Louise M. Canfield

Carotenoids as Cellular Antioxidants

Abstract Consumption of carotenoids is associated with an enhanced immune response and protection against neoplasia and atherosclerosis. Because these effects have been achieved using carotenoids with no pro-vitamin A activity, they are assumed to be due to the antioxidant properties of carotenoids. Carotenoids protect against photosensitized oxidation by quenching singlet oxygen. In addition, β-carotene reacts chemically with peroxyl radicals to produce epoxide and apocarotenal products. To investigate the potential significance of these reactions to biological systems, we have used soybean lipoxygenase to generate peroxyl radical enzymatically. β-Carotene inhibits the oxidation of linoleic acid by soybean lipoxygenase as well as the formation of the hydroperoxide product. In addition, the absorption of β-carotene is diminished (bleached) by soybean lipoxygenase. The potential significance of these antioxidant reactions of carotenoids to biological function is discussed.

Red palm oil in the maternal diet increases provitamin A carotenoids in breastmilk and serum of the mother-infant dyad

Background Despite vitamin A supplementation programs, vitamin A deficiency in children remains a public health concern in Honduras. Aim of the study We investigated the effectiveness of short-term dietary supplementation of mothers with red palm oil as a strategy for improving the vitamin A status of the mother-infant dyad. Methods Lactating mothers in Colonia Los Pinos, a barrio of Tegucigalpa, Honduras, consumed a total of 90-mg β-carotene as red palm oil (n = 32) supplements (n = 36) or placebo (n = 18) in six equal doses over 10 days. Carotenoids and retinol in maternal and infant serum, and breastmilk carotenoids and retinol were measured before and after supplementation. Maternal diet was evaluated by 24-hour recall. Results Maternal serum α-carotene and β-carotene concentrations were increased 2 fold by palm oil compared with 1.2 fold by β-carotene supplements. Changes were significantly different in infant serum α-carotene but not β-carotene among the three experimental groups. Increases in breastmilk β-carotene were greater for the palm oil group (2.5 fold) than for the β-carotene supplement group (1.6 fold) and increases in milk α-carotene concentrations (3.2 fold) were slightly greater than those of β-carotene. There were also small but significant changes among groups in breastmilk lutein and lycopene. Breastmilk retinol was not significantly different among the groups over the treatment period. Conclusions Red palm oil in the maternal diet increases provitamin A carotenoids in breastmilk and serum of the mother-infant dyad. The use of dietary red palm oil to improve the vitamin A status of this population should be further investigated.

Simultaneous quantitation and separation of carotenoids and retinol in human milk by high-performance liquid chromatography.

Publisher Summary This chapter discusses methods for separation, determination, and quantitation of the major carotenoids (and retinol) in mature breast milk. Two methods are presented in the chapter: (1) a saponification procedure that converts all the retinyl esters to free retinol, facilitating the simultaneous quantitation of total retinol and β-carotene and (2) a saponification procedure for the separation and quantitation of the more labile carotenoids α-carotene and lycopene. Carotenoids have received the attention of researchers in various fields because of the role carotenoids play as precursors of retinol, as antioxidants, and as effectors of immune function. The effects of β -carotene on the immune system is of particular importance to infants, as these infants rely almost entirely on breast milk as a source of nutrients. This chapter presents two sample preparation methods for the analysis of carotenoids and retinol in mature breast milk by high-performance liquid chromatography (HPLC): (1) a method for the simultaneous analysis of retinol and β-carotene and (2) a method for the analysis of the more labile carotenoids, such as lycopene and α-carotene. These methods can reproduce quantitate β carotene, α-carotene, lycopene, and lutein. Depending on the HPLC system utilized, total xanthophylls can be further separated, generating values for β -cryptoxanthin and zeaxanthin.

Antioxidant activity of carotenoids: An electron-spin resonance study on ?-carotene and lutein interaction with free radicals generated in a chemical system

β‐Carotene is thought to be a chain‐breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron‐spin resonance (ESR) spectroscopy coupled to the spin‐trapping technique, we have studied the effect of β‐carotene and lutein on the radical adducts of the spin‐trap PBN (N‐t ‐butyl‐α‐phenylnitrone) generated by the metal‐ion breakdown of different tert ‐butyl hydroperoxide (t BOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of t BOOH (1 M), were quenched by β‐carotene or lutein, in competition with the spin‐trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low t BOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by β‐carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of α‐tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical‐trapping activity of β‐carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems.

Quantitation of and inter/intra-individual variability in major carotenoids of mature human milk

Abstract The quantitation of carotenoids from milk has been technically challenging due to the small quantities present, insolubility and instability of the carotenoids, and significant inter- and intra-individual variation. Here we report methods for extraction and quantitation by high pressure liquid chromatography of the major carotenoids, β-Cryptoxanthin, lycopene, α-carotene, and β-carotene in a normal population of lactating women. Methodology to evaluate and protocol to accommodate the significant variation in carotenoids of human milk is also presented. Carotenoids present in the highest concentrations in breastmilk of these mothers were lycopene and β-carotene at 31.2 and 45.9 n m respectively, α-carotene and β-carotene concentrations in this population of mothers showed the greatest variation, ranging as much as 20-fold between individuals. The remaining carotenoids varied fourfold to sixfold across individuals. Within individuals, carotenoid concentrations varied from twofold to fivefold when measured on separate days. For an individual on a single day, breastmilk carotenoid concentrations vary as much as fourfold and are strongly correlated with lipid concentrations. For routine field collections, a collection protocol was developed that will approximate 24-hr collections of a population of mothers within 10%.

Enzymatic hydrolysis, extraction, and quantitation of retinol and major carotenoids in mature human milk 1

Abstract An improved method for enzymatic hydrolysis, extraction, and high performance liquid chromatography (HPLC) separation of major carotenoids and retinol of mature human milk is described. After pretreatment with bile salts and protease, carotenoids and retinol are released by lipase treatment followed with brief chemical saponification. The HPLC method provides enhanced peak resolution and improved elution profiles. The procedure is nondestructive to provitamin-A carotenoids while effectively hydrolyzing retinyl esters. Using this method, we were able to significantly increase the recovery of both β-carotene and retinol from human milk over previously published procedures. The method is sensitive to picomolar quantities of carotenoids and retinol. Retinol, lutein/zeaxanthin, β-cryptoxanthin, lycopene, α-carotene, and β-carotene are effectively recovered and quantitated from a single 1-mL sample of milk.

Inhibition of growth and cholesterol synthesis in breast cancer cells by oxidation products of β-carotene

Abstract We have isolated and chemically characterized a polar oxidation product of β-carotene and tested the effect of a highly enriched fraction containing this compound on the growth and metabolism of breast cancer (MCF-7) cells. This fraction strongly inhibits cell growth and cholesterol synthesis in MCF-7 cells. Pretreatment of the cells with mevalonate overcomes inhibition of cell growth by the oxidized fraction. Addition of the antioxidant butylated hydroxytoluene protects against inhibition of the growth of MCF-7 cells by β-carotene but not by the oxidized fraction. Pretreatment of cells with mevalonate overcomes inhibition of cell growth by oxidation products of β-carotene but not by retinoic acid. The oxidized fraction neither stimulates activity nor inhibits binding of retinoic acid to its nuclear receptors (RXR-α, RXR-β, RXR-γ, RAR-α, RAR-β, RAR-γ, and peroxisome proliferation receptors) in transfection assays. Mevalonate does not protect retinoic acid-induced growth inhibition of MCF-7 cells. The major compound in the inhibitory fraction was identified by mass spectrometry and nuclear magnetic resonance spectroscopy as 5,8-endoperoxy-2,3-dihydro-β-apocarotene-13-one. Our data suggest that the β-carotene oxidation products we have isolated represent a class of compounds not previously described with potential antineoplastic activity.

State of the art vitamin K in human milk

Breast-feeding is the sole source of vitamin K for most of the worlds children and breast-fed infants are at risk for vitamin K-responsive hemorrhagic disease of the newborn (HDNB). Recent advances in high performance liquid chromatography methodology have made possible the first quantitative studies of vitamin K in human milk. Although much progress has been made, much remains to be done. Innovative improvements in methodology are needed, as detection of nanogram quantities of vitamin K in milk is at the limit of current methodology. Additional studies are needed over the lactation period. A better understanding of colostrum is needed with regard to other nutrients as well as vitamin K. Vitamin K in the milk of mothers who gave birth prematurely has not been measured. The significance of menaquinones as a vitamin K source to the infant is undetermined. The mechanism regulating vitamin K secretion into milk has not been investigated. The localization of vitamin K in milk is undetermined as is the relationship of vitamin K to other milk lipids. The effects of fat-soluble vitamins in the diet on vitamin K concentrations in milk is unknown. The pharmacokinetics of vitamin K supplementation of mothers is particularly important in cultures where vitamin K is not routinely administered at birth. Finally, most critical at this point is our ignorance about the relationship of the maternal vitamin K status to the vitamin K status of the infant. As breast milk is the sole source of vitamin K for most of the worlds infants, HDNB remains a very real threat to the health of infants and warrants concentrated study.

Quantitation of vitamin K in human milk

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co-migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K1 was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post-partrum. Vitamin K1 was present at 2.94±1.94 and 3.15±2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.

Short-term β-carotene supplementation of lactating mothers consuming diets low in vitamin A☆

We have previously shown that beta-carotene supplementation of the diets of healthy U.S. mothers increases serum and milk beta-carotene concentrations. Building on these results, we investigated the possibility that beta-carotene supplementation could enhance the vitamin A status of mothers and their nursing infants. Three 30-mg doses of beta-carotene were administered on 3 consecutive days to 44 lactating mothers who had vitamin-A-poor diets. Concentrations of maternal serum and milk carotenoids and retinol were evaluated at baseline and after 2 and 3 days of supplementation. Infant serum carotenoids and retinol were measured at baseline and 2 days following maternal supplementation. beta-Carotene supplementation markedly elevated maternal serum and milk beta-carotene concentrations (nine- and sevenfold, respectively) and resulted in smaller, transient increases of alpha-carotene, lycopene, and beta-cryptoxanthin concentrations in maternal serum. Maternal serum and milk retinol were unchanged in response to the treatment. In contrast, maternal beta-carotene supplementation significantly increased infant serum retinol (P </= 0.001) and beta-carotene concentrations remained unchanged. These results imply that breast milk beta-carotene can supply retinol for the nursing infant. Further research is needed to identify the site of bioconversion of milk-derived beta-carotene to retinol and to describe the factors that regulate this process.