Biomarkers of Nutrition for Development (BOND)-Vitamin A Review.

The Biomarkers of Nutrition for Development (BOND) project is designed to provide evidence-informed advice to anyone with an interest in the role of nutrition in health. The BOND program provides information with regard to selection, use, and interpretation of biomarkers of nutrient exposure, status, function, and effect, which will be especially useful for readers who want to assess nutrient status. To accomplish this objective, expert panels are recruited to evaluate the literature and to draft comprehensive reports on the current state of the art with regard to specific nutrient biology and available biomarkers for assessing nutritional status at the individual and population levels. Phase I of the BOND project includes the evaluation of biomarkers for 6 nutrients: iodine, folate, zinc, iron, vitamin A, and vitamin B-12. This review of vitamin A is the current article in this series. Although the vitamin was discovered >100 y ago, vitamin A status assessment is not trivial. Serum retinol concentrations are under homeostatic control due in part to vitamin A's use in the body for growth and cellular differentiation and because of its toxic properties at high concentrations. Furthermore, serum retinol concentrations are depressed during infection and inflammation because retinol-binding protein (RBP) is a negative acute-phase reactant, which makes status assessment challenging. Thus, this review describes the clinical and functional indicators related to eye health and biochemical biomarkers of vitamin A status (i.e., serum retinol, RBP, breast-milk retinol, dose-response tests, isotope dilution methodology, and serum retinyl esters). These biomarkers are then related to liver vitamin A concentrations, which are usually considered the gold standard for vitamin A status. With regard to biomarkers, future research questions and gaps in our current understanding as well as limitations of the methods are described.

Current Capabilities and Limitations of Stable Isotope Techniques and Applied Mathematical Equations in Determining Whole-Body Vitamin A Status.

BACKGROUND: Retinol isotope dilution (RID) methodology provides a quantitative estimate of total body vitamin A (VA) stores and is the best method currently available for assessing VA status in adults and children. The methodology has also been used to test the efficacy of VA interventions in a number of low-income countries. Infections, micronutrient deficiencies (eg, iron and zinc), liver disease, physiological age, pregnancy, and lactation are known or hypothesized to influence the accuracy of estimating total body VA stores using the isotope dilution technique. OBJECTIVE: Our objectives were to review the strengths and limitations of RID methods, to discuss what is known about the impact of various factors on results, and to summarize contributions of model-based compartmental analysis to assessing VA status. METHODS: Relevant published literature is reviewed and discussed. RESULTS: Various equations and compartmental modeling have been used to estimate the total body VA stores using stable isotopes, including a newer 3-day equation that provides an estimate of total body VA stores in healthy adults. At present, there is insufficient information on absorption of the isotope tracer, and there is a need to further investigate how various factors impact the application of RID techniques in field studies. CONCLUSIONS: Isotope dilution methodology can provide useful estimates of total body VA stores in apparently healthy populations under controlled study conditions. However, more research is needed to determine whether the method is suitable for use in settings where there is a high prevalence of infection, iron deficiency, and/or liver disease.

Are dietary bioactives ready for recommended intakes?

Research has shown that numerous dietary bioactive components that are not considered essential may still be beneficial to health. The dietary reference intake (DRI) process has been applied to nonessential nutrients, such as fiber, yet the majority of bioactive components await a recommended intake. Despite a plethora of new research over the past several years on the health effects of bioactives, it is possible that the field may never reach a point where the current DRI framework is suitable for these food components. If bioactives are to move toward dietary guidance, they will likely require an alternative path to get there.

Application of Key Events Dose Response Framework to defining the upper intake level of leucine in young men.

Leucine is sold in large doses in health food stores and is ingested by weight-training athletes. The safety of ingestion of large doses of leucine is unknown. Before designing chronic high-dose leucine supplementation experiments, we decided to determine the effect of graded doses of leucine in healthy participants. The Key Events Dose Response Framework is an organizational and analytical framework that dissects the various biologic steps (key events) that occur between exposure to a substance and an eventual adverse effect. Each biologic event is looked at for its unique dose-response characteristics. For nutrients, there are a number of biologic homeostatic mechanisms that work to keep circulating/tissue levels in a safe, nontoxic range. If a response mechanism at a particular key event is especially vulnerable and easily overwhelmed, this is known as a determining event, because this event drives the overall slope or shape of the dose-response relationship. In this paper, the Key Events Dose Framework has been applied to the problem of leucine toxicity and leucine's tolerable upper level. After analyzing the experimental data vis a vis key events for leucine leading to toxicity, it became evident that the rate of leucine oxidation was the determining event. A dose-response study has been conducted to graded intakes of leucine in healthy human adult male volunteers. All participants were started at the mean requirement level of leucine [50 mg/(kg · d)] and the highest leucine intake was 1250 mg/( kg · d), which is 25 times the mean requirement. No gut intolerance was seen. Blood glucose fell progressively but remained within normal values without any changes in plasma insulin. Maximal leucine oxidation levels occurred at an intake of 550 mg leucine/( kg · d), after which plasma leucine progressively increased and plasma ammonia also increased in response to leucine intakes >500 mg/( kg · d). Thus, the "key determining event" appears to be when the participants reach their maximal leucine oxidation level, after which the risk of metabolic adverse effects progressively increased.

ß-Carotene in Golden Rice is as good as ß-carotene in oil at providing vitamin A to children.

BACKGROUND: Golden Rice (GR) has been genetically engineered to be rich in ß-carotene for use as a source of vitamin A. OBJECTIVE: The objective was to compare the vitamin A value of ß-carotene in GR and in spinach with that of pure ß-carotene in oil when consumed by children. DESIGN: Children (n = 68; age 6-8 y) were randomly assigned to consume GR or spinach (both grown in a nutrient solution containing 23 atom% ²H2O) or [²H8]ß-carotene in an oil capsule. The GR and spinach ß-carotene were enriched with deuterium (²H) with the highest abundance molecular mass (M) at M(ß-C)+²H10. [¹³C10]Retinyl acetate in an oil capsule was administered as a reference dose. Serum samples collected from subjects were analyzed by using gas chromatography electron-capture negative chemical ionization mass spectrometry for the enrichments of labeled retinol: M(retinol)+4 (from [²H8]ß-carotene in oil), M(retinol)+5 (from GR or spinach [²H10]ß-carotene), and M(retinol)+10 (from [¹³C10]retinyl acetate). RESULTS: Using the response to the dose of [¹³C10]retinyl acetate (0.5 mg) as a reference, our results (with the use of AUC of molar enrichment at days 1, 3, 7, 14, and 21 after the labeled doses) showed that the conversions of pure ß-carotene (0.5 mg), GR ß-carotene (0.6 mg), and spinach ß-carotene (1.4 mg) to retinol were 2.0, 2.3, and 7.5 to 1 by weight, respectively. CONCLUSIONS: The ß-carotene in GR is as effective as pure ß-carotene in oil and better than that in spinach at providing vitamin A to children. A bowl of ~100 to 150 g cooked GR (50 g dry weight) can provide ~60% of the Chinese Recommended Nutrient Intake of vitamin A for 6-8-y-old children.

Spirulina is an effective dietary source of zeaxanthin to humans.

Zeaxanthin is a predominant xanthophyll in human eyes and may reduce the risk of cataracts and age-related macular degeneration. Spirulina is an algal food that contains a high concentration of zeaxanthin. In order to determine the zeaxanthin bioavailability of spirulina for dietary supplementation in humans, spirulina was grown in nutrient solution with ²H2O for carotenoid labelling. Single servings of ²H-labelled spirulina (4.0-5.0 g) containing 2.6-3.7 mg zeaxanthin were consumed by fourteen healthy male volunteers (four Americans and ten Chinese) with 12 g dietary fat. Blood samples were collected over a 45 d period. The serum concentrations of total zeaxanthin were measured using HPLC, and the enrichment of labelled zeaxanthin was determined using LC-atmospheric pressure chemical ionisation-MS (LC-APCI-MS). The results showed that intrinsically labelled spirulina zeaxanthin in the circulation was detected at levels as low as 10 % of the total zeaxanthin for up to 45 d after intake of the algae. A single dose of spirulina can increase mean serum zeaxanthin concentration in humans from 0.06 to 0.15 µmol/l, as shown in our study involving American and Chinese volunteers. The average 15 d area under the serum zeaxanthin response curve to the single dose of spirulina was 293 nmol × d/µmol (range 254-335) in American subjects, and 197 nmol × d/µmol (range 154-285) in Chinese subjects. It is concluded that the relative bioavailability of spirulina zeaxanthin can be studied with high sensitivity and specificity using ²H labelling and LC-APCI-MS methodology. Spirulina can serve as a rich source of dietary zeaxanthin in humans.

Carotenoids and total phenolic contents in plant foods commonly consumed in Korea.

Phytochemicals are reported to provide various biological functions leading to the promotion of health as well as the reduced risk of chronic diseases. Fat-soluble plant pigments, carotenoids, are extensively studied micronutrient phytochemicals for their potential health benefits. It is noteworthy that specific carotenoids may be responsible for different protective effects against certain diseases. In addition, each carotenoid can be obtained from different types of plant foods. Considering the fact that the phytochemical content in foods can vary according to, but not limited to, the varieties and culture conditions, it is important to establish a database of phytochemicals in locally produced plant foods. Currently, information on individual carotenoid content in plant foods commonly consumed in Korea is lacking. As the first step to support the production and consumption of sustainable local plant foods, carotenoids and total phenolic contents of plant foods commonly consumed in Korea are presented and their potential biological functions are discussed in this review.

Combined α-tocopherol and ascorbic acid protects against smoke-induced lung squamous metaplasia in ferrets.

Many epidemiological studies show the benefit of fruits and vegetables on reducing risk of lung cancer, the leading cause of cancer death in the United States. Previously, we demonstrated that cigarette smoke exposure (SM)-induced lung lesions in ferrets were prevented by a combination of low dose of ß-carotene, α-tocopherol (AT), and ascorbic acid (AA). However, the role of a combination of AT and AA alone in the protective effect on lung carcinogenesis remains to be examined. In the present study, we investigated whether the combined AT (equivalent to ∼100 mg/day in the human) and AA (equivalent to ∼210 mg/day) supplementation prevents against SM (equivalent to 1.5 packs of cigarettes/day) induced lung squamous metaplasia in ferrets. Ferrets were treated for 6 weeks in the following three groups (9 ferrets/group): (i) Control (no SM, no AT+AA), (ii) SM alone, and (iii) SM+AT+AA. Results showed that SM significantly decreased concentrations of retinoic acid, AT, and reduced form of AA, not total AA, retinol and retinyl palmitate, in the lungs of ferrets. Combined AT+AA treatment partially restored the lowered concentrations of AT, reduced AA and retinoic acid in the lungs of SM-exposed ferrets to the levels in the control group. Furthermore, the combined AT+AA supplementation prevented SM-induced squamous metaplasia [0 positive/9 total ferrets (0%) vs. 5/8 (62%); p<0.05] and cyclin D1 expression (p<0.05) in the ferret lungs, in which both were positively correlated with expression of c-Jun expression. Although there were no significant differences in lung microsomal malondialdehyde (MDA) levels among the three groups, we found a positive correlation between MDA levels and cyclin D1, as well as c-Jun expressions in the lungs of ferrets. These data indicate that the combination of antioxidant AT+AA alone exerts protective effects against SM-induced lung lesions through inhibiting cyclin D1 expression and partially restoring retinoic acid levels to normal.

Economic analysis of nutrition interventions for chronic disease prevention: methods, research, and policy.

Increased interest in the potential societal benefit of incorporating health economics as a part of clinical translational science, particularly nutrition interventions, led the Office of Dietary Supplements at the National Institutes of Health to sponsor a conference to address key questions about the economic analysis of nutrition interventions to enhance communication among health economic methodologists, researchers, reimbursement policy makers, and regulators. Issues discussed included the state of the science, such as what health economic methods are currently used to judge the burden of illness, interventions, or healthcare policies, and what new research methodologies are available or needed to address knowledge and methodological gaps or barriers. Research applications included existing evidence-based health economic research activities in nutrition that are ongoing or planned at federal agencies. International and US regulatory, policy, and clinical practice perspectives included a discussion of how research results can help regulators and policy makers within government make nutrition policy decisions, and how economics affects clinical guideline development.

Yellow maize with high ß-carotene is an effective source of vitamin A in healthy Zimbabwean men.

BACKGROUND: The bioconversion efficiency of yellow maize ß-carotene to retinol in humans is unknown. OBJECTIVE: The objective of this study was to determine the vitamin A value of yellow maize ß-carotene in humans. DESIGN: High ß-carotene-containing yellow maize was grown in a hydroponic medium with 23 atom% (2)H(2)O during grain development. Yellow maize ß-carotene showed the highest abundance of enrichment as [(2)H(9)]ß-carotene. Eight healthy Zimbabwean men volunteered for the study. On day 1 after a fasting blood draw, subjects consumed 300 g yellow maize porridge containing 1.2 mg ß-carotene, 20 g butter, and a 0.5-g corn oil capsule. On day 8, fasting blood was drawn, and subjects consumed 1 mg [(13)C(10)]retinyl acetate in a 0.5-g corn oil capsule and 300 g white maize porridge with 20 g butter. Thirty-six blood samples were collected from each subject over 36 d. Concentrations and enrichments of retinol and ß-carotene in labeled doses and serum were determined with the use of HPLC, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. RESULTS: The area under the curve (AUC) of retinol from 1.2 mg yellow maize ß-carotene was 72.9 nmol · d, and the AUC of retinol from 1 mg retinyl acetate (13)C(10) was 161.1 nmol · d. The conversion factor of yellow maize ß-carotene to retinol by weight was 3.2 ± 1.5 to 1. CONCLUSION: In 8 healthy Zimbabwean men, 300 g cooked yellow maize containing 1.2 mg ß-carotene that was consumed with 20.5 g fat showed the same vitamin A activity as 0.38 mg retinol and provided 40-50% of the adult vitamin A Recommended Dietary Allowance. This trial was registered at clinicaltrials.gov as NCT00636038.

ß-Cryptoxanthin supplementation prevents cigarette smoke-induced lung inflammation, oxidative damage, and squamous metaplasia in ferrets.

In epidemiologic studies, high intake of ß-cryptoxanthin has been associated with a decreased risk of lung cancer, particularly among current smokers. However, data are not available from well-controlled animal studies to examine the effects of ß-cryptoxanthin on cigarette smoke-induced lung lesions, and the biological mechanisms by which ß-cryptoxanthin might affect lung carcinogenesis. We evaluated the effects of ß-cryptoxanthin supplementation on cigarette smoke-induced squamous metaplasia, inflammation, and changes in protein levels of proinflammatory cytokine [tumor necrosis factor alpha (TNFα)] and transcription factors [nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1)], as well as on smoke-induced oxidative DNA damage [8-hydroxy-2'-deoxyguanosine (8-OHdG)] in the lung tissue of ferrets. Thirty-six male ferrets were assigned to cigarette smoke exposure or no exposure and to low-dose, or high-dose ß-cryptoxanthin, or no dose (2 × 3 factorial design) for 3 months. ß-Cryptoxanthin supplementation dose-dependently increased plasma and lung ß-cryptoxanthin levels in ferrets, whereas cigarette smoke exposure lowered plasma and lung ß-cryptoxanthin levels. ß-Cryptoxanthin at both doses significantly decreased smoke-induced lung squamous metaplasia and inflammation. ß-Cryptoxanthin also substantially reduced smoke-elevated TNFα levels in alveolar, bronchial, bronchiolar, and bronchial serous/mucous gland epithelial cells and in lung macrophages. Moreover, ß-cryptoxanthin decreased smoke-induced activation of NF-κB, expression of AP-1 and levels of 8-OHdG. The beneficial effects of ß-cryptoxanthin were stronger for high-dose ß-cryptoxanthin than for low-dose ß-cryptoxanthin. Data from this study indicate that ß-cryptoxanthin provides a beneficial effect against cigarette smoke-induced inflammation, oxidative DNA damage and squamous metaplasia in the lungs.

Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and ß-cryptoxanthin by ferret carotene-9',10'-monooxygenase.

Xanthophyll carotenoids, such as lutein, zeaxanthin and ß-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,15'-monooxygenase (CMO1) has been shown to be involved in vitamin A formation, while recent studies suggest that carotene-9',10'-monooxygenase (CMO2) may have a broader substrate specificity than previously recognized. In this in vitro study, we investigated baculovirus-generated recombinant ferret CMO2 cleavage activity towards the carotenoid substrates zeaxanthin, lutein and ß-cryptoxanthin. Utilizing HPLC, LC-MS and GC-MS, we identified both volatile and non-volatile apo-carotenoid products including 3-OH-ß-ionone, 3-OH-α-ionone, ß-ionone, 3-OH-α-apo-10'-carotenal, 3-OH-ß-apo-10'-carotenal, and ß-apo-10'-carotenal, indicating cleavage at both the 9,10 and 9',10' carbon-carbon double bond. Enzyme kinetic analysis indicated the xanthophylls zeaxanthin and lutein are preferentially cleaved over ß-cryptoxanthin, indicating a key role of CMO2 in non-provitamin A carotenoid metabolism. Furthermore, incubation of 3-OH-ß-apo-10'-carotenal with CMO2 lysate resulted in the formation of 3-OH-ß-ionone. In the presence of NAD(+), in vitro incubation of 3-OH-ß-apo-10'-carotenal with ferret hepatic homogenates formed 3-OH-ß-apo-10'-carotenoic acid. Since apo-carotenoids serve as important signaling molecules in a variety of biological processes, enzymatic cleavage of xanthophylls by mammalian CMO2 represents a new avenue of research regarding vertebrate carotenoid metabolism and biological function.

Dietary lycopene and tomato extract supplementations inhibit nonalcoholic steatohepatitis-promoted hepatocarcinogenesis in rats.

Epidemiological and experimental studies provide supportive evidence that lycopene (LY), a major carotenoid from tomatoes and tomato products, may act as a chemopreventive agent against certain types of cancers. We recently showed that high-fat diet (HFD)-induced nonalcoholic steatohepatitis (NASH) promoted diethylnitrosamine (DEN)-initiated hepatocarcinogenesis in a rat model. Using this model, we investigated the efficacy of an equivalent dosage of dietary LY from either a pure compound or a tomato extract (TE) against NASH-promoted hepatocarcinogenesis. Six groups of rats were injected with DEN and then fed either Lieber-DeCarli control diet or HFD with or without LY or TE for 6 weeks. Results showed that both LY and TE supplementations significantly decreased the number of altered hepatic foci expressing the placental form of glutathione S-transferase in the livers of HFD-fed rats. This was associated with significantly lower proliferating cell nuclear antigen positive hepatocytes and cyclinD1 protein, as well as decreased activation of extracellular signal-regulated kinase and nuclear NF-kappaB. Although both LY and TE supplementations reduced HFD-induced lipid peroxidation in the livers, we observed significantly decreased cytochrome P450 2E1, inflammatory foci and mRNA expression of proinflammatory cytokines (TNF-alpha, IL-1beta and IL-12) in the HFD+TE fed group but increased nuclear NF-E2-related factor-2 and heme oxygenase-1 proteins in the HFD+LY fed group, relative to HFD feeding alone. These data indicate that LY and TE can inhibit NASH-promoted hepatocarcinogenesis mainly as a result of reduced oxidative stress, which could be fulfilled through different mechanisms.

Supplementation with lutein or lutein plus green tea extracts does not change oxidative stress in adequately nourished older adults.

Epigallocatechin gallate, a major component of green tea polyphenols, protects against the oxidation of fat-soluble antioxidants including lutein. The current study determined the effect of a relatively high but a dietary achievable dose of lutein or lutein plus green tea extract on antioxidant status. Healthy subjects (50-70 years) were randomly assigned to one of two groups (n=20 in each group): (1) a lutein (12 mg/day) supplemented group or (2) a lutein (12 mg/day) plus green tea extract (200 mg/day) supplemented group. After 2 weeks of run-in period consuming less than two servings of lightly colored fruits and vegetables in their diet, each group was treated for 112 days while on their customary regular diets. Plasma carotenoids including lutein, tocopherols, flavanols and ascorbic acid were analyzed by HPLC-UVD and HPLC-electrochemical detector systems; total antioxidant capacity by fluorometry; lipid peroxidation by malondialdehyde using a HPLC system with a fluorescent detector and by total hydroxyoctadecadienoic acids using a GC/MS. Plasma lutein, total carotenoids and ascorbic acid concentrations of subjects in either the lutein group or the lutein plus green tea extract group were significantly increased (P<.05) at 4 weeks and throughout the 16-week study period. However, no significant changes from baseline in any biomarker of overall antioxidant activity or lipid peroxidation of the subjects were seen in either group. Our results indicate that an increase of antioxidant concentrations within a range that could readily be achieved in a healthful diet does not affect in vivo antioxidant status in normal healthy subjects when sufficient amounts of antioxidants already exist.

Profiling histidine dipeptides in plasma and urine after ingesting beef, chicken or chicken broth in humans.

The in vitro metabolic stability of histidine-dipeptides (HD), carnosine (CAR) and anserine (ANS), in human serum, and their absorption kinetics after ingesting pure carnosine or HD rich foods in humans have been investigated. Healthy women (n = 4) went through four phases of taking one dose of either 450 mg of pure carnosine, 150 g beef (B), 150 g chicken (C), or chicken broth (CB) from 150 g chicken with a >2-week washout period between each phase. Blood samples were collected at 0, 30, 60, 100, 180, 240, and 300 min, and urine samples before and after (up to 7 h) ingesting pure carnosine or food. Both plasma and urine samples were analyzed for HD concentrations using a sensitive and selective LC-ESI-MS/MS method. CAR was undetectable in plasma after ingesting pure carnosine, B, C or CB. By contrast, plasma ANS concentration was significantly increased (P < 0.05) after ingesting C or CB, respectively. Urinary concentrations of both CAR and ANS were 13- to 14-fold increased after ingesting B, and 14.8- and 243-fold after CB ingestion, respectively. Thus, dietary HD, which are rapidly hydrolyzed by carnosinase in plasma, and excreted in urine, may act as reactive carbonyl species sequestering agents.