Use of a "Super-child" Approach to Assess the Vitamin A Equivalence of Moringa oleifera Leaves, Develop a Compartmental Model for Vitamin A Kinetics, and Estimate Vitamin A Total Body Stores in Young Mexican Children.

Background: Worldwide, an estimated 250 million children <5 y old are vitamin A (VA) deficient. In Mexico, despite ongoing efforts to reduce VA deficiency, it remains an important public health problem; thus, food-based interventions that increase the availability and consumption of provitamin A-rich foods should be considered.Objective: The objectives were to assess the VA equivalence of 2H-labeled Moringa oleifera (MO) leaves and to estimate both total body stores (TBS) of VA and plasma retinol kinetics in young Mexican children.Methods: ß-Carotene was intrinsically labeled by growing MO plants in a 2H2O nutrient solution. Fifteen well-nourished children (17-35 mo old) consumed puréed MO leaves (1 mg ß-carotene) and a reference dose of [13C10]retinyl acetate (1 mg) in oil. Blood (2 samples/child) was collected 10 times (2 or 3 children each time) over 35 d. The bioefficacy of MO leaves was calculated from areas under the composite "super-child" plasma isotope response curves, and MO VA equivalence was estimated through the use of these values; a compartmental model was developed to predict VA TBS and retinol kinetics through the use of composite plasma [13C10]retinol data. TBS were also estimated with isotope dilution.Results: The relative bioefficacy of ß-carotene retinol activity equivalents from MO was 28%; VA equivalence was 3.3:1 by weight (0.56 µmol retinol:1 µmol ß-carotene). Kinetics of plasma retinol indicate more rapid plasma appearance and turnover and more extensive recycling in these children than are observed in adults. Model-predicted mean TBS (823 µmol) was similar to values predicted using a retinol isotope dilution equation applied to data from 3 to 6 d after dosing (mean ± SD: 832 ± 176 µmol; n = 7).Conclusions: The super-child approach can be used to estimate population carotenoid bioefficacy and VA equivalence, VA status, and parameters of retinol metabolism from a composite data set. Our results provide initial estimates of retinol kinetics in well-nourished young children with adequate VA stores and demonstrate that MO leaves may be an important source of VA.

Identification of Enzymatic Cleavage Products of ß-Carotene-Rich Extracts of Kale and Biofortified Maize.

Provitamin A carotenoids in plant foods provide more than 80% of vitamin A intake for people in developing countries. Therefore, the conversion efficiency of ß-carotene to vitamin A is important, as it determines the effectiveness of plant foods as sources of vitamin A in humans. The objective of this study was to determine the effect of plant food antioxidants such as α-tocopherol, γ-tocopherol, α-tocotrienol, γ-tocotrienol and total γ-oryzanol on the cleavage of ß-carotene in vitro. Rat intestinal mucosa post mitochondrial fractions were incubated with ß-carotene-rich extracts of kale and biofortified maize for an hour at 37°C. Rat intestinal mucosa post mitochondrial fractions were also incubated with ß-carotene in the presence of either α-tocopherol, γ-tocopherol, α-tocotrienol, γ-tocotrienol or γ-oryzanol for 60 min at 37°C. The ß-carotene cleavage products were extracted and analyzed by an HPLC equipped with a C18 column at 340nm and 450nm. When ß-carotene alone was incubated without intestinal mucosa homogenate (control), no cleavage products were detected. When ß-carotene alone was incubated with intestinal mucosa homogenate, ß-apo-13-carotenone, ß-apo-14-carotenal, retinal, retinol and retinoic acid were formed. However, incubation of ß-carotene with either α-tocopherol, γ-tocopherol or α-tocotrienol resulted in a 10 fold inhibition of ß-apo-14-carotenal and ß-apo-13-carotenone formation. Antioxidant rich biofortified maize extract incubated with postmitochondrial fraction produced less ß-apo-13-carotenone compared to the kale extract. These results suggest that antioxidants inhibit the cleavage of ß-carotene and the formation of excentric cleavage products (ß-apo-13-carotenone, ß-apo-14-carotenal).

Genetic variation of carotenoids, vitamin E and phenolic compounds in Provitamin A biofortified maize.

BACKGROUND: Biofortified maize is not only a good vehicle for provitamin A carotenoids for vitamin A deficient populations in developing countries but also a source of vitamin E, tocochromanols and phenolic compounds, which have antioxidant properties. Using high-performance liquid chromatography and a total antioxidant performance assay, the present study analyzed the antioxidant variation and antioxidant activity of 36 provitamin A improved maize hybrids and one common yellow maize hybrid. RESULTS: The ranges of major carotenoids in provitamin A carotenoids biofortified maize were zeaxanthin [1.2-13.2 µg g-1 dry weight (DW)], ß-cryptoxanthin (1.3-8.8 µg g-1 DW) and ß-carotene (1.3-8.0 µg g-1 DW). The ranges of vitamin E compounds identified in provitamin A carotenoids biofortified maize were α-tocopherol (3.4-34.3 µg g-1 DW), γ-tocopherol (5.9-54.4 µg g-1 DW), α-tocotrienol (2.6-19.5 µg g-1 DW) and γ-tocotrienol (45.4 µg g-1 DW). The ranges of phenolic compounds were γ-oryzanol (0.0-0.8 mg g-1 DW), ferulic acid (0.4-3.6 mg g-1 DW) and p-coumaric acid (0.1-0.45 mg g-1 DW). There was significant correlation between α-tocopherol and cis isomers of ß-carotene (P < 0.01). Tocotrienols were correlated with α-tocopherol and γ-oryzanol (P < 0.01). CONCLUSION: Genotype was significant in determining the variation in ß-cryptoxanthin, ß-carotene, α-tocopherol and γ-tocopherol contents (P < 0.01). A genotype × environment interaction was observed for γ-tocopherol content (P < 0.01). © 2016 Society of Chemical Industry.

Retinaldehyde represses adipogenesis and diet-induced obesity.

The metabolism of vitamin A and the diverse effects of its metabolites are tightly controlled by distinct retinoid-generating enzymes, retinoid-binding proteins and retinoid-activated nuclear receptors. Retinoic acid regulates differentiation and metabolism by activating the retinoic acid receptor and retinoid X receptor (RXR), indirectly influencing RXR heterodimeric partners. Retinoic acid is formed solely from retinaldehyde (Rald), which in turn is derived from vitamin A. Rald currently has no defined biologic role outside the eye. Here we show that Rald is present in rodent fat, binds retinol-binding proteins (CRBP1, RBP4), inhibits adipogenesis and suppresses peroxisome proliferator-activated receptor-gamma and RXR responses. In vivo, mice lacking the Rald-catabolizing enzyme retinaldehyde dehydrogenase 1 (Raldh1) resisted diet-induced obesity and insulin resistance and showed increased energy dissipation. In ob/ob mice, administrating Rald or a Raldh inhibitor reduced fat and increased insulin sensitivity. These results identify Rald as a distinct transcriptional regulator of the metabolic responses to a high-fat diet.

Vitamin A value of plant food provitamin A - evaluated by the stable isotope technologies.

Humans need vitamin A and obtain essential vitamin A by conversion of plant foods rich in provitamin A and/or absorption of preformed vitamin A from foods of animal origin. The determination of the vitamin A value of plant foods rich in provitamin A is important but has challenges. The aim of this paper is to review the progress over last 80 years following the discovery on the conversion of ß-carotene to vitamin A and the various techniques including stable isotope technologies that have been developed to determine vitamin A values of plant provitamin A (mainly ß-carotene). These include applications from using radioactive ß-carotene and vitamin A, depletion-repletion with vitamin A and ß-carotene, and measuring postprandial chylomicron fractions after feeding a ß-carotene rich diet, to using stable isotopes as tracers to follow the absorption and conversion of plant food provitamin A carotenoids (mainly ß-carotene) in humans. These approaches have greatly promoted our understanding of the absorption and conversion of ß-carotene to vitamin A. Stable isotope labeled plant foods are useful for determining the overall bioavailability of provitamin A carotenoids from specific foods. Locally obtained plant foods can provide vitamin A and prevent deficiency of vitamin A, a remaining worldwide concern.

Vitamin A-fortified milk increases total body vitamin A stores in Mexican preschoolers.

Vitamin A (VA) deficiency (VAD) continues to be a major nutritional problem in developing countries, including Central America. In Mexico, milk is a well-accepted vehicle for the administration of micronutrients, including VA, to preschoolers. Thus, we conducted a randomized, controlled, clinical trial to investigate the efficacy of daily consumption of 250 mL of VA-fortified milk (which provided 196 retinol equivalents/d) for 3 mo on VA stores in mildly to moderately VAD (serum retinol concentration 0.35-0.7 µmol/L) preschoolers who were not enrolled in a food assistance program. Twenty-seven mildly to moderately VAD children were randomly assigned based on screening measurements to either the intervention (n = 14) or control group (n = 13) (children in the control group did not receive placebo). All children in the control group and 79% (n = 11) of the children in the intervention group completed the study. The total body VA (TBVA) pool size was estimated using the deuterated retinol dilution technique before and after the intervention. After 3 mo, median changes in the serum retinol concentration for the intervention and control groups were 0.13 and -0.21 µmol/L, respectively (P = 0.009). Median changes in the TBVA stores were 0.06 and 0.01 mmol, respectively (P = 0.006) and estimated median changes in the liver VA concentration were 0.09 and 0.01 µmol/g, respectively (P = 0.002). The VA-fortified milk was well accepted among preschoolers and significantly increased TBVA stores, liver VA stores, and serum retinol concentration, indicating that it may be an effective means to ameliorate VAD in young Mexican children.

Retinoids, carotenoids, and tocopherols in breast adipose tissue and serum of benign breast disease and breast cancer patients.

Various retinoic acid (RA) isomers (all-trans, 13-cis, 11-cis, and 9-cis) as well as retinol, carotenoids, and tocopherol concentrations were determined in both serum and breast adipose tissue of 22 benign breast disease patients and 52 breast cancer patients categorized into 4 stages by malignancy. Serum RA isomers were analyzed by a newly developed sensitive method combining a high-performance liquid chromatography and a gas chromatography-mass spectrometry, and retinol, carotenoid, and tocopherol concentrations using a high-performance liquid chromatography system. The breast cancer patients showed significantly lower serum retinol, whereas significantly higher breast adipose tissue retinol concentration than those of benign breast disease patients. Although breast cancer patients showed significantly higher serum all-trans and 13-cis RA concentrations, 11-cis RA in breast adipose tissue was significantly lower in the breast cancer patients than those of benign breast disease patients and it was associated with the stage of malignancy. The current study indicates that the retinol and RA isomers in the target tissue of breast tumor patients are not reflecting their concentrations in circulation. The mechanisms of tissue specific uptake of RA isomers and their functions warrant further studies.

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.

Plasma levels of retinoids, carotenoids and tocopherols in patients with mild obstructive sleep apnoea.

BACKGROUND AND OBJECTIVE: OSA is associated with increased incidence of cardiovascular diseases. Pathogenic mechanisms of vascular diseases include thickened vascular walls due to the increased number of smooth muscle cells (SMC). Retinoic acid (RA) suppresses the growth of SMC, and reduced retinoid levels are associated with vascular diseases. Oxidant signalling promotes SMC growth, thus antioxidant levels may also influence the development of cardiovascular diseases. The present study tested the hypothesis that plasmas from OSA patients contain altered levels of retinoids, carotenoids and tocopherols. METHODS: Plasma samples were taken before and after sleep from patients with OSA (mostly mild) without known cardiovascular diseases and from control subjects. Levels of retinoids, carotenoids and tocopherols were measured using sensitive gas chromatograph-mass spectrometry and high pressure liquid chromatography methods and total antioxidant capacity was assessed fluorometrically. RESULTS: Results showed that plasmas from patients with OSA had significantly lower retinyl palmitate and 9-cis RA compared with control subjects, while levels of retinol, all-trans RA and 13-cis RA were indifferent. All-transbeta-carotene and 9-cisbeta-carotene were also lower in OSA patients. Levels of all-trans RA and 13-cis RA in OSA patients were reduced after sleep compared with before sleep. OSA patients showed significantly higher delta-tocopherol compared with controls. Treatment of cultured human vascular SMC with post-sleep OSA patient plasmas promoted cell growth, but not in controls. CONCLUSIONS: Mild OSA exhibits altered levels of specific retinoids, carotenoids and tocopherols, which may be markers and/or mediators for the increased susceptibility of patients to vascular diseases.

Kinetic analysis shows that vitamin A disposal rate in humans is positively correlated with vitamin A stores.

Vitamin A (VA) kinetics, storage, and disposal rate were determined in well-nourished Chinese and U.S. adults using model-based compartmental analysis. [(2)H(8)]Retinyl acetate (8.9 micromol) was orally administered to U.S. (n = 12; 59 +/- 9 y; mean +/- SD) and Chinese adults (n = 14; 54 +/- 4 y) and serum tracer and VA concentrations were measured from 3 h to 56 d. Using the Windows version of the Simulation, Analysis and Modeling software, we determined that the average time from dosing until appearance of labeled retinol in serum was greater in U.S. subjects (40.6 +/- 8.47 h) than in Chinese subjects (32.2 +/- 5.84 h; P < 0.01). Model-predicted total traced mass (898 +/- 637 vs. 237 +/- 109 micromol), disposal rate (14.7 +/- 5.87 vs. 5.58 +/- 2.04 micromol/d), and system residence time (58.9 +/- 28.7 vs. 42.9 +/- 14.6 d) were greater in U.S. than in Chinese subjects (P < 0.05). The model-predicted VA mass and VA mass estimated by deuterated retinol dilution at 3 and 24 d did not differ. VA disposal rate was positively correlated with VA traced mass in Chinese (R(2) = 0.556), U.S. (R(2) = 0.579), and all subjects (R(2) = 0.808). Additionally, VA disposal rate was significantly correlated with serum retinol pool size (R(2) = 0.227) and retinol concentration (R(2) = 0.330) in all subjects. Our results support the hypothesis that VA stores are the principle determinant of VA disposal rate in healthy, well-nourished adults.

Determination of 9-cis beta-carotene and zeta-carotene in biological samples.

Concentrations of 9-cis beta-carotene (9-cis betaC) and zeta-carotene (zetaC) in biological samples may provide crucial information on the biological activities of these carotenoids. However, in high-performance liquid chromatography (HPLC) these carotenoids are often co-eluted. Therefore, there is an urgent need to develop a method for 9-cis betaC and zetaC quantitation. Both 9-cis betaC and zetaC have peak absorbance at 400 and 450 nm, respectively, whereas only 9-cis betaC has peak absorbance at 475 nm. We developed a HPLC method to quantitate 9-cis betaC and zetaC by using peak absorbance ratios. The 9-cis betaC/zetaC peak area was monitored at 475, 450 and 400 nm. The 9-cis betaC was quantified by using absorbance value at 475 nm; zetaC was then calculated from the 9-cis betaC/zetaC peak at 400 nm by subtracting 9-cis betaC contribution at 400 nm using the 400-nm/475-nm peak absorbance ratio of 9-cis betaC (0.39). This method was applied to determine 9-cis betaC and zetaC concentrations in serum and breast milk samples (n=12) from American lactating women and serum and breast adipose tissue samples (n=16) from Korean women with either benign or malignant breast tumors. 9-cis betaC concentrations in serum and breast milk of American women, and serum and adipose tissue of Korean women were 7.1+/-0.8 and 1.1+/-0.2 nM, and 15.6+/-1.1 nM and 0.2+/-0.1 nmol/g, respectively. zetaC concentrations in the above samples were 54.2+/-7.2 and 8.3+/-1.8 nM, and 49.0+/-3.9 nM and 0.3+/-0.1 nmol/g, respectively.

Asymmetric cleavage of beta-carotene yields a transcriptional repressor of retinoid X receptor and peroxisome proliferator-activated receptor responses.

beta-Carotene and its metabolites exert a broad range of effects, in part by regulating transcriptional responses through specific nuclear receptor activation. Symmetric cleavage of beta-carotene can yield 9-cis retinoic acid (9-cisRA), the natural ligand for the nuclear receptor RXR, the obligate heterodimeric partner for numerous nuclear receptor family members. A significant portion of beta-carotene can also undergo asymmetric cleavage to yield apocarotenals, a series of poorly understood naturally occurring molecules whose biologic role, including their transcriptional effects, remains essentially unknown. We show here that beta-apo-14'-carotenal (apo14), but not other structurally related apocarotenals, represses peroxisome proliferator-activated receptors (PPAR) and RXR activation and biologic responses induced by their respective agonists both in vitro and in vivo. During adipocyte differentiation, apo14 inhibited PPARgamma target gene expression and adipogenesis, even in the presence of the potent PPARgamma agonist BRL49653. Apo14 also suppressed known PPARalpha responses, including target gene expression and its known antiinflammatory effects, but not if PPARalpha agonist stimulation occurred before apo14 exposure and not in PPARalpha-deficient cells or mice. Other apocarotenals tested had none of these effects. These data extend current views of beta-carotene metabolism to include specific apocarotenals as possible biologically active mediators and identify apo14 as a possible template for designing PPAR and RXR modulators and better understanding modulation of nuclear receptor activation. These results also suggest a novel model of molecular endocrinology in which metabolism of a parent compound, beta-carotene, may alternatively activate (9-cisRA) or inhibit (apo14) specific nuclear receptor responses.

Determination of carotenoids in yellow maize, the effects of saponification and food preparations.

Maize is an important staple food consumed by millions of people in many countries. Yellow maize naturally contains carotenoids which not only provide provitamin A carotenoids but also xanthophylls, which are known to be important for eye health. This study was aimed at 1) evaluating the effect of saponification during extraction of yellow maize carotenoids, 2) determining the major carotenoids in 36 genotypes of yellow maize by high-performance liquid chromatography with a C30 column, and 3) determining the effect of cooking on the carotenoid content of yellow maize. The major carotenoids in yellow maize were identified as all-trans lutein, cis-isomers of lutein, all-trans zeaxanthin, alpha- and beta-cryptoxanthin, all-trans beta-carotene, 9-cis beta-carotene, and 13-cis beta-carotene. Our results indicated that carotenoid extraction without saponification showed a significantly higher yield than that obtained using saponification. Results of the current study indicate that yellow maize is a good source of provitamin A carotenoids and xanthophylls. Cooking by boiling yellow maize at 100 degrees C for 30 minutes increased the carotenoid concentration, while baking at 450 degrees F for 25 minutes decreased the carotenoid concentrations by almost 70% as compared to the uncooked yellow maize flour.

Doxorubicin as an antioxidant: maintenance of myocardial levels of lycopene under doxorubicin treatment.

The mechanism of doxorubicin-induced cardiotoxicity remains controversial. Wistar rats (n=96) were randomly assigned to a control (C), lycopene (L), doxorubicin (D), or doxorubicin+lycopene (DL) group. The L and DL groups received lycopene (5 mg/kg body wt/day by gavage) for 7 weeks. The D and DL groups received doxorubicin (4 mg/kg body wt intraperitoneally) at 3, 4, 5, and 6 weeks and were killed at 7 weeks for analyses. Myocardial tissue lycopene levels and total antioxidant performance (TAP) were analyzed by HPLC and fluorometry, respectively. Lycopene metabolism was determined by incubating (2)H(10)-lycopene with intestinal mucosa postmitochondrial fraction and lipoxygenase and analyzed with HPLC and APCI mass spectroscopy. Myocardial tissue lycopene levels in DL and L were similar. TAP adjusted for tissue protein were higher in myocardium of D than those of C (P=0.002). Lycopene metabolism study identified a lower oxidative cleavage of lycopene in D as compared to those of C. Our results showed that lycopene was not depleted in myocardium of lycopene-supplemented rats treated with doxorubicin and that higher antioxidant capacity in myocardium and less oxidative cleavage of lycopene in intestinal mucosa of doxorubicin-treated rats suggest an antioxidant role of doxorubicin rather than acting as a prooxidant.

Effect of lycopene on doxorubicin-induced cardiotoxicity: an echocardiographic, histological and morphometrical assessment.

Doxorubicin is an excellent chemotherapeutic agent utilized for several types of cancer but the irreversible doxorubicin-induced cardiac damage is the major limitation for its use. Oxidative stress seems to be associated with some phase of the toxicity mechanism process. To determine if lycopene protects against doxorubicin-induced cardiotoxicity, male Wistar rats were randomly assigned either to control, lycopene, doxorubicin or doxorubicin + lycopene groups. They received corn oil (control, doxorubicin) or lycopene (5 mg/kg body weight a day) (lycopene, doxorubicin + lycopene) by gavage for a 7-week period. They also received saline (control, lycopene) or doxorubicin (4 mg/kg) (doxorubicin, doxorubin + lycopene) intraperitoneally by week 3, 4, 5 and 6. Animals underwent echocardiogram and were killed for tissue analyses by week 7. Mean lycopene levels (nmol/kg) in liver were higher in the doxorubicin + lycopene group (5822.59) than in the lycopene group (2496.73), but no differences in lycopene were found in heart or plasma of these two groups. Lycopene did not prevent left ventricular systolic dysfunction induced by doxorubicin. However, morphologic examination revealed that doxorubicin-induced myocyte damage was significantly suppressed in rats treated with lycopene. Doxorubicin treatment was followed by increase of myocardium interstitial collagen volume fraction. Our results show that: (i) doxorubicin-induced cardiotoxicity was confirmed by echocardiogram and morphological evaluations; (ii) lycopene absorption was confirmed by its levels in heart, liver and plasma; (iii) lycopene supplementation provided myocyte protection without preventing interstitial collagen accumulation increase; (iv) doxorubicin-induced cardiac dysfunction was not prevented by lycopene supplementation; and (v) lycopene depletion was not observed in plasma and tissues from animals treated with doxorubicin.

Peanut butter increases the bioavailability and bioconversion of kale ß-carotene to vitamin A.

BACKGROUND AND OBJECTIVES: Kale is a rich source of provitamin A- ß-carotene. This study used intrinsically labeled kale [2H9] ß-carotene to determine the effect of peanut butter on the bioconversion of kale ß-carotene to vitamin A in preschool children. METHODS AND STUDY DESIGN: Preschool children (n=37; age 12-36 mo) were randomly assigned to 50 g cooked kale (1.5 mg ß-carotene content) with either 33 g peanut butter (PBG) or with 16 g lard (LG) and a reference dose of 1 mg [13C10] retinyl acetate capsule. Blood samples were processed to serum and analyzed by Negative Chemical Ionization-Gas Chromatography Mass Spectrometry (NCI-GCMS) for the enrichments of [2H] retinol from kale [2H9] ß-carotene and [13C10] retinol from reference dose. RESULTS: The area under curves (AUCs) of molar enrichment at days 1, 2, 3, 6, 15, and 21 after the labeled doses was 56.3±10.5 and 84.8±16.2 (nmole) for [2H] retinol from LG and PBG kale [2H9] ß-carotene, respectively. The AUC of [13C10] retinol from reference dose was 432.6±54.9 (LG) and 560.3±156.7 (nmole) (PBG), respectively. The calculated ß-carotene conversion factors were 13.4±3.1 and 11.0±3.9 to 1 (p>0.05) by weight for LG and PBG, respectively. CONCLUSIONS: This study showed that peanut butter enhances the vitamin A value of kale.

Retinoids and pulmonary hypertension.

BACKGROUND: Retinoic acid has antimitogenic effects on smooth muscle cells. Studies on the systemic circulation suggest that it may reduce vascular thickening. Relationships between retinoids and pulmonary hypertension/pulmonary vascular remodeling, however, have not been explored. Thus, the present study examined retinoid levels in plasma of patients with idiopathic pulmonary arterial hypertension and the effects of retinoic acid on human pulmonary artery smooth muscle cell growth. METHODS AND RESULTS: We measured retinoid levels by gas chromatograph-mass spectrometer technique in plasma of idiopathic pulmonary arterial hypertension patients and in age- and sex-matched healthy control subjects. Patients had significantly lower levels of all-trans retinoic acid and 13-cis retinoic acid than control subjects but similar 9-cis retinoic acid and retinol levels. In cultured human pulmonary artery smooth muscle cells, all-trans retinoic acid suppressed serotonin-induced cell growth. These cells were found to express the retinoid acid receptors RARalpha, RARbeta, RARgamma, RXRalpha, and RXRbeta. Gene array analysis showed that retinoic acid induces the expression of GADD45A, a known cell growth suppressor. Contrary to expectations, plasma from pulmonary hypertension patients suppressed cell growth, likely influenced by factors other than retinoids. CONCLUSIONS: Idiopathic pulmonary arterial hypertension patients have reduced retinoic acid levels, and retinoic acid treatment can elicit growth-inhibitory signals in pulmonary artery smooth muscle cells in vitro. Thus, retinoic acid may influence pulmonary vascular remodeling in humans.

Spinach or carrots can supply significant amounts of vitamin A as assessed by feeding with intrinsically deuterated vegetables.

BACKGROUND: The vitamin A value of spinach and carrots needs to be measured directly. OBJECTIVE: The objective was to determine the vitamin A value of intrinsically labeled dietary spinach and carrots in humans. DESIGN: Spinach and carrots were intrinsically labeled by growing these plants in 25 atom% 2H2O nutrient solution. Growth in this medium yielded a range of trans beta-carotene (tbeta-carotene) isotopomers with a peak enrichment at molecular mass plus 10 mass units. Seven men with a mean (+/-SD) age of 59.0 +/- 6.3 y and a body mass index (in kg/m2) of 25.7 +/- 1.5 consumed puréed spinach (300 g, 20.8 micromol tbeta-carotene equivalents) or carrots (100 g, 19.2 micromol tbeta-carotene equivalents) with a standardized liquid diet (no extra fiber) in random order 4 mo apart. Seven women with a mean (+/-SD) age of 55.5 +/- 6.3 y and a body mass index of 26.4 +/- 4.2 consumed puréed spinach only (300 g, 20.0 micromol tbeta-carotene equivalents). A reference dose of [13C8]retinyl acetate (8.9 micromol) in oil was given to each subject 1 wk after each vegetable dose. Blood samples were collected over 35 d. RESULTS: Areas under the curve for total labeled serum beta-carotene responses were 42.4 +/- 8.5 nmol.d per micromol spinach beta-carotene and 119.8 +/- 23.0 nmol.d per micromol carrot beta-carotene (P < 0.01). Compared with the [13C8]retinyl acetate reference dose, spinach tbeta-carotene conversion to retinol was 20.9 +/- 9.0 to 1 (range: 10.0-46.5 to 1) and carrot tbeta-carotene conversion to retinol was 14.8 +/- 6.5 to 1 (range: 7.7-24.5 to 1) by weight. CONCLUSIONS: Spinach and carrots can provide a significant amount of vitamin A even though the amount is not as great as previously proposed. Food matrices greatly affect the bioavailability of plant carotenoids, their efficiency of conversion to vitamin A, or both.

Bioavailability of synthetic and biosynthetic deuterated lycopene in humans.

Current knowledge of the bioavailability of lycopene in humans is limited due to the inability to distinguish newly administered lycopene from the body reserves of lycopene. A quantitative method to assess the absorption and relative bioavailability of newly absorbed synthetic or natural lycopene was developed using two deuterated lycopene sources, in conjunction with an advanced LC/APCI-MS (liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry) to analyze newly absorbed lycopene in blood samples of study subjects. Two subjects (1 male and 1 female) consumed hydroponically grown tomatoes containing deuterium-enriched lycopene (80-84 g wet weight tomato containing 16.3 and 17.4 micromol lycopene, respectively) and two subjects (1 male, and 1 female) consumed 11 micromol synthetic (2)H(10) lycopene in 6 g of corn oil. Tomatoes were steamed and pureed. The doses were given together with a liquid formulated drink with 25% energy from fat. Our results showed that up to 34 days after taking an oral (2)H(10) lycopene dose (synthetic or from tomato) with a liquid formula drink, the area under the curve of the average serum percent enrichment response of synthetic lycopene reached 33.9 (+/-1.7) nmol-day/micromol lycopene in the dose, whereas that of lycopene from the tomato dose was 11.8 (+/-0.3) nmol-day/mumol lycopene in the dose. Our study provides evidence that the absorption of physiological levels of lycopene in intrinsically labeled tomatoes can be studied in humans. From these preliminary investigations, we find that the bioavailability of synthetic lycopene in oil appears to be about three times higher than that of lycopene from steamed and pureed tomatoes.

Structure determination of partially deuterated carotenoids from intrinsically labeled vegetables by HPLC-MS and 1H NMR.

The structures of biosynthetic deuterated carotenoids in labeled vegetables were investigated: (all-E)-lutein and (all-E)-beta-carotene from spinach, and (all-E)-beta-carotene and (all-E)-alpha-carotene from carrots. The vegetables were grown hydroponically using a nutrient solution enriched with deuterium oxide (D(2)O) and were extracted using matrix solid-phase dispersion (MSPD). Deuterium enrichment in the carotenoid molecules was determined by liquid chromatography-mass spectrometry (LC-MS). (all-E)-Lutein and (all-E)-beta-carotene in spinach showed partial deuteration from (2)H(1) to (2)H(12), with the abundance maximum at (2)H(5). (all-E)-beta-Carotene and (all-E)-alpha-carotene from carrots showed partial deuteration from (2)H(1) to (2)H(17), with the abundance maximum at (2)H(11). The (1)H NMR spectra of the four deuterated carotenoids showed additional signals for all methyl groups and decreased signal intensity for the olefinic protons and the methylene protons in the ring. These differences are due to isotopic effects and are based on the substitution of protons by deuterium atoms. The deuteration was distributed randomly throughout the carotenoid molecules.