Safety, Tolerability, and Pharmacokinetics of ß-Cryptoxanthin Supplementation in Healthy Women: A Double-Blind, Randomized, Placebo-Controlled Clinical Trial.

BACKGROUND: ß-cryptoxanthin is a dietary carotenoid for which there have been few studies on the safety and pharmacokinetics following daily oral supplementation. METHODS: 90 healthy Asian women between 21 and 35 years were randomized into three groups: 3 and 6 mg/day oral ß-cryptoxanthin, and placebo. At 2, 4, and 8 weeks of supplementation, plasma carotenoid levels were measured. The effects of ß-cryptoxanthin on blood retinoid-dependent gene expression, mood, physical activity and sleep, metabolic parameters, and fecal microbial composition were investigated. RESULTS: ß-cryptoxanthin supplementation for 8 weeks (3 and 6 mg/day) was found to be safe and well tolerated. Plasma ß-cryptoxanthin concentration was significantly higher in the 6 mg/day group (9.0 ± 4.1 µmol/L) compared to 3 mg/day group (6.0 ± 2.6 µmol/L) (p < 0.03), and placebo (0.4 ± 0.1 µmol/L) (p < 0.001) after 8 weeks. Plasma all-trans retinol, α-cryptoxanthin, α-carotene, ß-carotene, lycopene, lutein, and zeaxanthin levels were not significantly changed. No effects were found on blood retinol-dependent gene expression, mood, physical activity and sleep, metabolic parameters, and fecal microbial composition. CONCLUSIONS: Oral ß-cryptoxanthin supplementation over 8 weeks lead to high plasma concentrations of ß-cryptoxanthin, with no impact on other carotenoids, and was well tolerated in healthy women.

Higher maternal plasma ß-cryptoxanthin concentration is associated with better cognitive and motor development in offspring at 2 years of age.

PURPOSE: Current literature on the roles of α-, ß-carotene and ß-cryptoxanthin in neurocognitive function has largely focused on preventing cognitive decline in older people, and less on neuro-development in children. We examined the relations of maternal plasma carotenoids concentrations with offspring cognitive development up to age 4.5 years in the Growing Up in Singapore Towards healthy Outcomes mother-offspring cohort study. METHODS: Maternal plasma α-, ß-carotene and ß-cryptoxanthin concentrations at delivery were determined by ultra-performance liquid chromatography. Children's cognition was assessed at ages 2 (Bayley Scales of Infant and Toddler Development) and 4.5 (Kaufman Brief Intelligence Test) years. Associations were examined in 419 mother-offspring pairs using linear regressions adjusting for key confounders. RESULTS: Median and interquartile range of maternal plasma concentrations (mg/L) were: α-carotene 0.052 (0.032, 0.081), ß-carotene 0.189 (0.134, 0.286), and ß-cryptoxanthin 0.199 (0.123, 0.304). In 2 years old children, higher maternal carotenoids [per standard deviation (SD) log-concentration] were positively associated with neurocognitive functions: ß-cryptoxanthin with higher scores in cognitive [ß = 0.18, (0.08, 0.28) SD], receptive language [ß = 0.17 (0.07, 0.27) SD], fine motor [ß = 0.16 (0.05, 0.26) SD], and gross motor [ß = 0.16 (0.06, 0.27) SD] scales; ß-carotene with higher cognitive score [ß = 0.17 (0.05, 0.29) SD]. No significant associations were observed with neurocognitive functions at age 4.5 years. CONCLUSION: Our study provides novel data suggesting a potential role of prenatal carotenoids, particularly ß-cryptoxanthin, on early offspring cognitive and motor development. Whether the prenatal influences sustain beyond early childhood requires further investigation in longer term studies.

Maternal Lutein and Zeaxanthin Concentrations in Relation to Offspring Visual Acuity at 3 Years of Age: The GUSTO Study.

Lutein and zeaxanthin play important roles in visual functions, but their influence on early visual development is unclear. We related maternal lutein and zeaxanthin concentrations during pregnancy to offspring visual acuity (VA) in 471 mother-child pairs from the Growing Up in Singapore Towards healthy Outcomes (GUSTO) cohort. Maternal concentrations of plasma lutein and zeaxanthin were determined at delivery. We measured uncorrected distance of VA in 3-year old children using a LEA Symbols chart; readings were converted to the logarithm of Minimum Angle of Resolution (logMAR), with >0.3 logMAR indicating poor VA. Associations were examined using linear or Poisson regression adjusted for confounders. The median (inter-quartile range) of maternal lutein and zeaxanthin concentrations were 0.13 (0.09, 0.18) and 0.09 (0.07, 0.12) µmol/L, respectively. A total of 126 children had poor VA. The highest tertile of maternal zeaxanthin concentration was associated with 38% lower likelihood of poor VA in children (95% CI: 0.42, 0.93, p-Trends = 0.02). Higher maternal lutein concentrations were associated with a lower likelihood of poor VA in children (RR 0.60 (95% CI: 0.40, 0.88) for middle tertile; RR 0.78 (95% CI: 0.51, 1.19) for highest tertile (p-Quadratic = 0.02)). In conclusion, lutein and zeaxanthin status during pregnancy may influence offspring early visual development; but the results require confirmation with further studies, including more comprehensive measurements of macular functions.

Comprehensive high-performance liquid chromatographic method for the measurements of lipophilic antioxidants in human plasma.

Owing to the increasing interest in the health effects of antioxidant micronutrients on chronic diseases, a robust and rapid HPLC method for simultaneous measurement of coenzyme Q(10) (ubiquinone and ubiquinol), vitamin A (all-trans-retinol), vitamin E (tocopherols and tocotrienols) and carotenoids (lutein, zeaxanthin, beta-cryptoxanthin, lycopene and beta-carotene) was developed. Sample preparation and analytical conditions that would affect solubility and stability of these antioxidants were investigated and optimized. The mobile phase used was made up of acetonitrile, methanol, ethanol and tert-butanol without corrosive additives such as ammonium perchlorate and perchloric acid. Our results show that using two C(18) columns coupled with photodiode array, fluorescence and electrochemical detection, a comprehensive spectrum of 16 lipid-soluble antioxidants in 30 microL of plasma could be separated and quantified within 30 min. The chromatographic run time was about 3-fold faster and the sample size was about 5-fold smaller than when assays were performed separately using existing methods. The present method will be useful for dietary habit studies and for antioxidant status investigations.

Peroxyl radical scavenging capacity, polyphenolics, and lipophilic antioxidant profiles of mulberry fruits cultivated in southern China.

Twenty-seven cultivars of mulberry fruits ( Morus atropurpurea Roxb) were analyzed for their total phenolic content, total anthocyanin content, and peroxyl radical scavenging capacities. The proanthocyanidin contents of the fruit were also quantified using 4-dimethylamino-cinnamaldehyde assay, and characterization was attempted using electrospray ionization mass spectra. The phenolic compounds of mulberry fruits were characterized using HPLC with ESI-MS and diode array detection. Results showed that the content of mulberry fruits varied with different cultivars with total phenolic content, total anthocyanin content, total proanthocyanidin content, and peroxyl radical scavenging capacities ranging from 0.060-0.244, 0.001-0.056, 0.001-0.015, and 0.301-1.728, respectively. Good correlations were observed among the phenolic, anthocyanin, and proanthocyanidin contents and the radical scavenging capacities of mulberry fruits. Mulberry fruits were found to contain low amount of proanthocyanidins. The high total phenolic content of mulberry fruits were mainly contributed by anthocyanins, rutin, and chlorogenic acids. The lipid soluble antioxidants are profiled by an HPLC method developed in-house, and the results of selected mulberry fruits revealed significant amounts of lutein and delta- and gamma-tocopherols but low alpha-tocopherol. Our results provide useful antioxidant nutritional information of a mulberry cultivar that has potential for large scale plantations.

Association of blood lead and homocysteine levels among lead exposed subjects in Vietnam and Singapore.

OBJECTIVES: Lead and homocysteine are both linked to cardiovascular disease. With this in mind, the authors evaluated the relation between blood lead and homocysteine in people aged 19-66 years in two Asian populations. METHODS: This cross-sectional study comprised 183 workers from a lead stabiliser factory in Singapore and 323 workers from a battery factory in Vietnam. Workers were occupationally exposed to lead. Blood lead was analysed using atomic absorption spectrophotometry while plasma homocysteine was measured using high performance liquid chromatography. RESULTS: Chinese subjects had the lowest blood lead levels while the Indians had the highest. Controlling for age, sex and race, an increase of 1 microg/dl in blood lead was associated with an increase of 0.04 micromol/l of homocysteine on the log scale. Gender and ethnicity seemed to be strongly associated with the relation between lead and homocysteine. The positive relation between lead and homocysteine among the Vietnamese subjects was significant (Pearson's r = 0.254, p<0.01). When blood lead levels were divided by quartiles, the correlation coefficient between blood lead levels in the 4th quartile and homocysteine among the Vietnamese was higher (r = 0.405, p<0.01). Overall, an increase of 1 microg/dl in blood lead in all the Vietnamese subjects was associated with an increase of 0.05 micromol/l increase in homocysteine on the log scale. However, in the 4th quartile, the same increase was associated with an increase of 0.41 micromol/l of homocysteine on the log scale. CONCLUSIONS: Blood lead was found to be associated with homocysteine levels in this Asian sample. Although we cannot determine causality from cross-sectional data, it is sensible to consider the probability that this relation could explain one of the mechanisms of the impact of lead on the cardiovascular system. More studies would be needed to confirm this inference.

A sensitive liquid chromatographic method for the spectrophotometric determination of urinary trans,trans-muconic acid.

Benzene is a human carcinogen and its metabolite, urinary trans,trans-muconic acid (ttMA), is a biomarker for risk assessment. However, most of the existing methods were not sensitive enough for monitoring of low level exposure. This paper describes a HPLC-UV method for ttMA determination with enhanced selectivity and sensitivity. A 30 mg OasisMAX cartridge was used to clean-up 50 microl of urine sample and gradient elution was performed on a Zorbax SB-C(18) column (30 degrees C). ttMA was detected at wavelength 263 nm using a UV diode array detector (DAD). The two mobile phases used were (A) 150 mM ortho-phosphoric acid containing of 9% (v/v) methanol; and (B) 125 mM ortho-phosphoric acid containing 30% (v/v) acetonitrile. The method was validated with 61 urine samples collected from non-occupationally benzene exposed individuals and 14 quality control specimens from an international quality assessment scheme. The urinary ttMA concentrations (mean+/-S.D.microg/g creatinine) were 90+/-34 for smokers (n=26), 49+/-39 for non-smokers (n=21) and 23+/-18 for non-smoking hospital staff (n=14). A correlation coefficient, r=0.99 was found with 14 external quality specimens for ttMA ranged from 0.4 to 6.8 mg/l. The recovery and reproducibility were generally over 90% and the detection limit was 5 microg/l.

Monomeric C18 chromatographic method for the liquid chromatographic determination of lipophilic antioxidants in plants.

Reversed-phase liquid chromatography was used to determine lipophilic antioxidants in plants using two monomeric C18 columns operated at 30 degrees C and 4 degrees C, with a column-switching technique and acetonitrile-methanol gradient elution. The chromatograms were extracted at different wavelengths using a UV diode array detector (DAD). A wide range of plant antioxidants, including nine carotenoids (neoxanthin, violaxanthin, lutein, zeaxanthin, beta-cryptoxanthin, lycopene, canthaxanthin, alpha-carotene and beta-carotene) together with all-trans-retinol, capsaicin, dihydrocapsaicin, chlorophyll a, and chlorophyll b can be separated within 50 min. Fluorometric detection was applied to quantify trace amounts of six vitamin E analogues (alpha-, delta- and gamma-tocopherols and tocotrienols). The detection limits were 0.2-0.4 microg/g for various xanthophylls and 0.04-0.10 microg/g for vitamin E analogues.

Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes.

Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0-3000 nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3-3.9 and 0.6-2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100 pg of uracil yielded a mean uracil measurement of 110.1 pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12 nM, and minima at 0 and 3000 nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000 nM folate for 10 days, P > 0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3 microg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans.

Simultaneous determination of tocotrienols, tocopherols, retinol, and major carotenoids in human plasma.

BACKGROUND: Epidemiologic evidence suggests that the concentrations of antioxidant vitamins in human plasma may play an important role in numerous chronic diseases, such as cancer and cardiovascular disease. However, methods for simultaneous measurement of these antioxidants are scarce. We developed and validated a new HPLC method for simultaneous determination of these vitamers in human plasma that uses a novel column-switching approach. METHODS: The new method uses liquid-liquid extraction and isocratic separation with two monomeric C(18) columns maintained at 35 and 4 degrees C coupled with ultraviolet-visible and fluorometric detection. This method could separate 14 vitamers and 3 internal standards within 27 min. No additional modifier was required; the mobile phase was acetonitrile-methanol (65:35 by volume), and the flow rate was 1 mL/min. RESULTS: For photodiode array detection, the detection limits (signal-to-noise ratio >3) were 0.02 mg/L for beta-carotene, lutein, zeaxanthin, and canthaxanthin; 0.01 mg/L for all-trans-retinol, beta-cryptoxanthin, alpha-carotene, and lycopene; and 0.1 mg/L for all tocopherols and tocotrienols. The detection limit was at least 25-fold lower (0.004 mg/L) when fluorometry was used for measurement of delta-, gamma-, and alpha-tocotrienol and delta-tocopherol compared with ultraviolet detection. The recovery and imprecision of the assay were generally >90% and <10%, respectively. CONCLUSIONS: This new method separates a wide range of fat-soluble antioxidant vitamins in human plasma, including six carotenoids, three isoforms of tocotrienols and tocopherols (delta-, gamma-, and alpha-), and all-trans-retinol. The overall findings suggest that our method is faster, more sensitive, and more comprehensive than existing methods.

Coenzyme Q10 and differences in coronary heart disease risk in Asian Indians and Chinese.

Indians or South Asians have been found to be particularly susceptible to coronary heart disease (CHD) in many countries. A novel risk factor for CHD may be coenzyme Q10 (CoQ10). In this study, plasma CoQ10 (including ubiquinol-10, CoQ10H2, and total CoQ10), various lipid parameters, and antioxidant levels were determined in a random sample of Indians and Chinese from the general population of Singapore. The reduced form of coenzyme Q10, CoQ10H2, and total Q10 concentrations in plasma were significantly lower in Indian males than Chinese males. Although no significant differences were found in plasma concentrations of total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL) between the two ethnic groups, the ratios of ubiquinol and total CoQ10 to triglycerides, total cholesterol, and LDL were significantly lower in Indian males than Chinese males. There were no significant ethnic differences in other antioxidant levels, including trans-retinol, alpha-tocopherol, and ascorbic acid. The consistently lower values of coenzyme Q10, especially its reduced form, in Indian males may contribute to the higher susceptibility of this ethnic group to coronary heart disease.