Oxygen requirements for vitamin K-dependent carboxylation and epoxide formation.

The requirement of vitamin K-dependent carboxylation for oxygen was determined. Carboxylation was not detected at oxygen concentrations less than 0.05 mM or in the absence of vitamin K epoxide formation. Epoxide formation was detectable at 0.05 mM and was maximal at 0.10 mM O2. Carboxylation increased with oxygen concentrations over the range of 0.10 to 0.25 mM. At oxygen concentrations at which epoxide formation was maximal, the ratio of epoxide formation to carboxylated product was approx. 3.5:1. The data are consistent with the hypothesis that an oxygenated vitamin K intermediate is required for carboxylation.

Vitamin K in colostrum and mature human milk over the lactation period--a cross-sectional study.

Vitamin K was quantitated in the milk of four groups of 15 mothers from 1 d to 6 mo postpartum in a cross-sectional study. Concentrations were 7.52 +/- 5.90 and 6.36 +/- 5.32 nmol/L (3.39 +/- 2.66 and 2.87 +/- 2.40 micrograms/L) in colostrum and mature milk, respectively. Differences between colostrum and mature milk or among samples of mature milk collected at 1, 3, and 6 mo were not statistically significant. Because of significantly increased volumes of milk over the lactation period, approximately twice as much vitamin K was delivered in mature milk as in colostrum. Within normal ranges, concentrations of vitamin K in milk were not predicted by dietary intake of vegetables or fat. Vitamin K was correlated with fat in colostrum and was localized in the lipid core of the milk fat globule but was not associated with membranes. Vitamin K in human milk is insufficient to meet recommended intakes for infants aged less than 6 mo. Population and clinical studies are needed to assess the vitamin K status of exclusively breast-fed infants and to evaluate current recommendations.

beta-Carotene in breast milk and serum is increased after a single beta-carotene dose.

Normal lactating mothers were administered a single dose of 60 or 210 mg beta-carotene and changes in serum and milk retinol, alpha-tocopherol, and carotenoids were monitored for 8 d. Average serum beta-carotene concentrations increased 4.1- and 4.0-fold after the 60- and 210-mg doses, respectively. Milk beta-carotene concentrations increased 4.1- and 3.0-fold after the 60- and 210-mg doses, respectively. Maximum serum concentrations were reached 24 h after both supplements, although concentrations of milk beta-carotene continued to rise for 2-3 d. After 8 d, both serum and milk beta-carotene continued to rise for 2-3 d. After 8 d, both serum and milk beta-carotene concentrations remained about twofold higher than baseline concentrations. Increases in serum or milk beta-carotene concentrations were not dose-dependent. Initial serum and milk concentrations of beta-carotene predicted increases after supplementation, and increases in serum beta-carotene concentrations predicted those in milk. Concentrations of milk carotenoids were less than one-tenth their respective concentrations in serum. Lutein, beta-cryptoxanthin, lycopene, alpha-carotene, retinol, and alpha-tocopherol concentrations in serum or milk did not change significantly after beta-carotene supplementation. Retinol esters account for most of the retinol equivalents in the milk of well-nourished mothers. Initial and maximum concentrations of beta-carotene in serum and milk were strongly correlated for individual mothers. Collectively, the data showed that a single 60-mg supplement of beta-carotene sustained elevated beta-carotene concentrations in serum and milk for > 1 wk in normal mothers but did not affect concentrations of other major carotenoids, retinol, or alpha-tocopherol.

Plasma response of children to short-term chronic beta-carotene supplementation.

Plasma concentrations of beta-carotene and retinol after supplementation of children with beta-carotene in supplements and in foods were compared in Guatemalan children. The children received 6 mg/d beta-carotene [1000 retinol equivalents (RE)] as purified supplements or as carrots for 20 d and the effects on plasma beta-carotene and retinol concentrations were assessed. Plasma concentrations of beta-carotene were increased by 0.59 +/- 0.65 and 0.60 +/- 0.67 mumol/L after supplementation with beta-carotene capsules for 10 and 20 d, respectively. Addition of cooked carrots to the diet resulted in no significant change in plasma beta-carotene. Fasting plasma retinol concentrations were 1.23 +/- 0.35 mumol/L and were not significantly changed in response to supplementation with either of the carotene treatments with retinyl palmitate (1000 RE/d) for 20 d. Possible explanations for the differences in effects of plasma concentrations between the two methods of supplementation are discussed.

Kinetics of the response of milk and serum beta-carotene to daily beta-carotene supplementation in healthy, lactating women.

Changes in concentrations of milk and serum carotenoids, retinol, and alpha-tocopherol of five healthy, well-nourished, lactating women were measured over a 28-d supplementation trial with 30 mg beta-carotene and for 4 wk thereafter. Beta-carotene supplementation increased mean beta-carotene concentrations in milk and serum 6.4- and 7.4-fold, respectively. Concentrations of other major carotenoids, retinol, and alpha-tocopherol did not change substantially in either milk or serum. Uptake of beta-carotene into both serum and milk followed apparent first-order kinetics, occurring more rapidly into serum (t(1/2) = 5.5 d) than into milk (t(1/2) = 9 d). After supplementation, milk and serum beta-carotene concentrations decayed slowly, reaching approximately twofold initial concentrations by 4 wk. Kinetics of uptake and decay in milk beta-carotene concentrations paralleled those in serum. The data show that short-term supplementation of healthy, lactating mothers with purified beta-carotene at approximately fivefold the average daily dietary intake substantially increased milk and serum beta-carotene concentrations while not interfering with concentrations of other carotenoids, retinol, or alpha-tocopherol in milk or serum. Thus, an increased intake of beta-carotene by healthy, lactating women increases the supply of milk beta-carotene available to their breast-feeding infants.

Plasma response to oral beta-carotene in Guatemalan schoolchildren.

The response to oral doses of beta-carotene (0 mg, n = 10; 15 mg, n = 20; and 30 mg, n = 21) was studied in 51 Guatemalan children aged 8-15 y, with mean fasting plasma retinol concentrations of 1.72 +/- 0.38 mumol/L. Beta-carotene was delivered with a chocolate drink containing 8.4 g fat. Serial blood sampling was performed at intervals up to 48 h. Circulating retinol concentrations remained relatively constant. The maximum increases in plasma beta-carotene after the 30- and 15-mg doses for all subjects occurred at 24 h and were 0.29 and 0.23 mumol/L, respectively. Time of maximum increase for individuals varied and average maxima over the 48-h period for individuals were 0.13 and 0.26 mumol/L for the 15- and 30-mg-treatment groups, respectively. Increased plasma beta-carotene concentrations were not predicted by recent intake of dietary vitamin A, fasting plasma concentrations, or anthropometric measurements.

Antioxidant nutrients: associations with persistent human papillomavirus infection.

Research from the past several years has definitively shown intermediate and high risk-type human papillomavirus (HPV) infection to play a significant role in cervical carcinogenesis. Persistent compared with intermittent infection appears to confer an elevated risk, and cofactors may be necessary to allow the virus to progress to cervical cancer. We explored the association between circulating concentrations of the antioxidant nutrients (alpha- and beta-carotene, lutein, lycopene, beta-cryptoxanthin, alpha-tocopherol, gamma-tocopherol, and ascorbate) and persistent HPV infection among 123 low-income Hispanic women who were all nonsmokers and were not currently using vitamin and mineral supplements. In addition, the association between these nutrients and grade of cervical pathology, independent of HPV status, was assessed. Intermediate and high risk-type HPV infection was assessed by the Digene Hybrid Capture System at two time points, 3 months apart. At the second interview, cytology, colposcopy, and a fasting blood draw were conducted. Mean concentrations of serum and plasma antioxidant nutrients were calculated within categories of HPV status (two times HPV negative, one time HPV positive, and two times HPV positive) and colposcopy. Adjusted mean concentrations of serum beta-carotene, beta-cryptoxanthin, lutein, and alpha- and gamma-tocopherol were on average 24% (P < 0.05) lower among women two times HPV positive compared with either two times HPV negative or one time HPV positive. Independent of HPV status, alpha-tocopherol was significantly inversely associated with grade of cervical dysplasia (normal, 21.57 microM; cervical intraepithelial neoplasia III, 17.27 microM). The results obtained in this study need to be confirmed in larger cohort studies with a longer follow-up period.

Short-term beta-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

Carotenoids of human colostrum.

Colostrum, the initial postpartum secretion of the breast, ordinarily has a distinct yellow color due to carotenoids of its fat globules. This pigmentation progressively diminishes as milk production increases during the first week of lactation. Identity of these carotenoids was investigated by means of thin-layer chromatography, high performance liquid chromatography and spectral analysis. Alpha- and beta-carotene, lycopene and beta-cryptoxanthin were revealed as major chromogens. A component corresponding to lutein and/or zeaxanthin was also detected by both chromatographic techniques. Extracts of 23 saponified colostrum samples from 10 donors revealed considerable variation in total carotenoid concentration (0.34-7.57 micrograms/ml of colostrum). Multiparous mothers had greater mean colostrum carotenoid concentrations than did the primiparae, 2.18 +/- 1.94 vs 1.14 +/- 1.32 micrograms/ml, respectively. Seven of the eight primiparous donors' samples had little or no yellow color. These findings imply a difference in carotenoid transport by breasts that have lactated as compared to those that have not. The interrelation of carotenoids, lactation and breast cancer is discussed.

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-partum. 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.

Vitamin K-dependent oxygenase/carboxylase; differential inactivation by sulfhydryl reagents.

Inhibition of vitamin K-dependent carboxylase and oxygenase by sulfhydryl reagents was compared. Formation of vitamin K epoxide and vitamin K-dependent carboxylation are both strongly (greater than 90%) inhibited by l mM p-hydroxy-mercuribenzoate, and this inhibition is reversed by dithiothreitol. Both activities are also effectively inhibited by N-ethylmaleimide (NEM). Preincubation with vitamin K hydroquinone prevents NEM inhibition of epoxide formation but not of carboxylation. These data argue that separate active sites are required to support vitamin K-dependent epoxide formation and carboxylation and that the binding site vitamin K oxygenase contains an active thiol group.

State of the art vitamin K in human milk.

Breast-feeding is the sole source of vitamin K for most of the world's 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 world's infants, HDNB remains a very real threat to the health of infants and warrants concentrated study.

Reaction of vitamin K and dithiothreitol on reversed-phase C18 high-performance liquid chromatographic columns.

A non-enzymatically catalyzed side reaction between dithiothreitol and vitamin K occurs on reversed-phase C18 high-performance liquid chromatographic columns. Excess dithiothreitol may be effectively removed from enzymatic reaction mixtures with N-ethylmaleimide. A low polarity gradient elution system is reported which gives quantitative recoveries (greater than 95%) of the vitamins in a concentration range useful for analysis of enzymatically catalyzed reactions.

Vitamin K-dependent carboxylase: evidence for a semiquinone radical intermediate.

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, Phe-Leu-Glu-Glu-Ile. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.

Effect of low lysine diet on rat liver nuclear metabolism.

Prolonged dietary lysine deficiency induced by feeding male weanling rats a diet low in lysine for 6 weeks resulted in substantial impairment of growth as compared with control rats and in alterations in the morphology of the nucleus of hepatocytes. Electronmicrographs of liver nuclei from lysine deficient rats showed an increased incidence of binucleated cells, reduction in numbers of nucleoli, changes in the position and size of nucleoli within the nucleus and altered patterns of chromatin aggregation in lysine deficient rats. Mass ratios of nonhistone proteins to DNA in liver chromatin from lysine deficient rats were higher than those of controls and electrophoretic patterns of chromatin nonhistone proteins were altered. In particular, one high molecular weight fraction of these proteins showing the same mobility as myosin was substantially enriched. The histone: DNA ratio and histone electrophoretic patterns, however, appeared to be unchanged. Incorporation of [3H] thymidine into DNA was increased, but the nuclear content of DNA was unchanged in lysine deficient animals.