaloids arecoline and arecaidine-guvacoline were detected in BCs exposed ex vivo to BQE up to 7 days (the last day tested) after exposure and in BCs from chewers up to 3 days (the last day tested) post chewing. DISCUSSION: The presence of alkaloid signals in BQ-exposed BCs verified BCs as a valid matrix and DART-MS as a suitable technique to measure biomarkers for AN chewing and provided reliable information on AN chewing timing. CONCLUSION: DART-MS analyses of BCs can be used to accurately determine areca alkaloids as AN chewing biomarkers up to 3 days post chewing and possibly longer.

Oxytocin analysis from human serum, urine, and saliva by orbitrap liquid chromatography-mass spectrometry.

Oxytocin (OT) is a neurohormone that has gained interest recently due to its emerging role in cognition and social/emotional behaviors, including possibly depression and autism. OT is commonly measured using enzyme- or radio-based immunoassays (RIA, ELISA), which lack specificity or are complicated to perform and involve hazardous radioactive material. We have developed a high resolution accurate-mass (HRAM) liquid chromatography-mass spectrometry (LC-MS) method that separates interferences and selectively and accurately quantitates native OT from human serum, urine, and saliva after solid phase extraction. The doubly protonated OT ion m/z 562.25503 was selected for quantitation due its high signal intensity. With our method lower limit of detection (LLOD) of 5-25 pg/mL, we measured native OT in serum from pregnant women (16-24 pg/mL) and rats (350 pg/mL), and in serum, urine, and saliva from a healthy male after intranasal (IN) OT application of 100 IU and 20 IU and from a healthy post-menopausal female after IN OT application of 100 IU. Peak levels were detected in serum, urine, and saliva 15-30 minutes after each dose then decreased to below detection limits 1-2 hours thereafter. We were unable to detect native OT in serum from non-pregnant/non-lactating/non-medicated women due to levels known to occur below 5 pg/mL. The fast elimination of OT we found is in excellent agreement with the pharmacokinetics of OT in other studies. The effects on the central nervous system occurring after IN OT administration remains to be determined.

Pilot study of the pharmacokinetics of betel nut and betel quid biomarkers in saliva, urine, and hair of betel consumers.

Approximately 600 million people worldwide practise the carcinogenic habit of betel nut/quid chewing. Carcinogenic N-nitroso compounds have been identified in saliva or urine of betel chewers and the betel alkaloid arecoline in hair from habitual betel quid chewers. However, the pharmacokinetic parameters of these compounds have been little explored. Assessment of betel use by biomarkers is urgently needed to evaluate the effectiveness of cessation programmes aimed at reducing betel consumption to decrease the burden of cancers in regions of high betel consumption. In the search for biomarkers of betel consumption, we measured by liquid chromatography-mass spectrometry (LC-MS) the appearance and disappearance of betel alkaloids (characteristic for betel nuts), N-nitroso compounds, and chavibetol (characteristic for Piper Betle leaves) in saliva (n=4), hair (n=2), and urine (n=1) of occasional betel nut/quid chewers. The betel alkaloids arecoline, guvacoline, guvacine, and arecaidine were detected in saliva of all four participants and peaked within the first 2 h post-chewing before returning to baseline levels after 8 h. Salivary chavibetol was detected in participants consuming Piper Betle leaves in their quid and peaked ~1 h post-chewing. Urinary arecoline, guvacoline, and arecaidine excretion paralleled saliva almost exactly while chavibetol glucuronide excretion paralleled salivary chavibetol. No betel nut related compounds were detected in the tested hair samples using various extraction methods. From these preliminary results, we conclude that betel exposure can only be followed on a short-term basis (≤8 h post-chewing) using the applied biomarkers from urine and saliva while the feasibility of using hair has yet to be validated. Copyright © 2015 John Wiley & Sons, Ltd.

Composition of betel specific chemicals in saliva during betel chewing for the identification of biomarkers.

Betel nut chewing causes cancer in humans, including strong associations with head and neck cancer in Guam. In the search for biomarkers of betel chewing we sought to identify chemicals specific for the 3 most commonly consumed betel preparations in Guam: nut ('BN'), nut + Piper betle leaf ('BL'), and betel quid ('BQ') consisting of nut + lime + tobacco + Piper betle leaf. Chemicals were extracted from the chewing material and saliva of subjects chewing these betel preparations. Saliva analysis involved protein precipitation with acetonitrile, dilution with formic acid followed by LCMS analysis. Baseline and chewing saliva levels were compared using t-tests and differences between groups were compared by ANOVA; p < 0.05 indicated significance. Predominant compounds in chewing material were guvacine, arecoline, guvacoline, arecaidine, chavibetol, and nicotine. In chewing saliva we found significant increases from baseline for guvacine (BN, BQ), arecoline (all groups), guvacoline (BN), arecaidine (all groups), nicotine (BQ), and chavibetol (BL, BQ), and significant differences between all groups for total areca-specific alkaloids, total tobacco-specific alkaloids and chavibetol. From this pilot study, we propose the following chemical patterns as biomarkers: areca alkaloids for BN use, areca alkaloids and chavibetol for BL use, and areca alkaloids plus chavibetol and tobacco-specific alkaloids for BQ use.

Pilot study for the establishment of biomarkers for radiation damage after computed tomography in children.

Computed tomography (CT) is an imaging modality that exposes patients to ionizing radiation (IR). We review and report findings from our pilot study evaluating whether blood markers are altered in 17 children undergoing medically indicated CT scans. Blood was drawn before ('pre-CT') and 1 hour after ('post-CT' CT scans. Plasma carotenoids, tocopherols, Q10, ascorbic acid (AA) and uric acid (UA) were analyzed by RP-HPLC with diode-array and electrochemical detection. Dehydroascorbic acid (DHAA) was calculated by subtraction from total AA. Total antioxidant capacity (TAC) was measured using the ORAC assay. Cytokines were quantified using a multiplex immunoassay. γ-H2AX foci were visualized using immunofluorescence. Mean pre- and post-CT changes were compared using t-tests; P-levels < .05 indicated significance. All major plasma lipid soluble antioxidant levels were lower post- vs pre-CT (P < .05) possibly from the scavenging of free radicals formed by CT-induced IR. Average AA levels increased (134%) while DHAA levels were decreased (29%) post-CT, probably due to intracellular recycling of AA from DHAA. TAC levels in lipophilic and hydrophilic extracts were unchanged, suggesting that other antioxidants may have assisted in free radical quenching, which would corroborate their lower concentrations post-CT. Cytokine levels were unchanged and dose-dependent increases in γ-H2AX foci, a measure of double strand DNA breaks, were observed (P = .046, n = 3 children). Our results suggest that CT-derived IR can influence the antioxidant system and may elicit detrimental responses on the cellular level of young children. When possible and if appropriate non-IR based techniques such as ultrasound or magnetic resonance imaging should be used.

Absorption, distribution, metabolism, and excretion of isoflavonoids after soy intake.

Soy is the major source of dietary exposure to isoflavonoids (IFLs). Accumulating evidence supports a role for soy and IFLs in the protection against many chronic diseases including cancer. After soy intake we found a biphasic IFL appearance pattern in plasma as well as in urine that we suggest to be due to IFL absorption in the small intestine (ca. 10%) during the first 2h after intake and IFL absorption in the large intestine (ca. 90%) 4-6 h after intake. While each IFL disappears from the circulation at different times excellent correlations between urinary and circulating IFL values were discovered and algorithms to convert urinary excretion values into circulating levels were established. We suggest the term 'apparent bioavailability' when using urinary data to describe IFL exposure. The IFL bioavailability was found to be influenced by gut bacteria, oral antibiotic treatment (OABX), and an individual's age and health status. While daidzein (DE) and genistein start to be absorbed minutes after intake, equol (EQ) appears in plasma only after a minimum of 8h following soy intake owing to the required transit time of DE to the colon where the conversion of DE to EQ takes place by intestinal microbiota. We have also shown that the apparent IFL bioavailability is higher in children than adults, higher in healthy versus non-healthy individuals, and decreased in children but increased in adults during OABX. Finally, we propose to use a urinary EQ/DE ratio of 0.018 with a DE threshold to identify EQ producers. With this cutoff definition we observed that EQ production is inconsistent over time in 5-30% of both premenopausal and postmenopausal women.

γ-H2AX foci are increased in lymphocytes in vivo in young children 1 h after very low-dose X-irradiation: a pilot study.

BACKGROUND: Computed tomography (CT) is an imaging modality involving ionizing radiation. The presence of γ-H2AX foci after low to moderate ionizing radiation exposure has been demonstrated; however it is unknown whether very low ionizing radiation exposure doses from CT exams can induce γ-H2AX formation in vivo in young children. OBJECTIVE: To test whether very low ionizing radiation doses from CT exams can induce lymphocytic γ-H2AX foci (phosphorylated histones used as a marker of DNA damage) formation in vivo in young children. MATERIALS AND METHODS: Parents of participating children signed a consent form. Blood samples from three children (ages 3-21 months) undergoing CT exams involving very low blood ionizing radiation exposure doses (blood doses of 0.22-1.22 mGy) were collected immediately before and 1 h post CT exams. Isolated lymphocytes were quantified for γ-H2AX foci by a technician blinded to the radiation status and dose of the patients. Paired t-tests and regression analyses were performed with significance levels set at P < 0.05. RESULTS: We observed a dose-dependent increase in γ-H2AX foci post-CT exams (P = 0.046) among the three children. Ionizing radiation exposure doses led to a linear increase of foci per cell in post-CT samples (102% between lowest and highest dose). CONCLUSION: We found a significant induction of γ-H2AX foci in lymphocytes from post-CT samples of three very young children. When possible, CT exams should be limited or avoided by possibly applying non-ionizing radiation exposure techniques such as US or MRI.

In vivo changes in plasma coenzyme Q10, carotenoid, tocopherol, and retinol levels in children after computer tomography.

BACKGROUND: Low dose X-irradiation (IR) from computer tomography (CT) can generate free radicals, which can damage biologically relevant molecules and ultimately lead to cancer. These effects are especially concerning for children owing to their higher radiosensitivity and longer life expectancy than adults. The lipid phase micronutrients (LPM) coenzyme Q10, carotenoids, E vitamers, and vitamin A are potent radical scavengers that can act as intracellular antioxidants. METHODS: We investigated changes in circulating levels of these LPM in 17 children (0.25-6 y) undergoing medically indicated CT scans involving relatively low IR doses. Blood was drawn before and 1h after CT scans and analyzed using HPLC with electrochemical and UV/VIS detection. RESULTS: We found significant decreases (p<0.05) in post-CT plasma levels in several LPM which suggests that these LPM can serve as biodosimeters and may protect against damage from IR during clinical procedures such as CT. The strongest predictors for pre- to post-CT changes for many LPM were their baseline levels. CONCLUSION: Future larger studies are warranted to confirm our findings and to test whether high circulating antioxidant levels protect against IR damage in vivo with an ultimate goal of establishing prophylactic modalities for CT-induced IR damage.

Vitamin D deficiency in cord plasma from multiethnic subjects living in the tropics.

BACKGROUND: Vitamin D deficiency is commonly reported in high-latitude areas and in dark-pigmented individuals. However, nothing is known about vitamin D in cord blood from multiethnic subjects living in the tropics. OBJECTIVE: Our study objective was to determine the prevalence of vitamin D deficiency in summer and winter in cord blood from multiethnic individuals in Hawai'i where sufficient sun irradiance occurs year-round for cutaneous vitamin D production. METHODS: 25-Hydroxyvitamin D (25(OH)D) levels were quantified by enzyme immunoassay in 100 cord plasma samples from apparently healthy full-term newborns and their mothers. Stratification was performed by birth season and ethnicity. RESULTS: Mean 25(OH)D levels were 24.5 ng/mL (9.1-68.3 ng/mL). Overall, 28% of samples were vitamin D deficient (<20 ng/mL) and 50% were insufficient (20-30 ng/mL). 25(OH)D levels (ng/mL) were highest in Caucasians (30.5, n = 19), followed by Asians (25.1, n = 43), Hispanics (21.5, n = 3), Pacific Islanders (20.0, n = 25), and African Americans (19.6, n = 2). Differences among groups were significant (p = 0.008). Cord plasmas from summer versus winter were higher overall (p = 0.001) and among Asians (p = 0.0003). Seasonal changes were correlated with sun irradiance overall (r = 0.43, p = 0.0001), among Caucasians (r = 0.45, p = 0.05), and among Asians (r = 0.45, p = 0.0001). CONCLUSION: Our results suggest that prenatal supplement recommendations of 400 IU vitamin D/day do not protect against vitamin D deficiency, even in subjects living in the tropics where ample sun irradiance exists for cutaneous vitamin D synthesis. The high prevalence of vitamin D deficiency we observed emphasizes the necessity for regular 25(OH)D monitoring, particularly during pregnancy and lactation, in dark-pigmented individuals, and during winter months.

Simultaneous analysis of circulating 25-hydroxy-vitamin D3, 25-hydroxy-vitamin D2, retinol, tocopherols, carotenoids, and oxidized and reduced coenzyme Q10 by high performance liquid chromatography with photo diode-array detection using C18 and C30 columns alone or in combination.

Circulating lipid-phase micronutrients (LPM) such as 25-hydroxylated D vitamers, retinol, tocopherols, carotenoids including their isomers, and coenzyme Q10 play important roles in health maintenance and disease prevention and can serve as useful biomarkers. We developed fast, affordable, and accurate HPLC assays that simultaneously measured all above LPM in a single run using UV/VIS detection at 265nm, 295nm, and 480nm with (1) a C18 column alone; (2) a C30 column alone; or (3) each of these columns connected in series. The C18 column alone could separate all major LPM of interest in less than 17min but insufficiently resolved the lycopene isomers, the 25-hydroxylated D vitamers, lutein from zeaxanthin and ß- from γ-tocopherol. The C30 column alone separated all LPM of interest including many isomeric analytes but failed to resolve the Q10 compounds, which co-eluted with carotenoids. Connecting the C18 and C30 columns in series with a detector after the C30 column and a pressure resistant detector between the columns resulted in ideal resolution and accurate quantitation of all LPM of interest but required software capable of processing the acquired data from both detectors. Connecting the C18 and C30 columns in series with exclusively one detector after the C30 column resulted in carotenoid-Q10 interferences, however, this was remedied by heart-cutting 2D-LC with a 6-port valve between the columns, which resolved all analytes in 42min. Faster run times led to some analytes not being resolved. Many variations of these methods are possible to meet the needs of individual requirements while minimizing sample material and turn-around-times.

Analysis of circulating lipid-phase micronutrients in humans by HPLC: review and overview of new developments.

Retinol, tocopherols, coenzyme Q10, carotenoids, and vitamin D are lipophilic compounds shown to function as important health-protective agents by mitigating the damaging effects of oxidative and other injury. Scientific interest in evaluating these compounds has resurfaced in recent years, particularly in the nutritional, clinical and epidemiologic fields, and has precipitated the development of a multitude of new analytical techniques. This review considers recent developments in HPLC-based assays since 2007 for the simultaneous determination of these lipid-phase compounds utilizing exclusively serum or plasma as these matrices are mostly used in clinical and epidemiological investigations. We also provide an overview of blood measurements for selected carotenoids, tocopherols, coenzyme Q10 and retinol from the last 15years of healthy umbilical cord blood, children, and adults.

Equol production changes over time in pre-menopausal women.

Equol (EQ) is a metabolite produced by gut bacteria through the chemical reduction of the soya isoflavone daidzein (DE), but only by 30-60% of the population. EQ is believed to provide benefits derived from soya intake and its production is widely viewed as a relatively stable phenomenon. In a randomised, cross-over intervention with soya foods, seventy-nine pre-menopausal women were challenged with a high-soya and a low-soya diet each for 6 months, separated by a 1-month washout period. Overnight urine was collected at three time points during each diet period and analysed for DE and EQ by liquid chromatography tandem MS. Remaining an EQ producer (EP) or non-producer (NP) or changing towards an EP or NP was assessed using an EQ:DE ratio of ≥0·018 combined with a DE threshold of ≥2 nmol/mg creatinine as a cut-off point. We observed 19 and 24% EP during the low-soya and high-soya diet periods, respectively, and found that 6-11% of our subjects changed EQ status 'within' each study period (on an average of 1·2 times), while 16% changed 'between' the two diet periods. The present finding challenges the widely held conviction that EQ production within an individual remains stable over time. The precise factors contributing to changes in EQ status, however, remain elusive and warrant further investigation.

Equol production changes over time in postmenopausal women.

Equol (EQ) is produced by intestinal bacteria from the soy isoflavone daidzein (DE) in 30%-60% of the population and is believed to provide benefits from soy intake. A robust EQ status definition is lacking, and it is uncertain whether EQ is formed consistently within an individual and ceases upon oral antibiotic treatment. In a randomized, double-blind, placebo-controlled soy intervention trial with 350 postmenopausal women, DE and EQ were analyzed by liquid chromatography/tandem mass spectrometry at baseline and every 6 months over 2.5 years in overnight urine, spot urine and plasma. Equol production changes and status (remaining an EQ producer or nonproducer or changing towards an EQ producer or nonproducer) were assessed. Equol status was determined most dependably by overnight urine applying as cutoff a ratio of EQ/DE≥0.018 with a DE threshold ≥2 nmol/mg creatinine: the soy and placebo groups had approximately 30% consistent EQ producers during the study, but 14% and 35%, respectively, changed EQ status (mean 1.4-1.7 times), while 27% and 17%, respectively, had antibiotic treatment (P<.01 for inverse association). No significant trend in change of EQ production or status was observed when overnight urine was limited to collections closest to before and after antibiotic treatment. Similarly, antibiotic type or class, duration, dose or time between antibiotic treatment and overnight urine collection showed no consistent influence on EQ production. Equol production can markedly change intraindividually over 2.5 years, and antibiotic treatment impacts it inconsistently. Factors other than antibiotic treatment must be considered as causes for EQ production changes.