Jennifer F. Lai

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.

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 β- 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.

Coenzyme Q10, carotenoid, tocopherol, and retinol levels in cord plasma from multiethnic subjects in Hawaii.

Abstract Coenzyme Q10 (Q10), carotenoids, tocopherols, and retinol are the major circulating lipid-phase micronutrients (LPM) known to help mitigate oxidative damage and prevent chronic diseases. However, the functions of these compounds in newborns are little understood. This is due, in part, to the paucity of studies reporting their concentrations in this population. We measured Q10, carotenoids, tocopherols, and retinol in cord plasma from 100 multiethnic subjects living in Hawaii using HPLC with diode array and electrochemical detection. Appropriate internal standards were used including, for the first time, custom designed oxidized (UN10) and reduced (UL10) Q10 analogues. These compounds reflected the oxidation of UL10 to UN10 that occurred during sample processing and analysis and thus permitted accurate adjustments of natively circulating Q10 levels. All LPM measured were much lower in cord than in peripheral plasma. Cord plasma levels of total carotenoids, tocopherols, and retinol were approximately 10-fold, 3- to 5-fold and 1.5- to 3-fold lower than those in children or women. Cord plasma levels of total Q10 (TQ10; median, 113 ng/mL) were approximately 2-fold or 7- to 9-fold lower than peripheral plasma levels of neonates or children and adults, respectively. In contrast, the UN10/TQ10 ratio was substantially higher in cord (24%) than in peripheral plasma of children (3–4%) or adults (9%). Among the 5 ethnic groups in our cohort, no differences were observed in the levels of UN10, UL10, or TQ10. However, significant differences in many of the LPM were observed between ethnicities. More research is needed to explain these phenomena.

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

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.

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.

Changes in Whole Blood Gene Expression after Computed Tomography in Children: A Pilot Study

Computed tomography (CT) exposes patients to ionizing X-irradiation (IR) that can alter the expression of genes responsible for controlling complex regulatory pathways. We sought to determine trends in the expression of 24 documented radiation-responsive genes linked to cancer in vivo. A total of 17 children (0.25–6 years old) undergoing medically indicated CT examinations with radiation doses ranging from 92.46 to 525.55 mGy cm (equivalent to effective doses of 0.78–11.30 mSv) were enrolled. Blood was drawn immediately before and 1 hour after their CT exams and mixed with an RNA additive for stabilization of gene expression. RNA samples of 14 of the 17 children were analyzed on a gene expression microarray. Absolute changes in gene expression were subtle, averaging less than 10%, but trends in expression changes of several genes were observed. ERP29, an endoplasmic reticulum chaperone, thought to be involved in the folding of secretory proteins, showed significant change in expression (8% decrease, P = 0.002) in the expected direction consistent with previous literature. PCNA expression increased linearly with CT dose (mGy cm) (P = 0.001). TP53 and FLT3LG expression increased linearly with effective dose (mSv) (P = 0.02 and P = 0.02). Previous IR exposure was associated with decreased GADD45A (P = 0.001) and FLT3LG (P = 0.03) and increased MDM2 expression (P = 0.02). We observed in this pilot study modest gene expression changes in the 24 IR-responsive candidate genes studied. Our results showed trends in gene expression changes, and they need to be confirmed in future studies with larger sample sizes to help develop risk assessments and preventive modalities for young patients undergoing CT.

Abstract 4348: Changes in leukocyte gene expression after computer tomography in children

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective: Computer tomography (CT) involves ionizing X-irradiation which can lead to DNA damage from free radicals. CT use has risen dramatically due to its high speed and diagnostic value but it leads to an estimated lifetime cancer risk of up to 4%, with highest risk for children. We tested in a pilot project whether expression of genes hypothesized to change upon ionizing radiation are altered in the leukocytes of young children undergoing CT exams that involved relatively low X-ray doses. Methods: 17 children aged 0.25-6 years and scheduled for medically indicated CT donated blood immediately before and 1 hour after a CT exam (median 340.9 mGy*cm equivalent to 2.3 mSv). Blood was collected into PaxGene tubes and RNA was extracted according to the suggested protocol, labeled and hybridized onto Affymetrix Gene 1.0 ST Arrays. Preliminary expression analysis was done on 24 genes suggested to fluctuate after ionizing radiation: GADD45, CDKN1A, ATM, ERP29, TP53, CDKN2A, MUC1, CDH6, DDB2, XPC, TNFRSF10B, FHL2, CCNG1, PCNA, CCNB1, MDM2, BAX, MAPK8, ALB, PPP1R14A, FLT3LG, HP, RPA2, NFKB1. Significance was determined by paired t-tests and stepwise multiple regression using the SAS 9.2 software. Results: In post versus pre CT leukocytes paired t-tests revealed that the expression of ERP29 decreased (p=0.002) but that the expression of the other 23 genes did not change significantly except for possible suggestive increases of XPC (p=0.11) and MUC1 (p=0.18) and decreases of PCNA (p=0.16) and PPP1R14A (p=0.16) expression. Stepwise multiple regression tests for predictors of pre to post changes revealed (i) that the CT dose (mGy*cm) was positively associated with PCNA expression (p=0.001), (ii) that the effective dose (mSv) was positively associated with TP53 (p=0.02) and FLT3LG (p=0.02) expression, and (iii) that the radiation history was associated with increased MDM2 (p=0.002) but negatively associated with PPP1R14A (p=0.05) and FLT3LG (p=0.03) expression. Stepwise multiple regression tests for predictors of baseline levels showed that the radiation history was associated with higher baseline expression of XPC (p=0.05), MDM2 (p=0.01), and BAX (p=0.001) but lower baseline expression of ALB (p=0.04). Conclusions: Few of the investigated 24 radiosensitive genes with the exception of ERP29 changed expression levels in the leukocytes of the 17 young children studied after these subjects received relatively low radiation doses. However, radiation dose dependent changes after CT were observed (PCNA, TP53, and FLT3LG expression). In addition, radiation history was found to be positively associated with baseline expression levels of XPC, MDM2, and BAX, but negatively with that of ALB. Genome wide analyses are currently under way to explore expression changes of other genes in these 17 subjects. Larger studies are needed to confirm these preliminary findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4348. doi:1538-7445.AM2012-4348

Abstract 4347: γH2AX foci formation in lymphocytes in vivo is increased one hour after very low dose X-radiation in three young children

Objective: Computer tomography (CT) involves ionizing X-irradiation which can cause DNA damage from free radicals. CT use has risen dramatically due to its high speed and diagnostic value but it leads to an estimated lifetime cancer risk of up to 4% with highest risk for children. Double strand breaks, the most serious DNA damage caused by X-rays and other types of ionizing radiation, leads to histone H2AX phosphorylation which causes recruitment of repair proteins to DNA damage sites. γH2AX foci therefore represent quantitatively double strand breaks and can be assayed by a fluorescent immunostaining procedure. This γH2AX assay has been shown to be useful as a reliable biodosimeter for X-irradiation in vitro, in animals, and in adults. Recently also children who received cardiac catheterization procedures involving fluoroscopy with intermediate X-irradiation doses showed a dose response in this assay. We tested whether the γH2AX assay can detect changes when children receive X-irradiation at very low doses ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4347. doi:1538-7445.AM2012-4347