Vitamin K status of premature infants: implications for current recommendations.

OBJECTIVE: Newborn infants are vitamin K deficient. Vitamin K status in full-term infants after intramuscular vitamin K supplementation at birth has been described. Similar information in growing premature infants has not been reported. The objective of this study was to assess vitamin K status in premature infants by measuring plasma vitamin K and plasma protein-induced in vitamin K absence (PIVKA II) from birth until 40 weeks' postconceptional age. METHODS: Premature infants (/=1000 g) via total parenteral nutrition. After hyperalimentation, most received vitamin K-fortified enteral feedings with the remainder receiving unfortified breast milk. Blood was obtained for PIVKA II in cord blood and for PIVKA II and vitamin K at 2 weeks and 6 weeks after birth and at 40 weeks' postconception. RESULTS: Of the 44 infants enrolled, 10 infants in each gestational age group completed the study. The patient characteristics for groups 1, 2, and 3 were as follows: gestational age, 26.3 +/- 1.7, 30.3 +/- 1.3, and 33.9 +/- 1.1 weeks; birth weight, 876 +/- 176, 1365 +/- 186, and 1906 +/- 163 g; and days of hyperalimentation, 28.9 +/- 16, 16.8 +/- 12, and 4.3 +/- 4 days, respectively. At 2 weeks of age, the vitamin K intake and plasma levels were highest in group 1 versus group 3 (intake: 71.2 +/- 39.6 vs 13.4 +/- 16.3 microg/kg/day; plasma levels: 130.7 +/- 125.6 vs 27.2 +/- 24.4 ng/mL). By 40 weeks' postconception, the vitamin K intake and plasma levels were similar in all 3 groups (group 1, 2, and 3: intake, 11.4 +/- 2.5, 15.4 +/- 6.0, and 10.0 +/- 7.0 microg/kg/day; plasma level, 5.4 +/- 3.8, 5.9 +/- 3.9, and 9.3 +/- 8.5 ng/mL). None of the postnatal plasma samples had any detectable PIVKA II. CONCLUSIONS: Premature infants at 2 weeks of age have high plasma vitamin K levels compared with those at 40 weeks' postconceptional age secondary to the parenteral administration of large amounts of vitamin K. By 40 weeks' postconception, these values are similar to those in term formula-fed infants. Confirming "adequate vitamin K status," PIVKA II was undetectable by 2 weeks of life in all of the premature infants. With the potential for unforeseen consequences of high vitamin K levels, consideration should be given to reducing the amount of parenteral vitamin K supplementation in the first few weeks of life in premature infants.vitamin K, PIVKA II, premature, total parenteral nutrition, enteral nutrition.

Vitamin K supplementation reduces serum concentrations of under-gamma-carboxylated osteocalcin in healthy young and elderly adults.

BACKGROUND: Subclinical vitamin K insufficiency, manifested by under-gamma-carboxylation of the bone matrix protein osteocalcin, may be common. OBJECTIVE: Our objective was to delineate the prevalence of submaximal gamma-carboxylation as assessed by response to phylloquinone supplementation and to evaluate the effect of this intervention on skeletal turnover in healthy North American adults. DESIGN: Healthy subjects (n = 219), approximately equally distributed by sex and age (18-30 y and >/=65 y), received daily phylloquinone (1000 microg) or placebo for 2 wk. Serum undercarboxylated osteocalcin (ucOC) and total osteocalcin, N:-telopeptides of type I collagen (NTx), bone-specific alkaline phosphatase (BSAP), and phylloquinone concentrations were measured at baseline and after weeks 1 and 2. RESULTS: At baseline, the mean serum phylloquinone concentration was lower in the young than in the old group; there was no effect of sex. Concomitantly, baseline %ucOC was highest in the young and lowest in the old men (P: < 0.0001) but did not differ significantly by age in women. After supplementation, serum phylloquinone concentration increased approximately 10-fold (P: < 0.0001) at week 1 (from 0.93 +/- 0.08 to 8.86 +/- 0.70 nmol/L, x+/- SEM); this was sustained through week 2. Among all supplemented groups, mean %ucOC decreased from 7.6% to 3. 4% without significant differences by age or sex; 102 of 112 subjects had a >1% decrease. Phylloquinone supplementation reduced serum osteocalcin but did not alter NTx or BSAP concentration. CONCLUSIONS: Usual dietary practices in this population did not provide adequate vitamin K for maximal osteocalcin carboxylation. Phylloquinone supplementation reduced serum osteocalcin concentration but did not alter other markers of serum bone turnover.

Comparison of phylloquinone bioavailability from food sources or a supplement in human subjects.

Phylloquinone (K) absorption was assessed in 22- to 30-y-old human subjects consuming a standard test meal [402 kcal (1682 kJ), 27% energy from fat]. The absorption of phylloquinone, measured over a 9-h period as the area under the curve (AUC), was higher (P < 0.01) after the consumption of a 500- microgram phylloquinone tablet [27.55 +/- 10.08 nmol/(L. h), n = 8] than after the ingestion of 495 microgram phylloquinone as 150 g of raw spinach [4.79 +/- 1.11 nmol/(L. h), n = 3]. Less phylloquinone (P < 0.05) was absorbed from 50 g of spinach (AUC = 2.49 +/- 1.11 nmol/(L. h) than from 150 g of spinach. Absorption of phylloquinone from fresh spinach (165 microgram K), fresh broccoli (184 microgram K) and fresh romaine lettuce (179 microgram K) did not differ. There was no difference in phylloquinone absorption from fresh or cooked broccoli or from fresh romaine lettuce consumed with a meal containing 30 or 45% energy as fat.

Conversion of dietary phylloquinone to tissue menaquinone-4 in rats is not dependent on gut bacteria.

The ability of male rats to accumulate menaquinone-4 (MK-4) in tissues when fed a vitamin K-deficient diet supplemented with intraperitoneal phylloquinone (K) as the sole source of vitamin K for 14 d was assessed. In both conventionally housed controls and gnotobiotic rats, supplementation with the equivalent of 1500 microg vitamin K/kg diet increased (P < 0.001) tissue MK-4 concentrations above those of controls fed a vitamin K-deficient diet. MK-4 concentrations were approximately 5 ng/g (11 pmol/g) in liver, 14 ng/g in heart, 17 ng/g in kidney, 50 ng/g in brain and 250 ng/g in mandibular salivary glands of gnotobiotic rats. MK-4 concentrations in conventionally housed rats were higher than in gnotobiotic rats in heart (P < 0.01), brain (P < 0.01) and kidney (P < 0.05) but lower in salivary gland (P < 0.05). Cultures of a kidney-derived cell line (293) converted K to the expoxide of MK-4 in a manner that was dependent on both time of incubation and concentration of vitamin K in the media. A liver-derived cell line (H-35) was less active in carrying out this conversion. These data offer conclusive proof that the tissue-specific formation of MK-4 from K is a metabolic transformation that does not require bacterial transformation to menadione as an intermediate in the process.

Improving the vitamin K status of breastfeeding infants with maternal vitamin K supplements.

OBJECTIVE: To increase the phylloquinone (vitamin K1) concentration of human milk with maternal oral phylloquinone supplements such that both the phylloquinone intake of breastfed infants and their serum concentrations of phylloquinone would approach those of formula-fed infants who are known to be at much less risk for hemorrhagic disease of the newborn. DESIGN: Two stages: stage I, longitudinal, randomized study of 6 weeks' duration; and stage II, longitudinal, randomized, double-blind, placebo-controlled study of 12 weeks' duration. SETTING: Patients from a private pediatric practice in Madison, WI. PATIENTS: Stage I: sequential sampling of 20 lactating mothers to determine the level of maternal supplementation needed in stage II. Ten mothers received 2.5 mg/d oral phylloquinone, and 10 mothers received 5.0 mg/d oral phylloquinone. Stage II: sequential sampling of 22 human milk-fed infants and lactating mothers. All infants received 1 mg of phylloquinone at birth. Eleven mothers received a placebo; 11 mothers received 5 mg/d phylloquinone. MEASUREMENTS AND RESULTS: In stage I, both 2.5 and 5.0 mg/d phylloquinone significantly increased the phylloquinone content of human milk at both 2 and 6 weeks. As expected, 5.0 mg had a greater effect (mean +/- SD, 58.96 +/- 25.39 vs 27.12 +/- 12.18 ng/mL at 2 weeks). In stage II, the vitamin K-supplemented group had significantly higher maternal serum phylloquinone concentrations, higher phylloquinone milk concentrations, and higher infant plasma phylloquinone concentrations at 2, 6, and 12 weeks compared with the placebo group. At 12 weeks infant phylloquinone intakes were significantly higher for the vitamin K group than the placebo group (9.37 +/- 4.55 vs 0.15 +/- 0.07 microgram/kg per day). This corresponded to a plasma phylloquinone concentration in the vitamin K group of 2.84 +/- 3.09 vs 0.34 +/- 0.57 ng/mL in the placebo group. At 12 weeks, the prothrombin times did not differ between the groups, but the des-gamma-carboxy-prothrombin (partially carboxylated prothrombin thought to be a measure of vitamin K deficiency) was significantly elevated in the placebo group compared with the vitamin K group (1.48 +/- 1.19 vs 0.42 +/- 0.55 ng/mL). CONCLUSION: In exclusively breastfed infants who receive intramuscular phylloquinone at birth, the vitamin K status as measured by plasma phylloquinone and des-gamma-carboxy-prothrombin concentrations is improved by maternal oral supplements of 5 mg/d phylloquinone through the first 12 weeks of life.

Vitamin K status influences brain sulfatide metabolism in young mice and rats.

The established role of vitamin K in nutrition is as a cofactor in the post-translational conversion of specific glutamyl to gamma-carboxyglutamyl (Gla) residues in a limited number of proteins. Administration of the vitamin K antagonist warfarin has previously been shown to decrease brain sulfatide concentrations and decrease brain galactocerebroside sulfotransferase (GST) activity in young mice. A dietary deficiency of vitamin K has now been shown to decrease (P < 0.01) brain sulfatide concentrations of 30-d-old mice significantly (by 21%). Male 21-d-old rats fed an excess of vitamin K for 7 or 14 d had 26 and 31% (P < 0.05) greater GST activity and 15 and 18% (P < 0.05) greater brain sulfatide concentrations, respectively, than controls fed a vitamin K-deficient diet. Male 21-d-old rats fed a diet containing 500 mg of phylloquinone/kg diet had an intermediate response and were vitamin K sufficient by normal criteria. The vitamin K response was observed when either phylloquinone or menaquinone-4 was fed as a source of the vitamin. These data suggest that in addition to its recognized role in Gla synthesis, vitamin K status is important in the maintenance of normal complex lipid sulfatide metabolism in young rats and mice.

Multi-site-specificity of the vitamin K-dependent carboxylase: in vitro carboxylation of des-gamma-carboxylated bone Gla protein and Des-gamma-carboxylated pro bone Gla protein.

The vitamin K-dependent carboxylase processes multiple glutamic acid residues to gamma-carboxyglutamic acid (Gla) residues in a limited number of proteins. The targeted proteins are synthesized with an amino-terminal propeptide which has been shown to play an important role in gamma-carboxylation. The specificity of the enzyme for each potential Gla site, the direction of carboxylation, and the influence of a bound propeptide on these events are not understood. Des-gamma-carboxy forms of bone Gla protein (BGP), which contain potential Gla residues at positions 17, 21, and 24, were employed as model substrates to determine the multi-site-specificity of the enzyme. Recombinant bovine des-gamma-carboxylated proBGP (rdproBGP) and heat-decarboxylated BGP (dBGP), lacking a propeptide, were used as substrates for a bovine liver carboxylase, and the in vitro reaction products were analyzed for the formation of 14CO2 Gla. The di-Gla species was found to be the predominant product of in vitro carboxylation of both rdproBGP and dBGP at less than saturating concentrations of each substrate. Carboxylation of both substrates occurred preferentially at the more C-terminal potential Gla sites, residues 21 and 24. A similar pattern of carboxylation was observed with a rat bone cell carboxylase, suggesting no species or tissue variation in the enzyme specificity. Some tricarboxylated product accumulated during carboxylation of rdproBGP but not dBGP, suggesting that the covalently bound propeptide directs more complete carboxylation of the Gla domain. In addition, monocarboxylated rdproBGP was found to accumulate in the absence but not in the presence of a free noncovalently attached propeptide, indicating that free propeptide affects more efficient carboxylation of rdproBGP.

Vitamin K-dependent carboxylase activity, prothrombin mRNA, and prothrombin production in two cultured rat hepatoma cell lines.

The presence of under-gamma-carboxylated forms of plasma prothrombin is a marker for human primary hepatocellular carcinoma. A rat hepatoma cell line (7777) which was previously shown to secrete undercarboxylated prothrombin when grown as a solid tumor has now been grown in monolayer culture. This cell line has a decreased activity of the microsomal vitamin K-dependent carboxylase when compared to a control (H4IIEC3) hepatoma line, does not increase intracellular prothrombin concentrations in response to vitamin K depletion, and secretes undercarboxylated prothrombin even when grown in vitamin K supplemented media. Prothrombin gene expression in the 7777 cell line, as measured by prothrombin mRNA levels, was not altered in the 7777 cell line. This cell line appears to be a model for assessing the cellular alterations responsible for undercarboxylated prothrombin excretion by human hepatocellular tumors.

Vitamin K status of lactating mothers, human milk, and breast-feeding infants.

Hemorrhagic disease of the newborn is a disease of breast-feeding newborns. There is little information on longitudinal breast milk concentrations of phylloquinone (vitamin K1) or the effects of maternal phylloquinone supplements on breast milk. In study part 1, 11 lactating mothers, who received 20 mg of phylloquinone orally, had rises in plasma (less than 1 to 64.2 +/- 31.5 ng/mL by 6 hours) and breast milk concentrations (from 1.11 +/- 0.82 to 130 +/- 188 ng/mL by 12 hours). In part 2, 23 lactating mothers and their infants were observed longitudinally along with a formula-fed control group of infants (n = 11). Mean breast milk concentrations of phylloquinone at 1, 6, 12, and 26 weeks were 0.64 +/- 0.43, 0.86 +/- 0.52, 1.14 +/- 0.72, and 0.87 +/- 0.50 ng/mL, respectively, in the infants fed human milk. Maternal phylloquinone intakes (72-hour dietary recalls) exceeded the recommended daily allowance of 1 microgram/kg per day. Infant phylloquinone intakes did not achieve the recommended daily allowance of 1 microgram/kg per day in any infant. Plasma phylloquinone concentrations in the infants fed human milk remained extremely low (mean less than 0.25 ng/mL) throughout the first 6 months of life compared with the formula-fed infants (4.39 to 5.99 ng/mL). In this small sample, no infant demonstrated overt vitamin K deficiency. Despite very low plasma phylloquinone concentrations, vitamin K supplements (other than in the immediate newborn period) cannot be recommended for exclusively breast-fed infants based on these data.

Vitamin K1 and K2 in infant human liver.

It is not known to what extent humans store vitamin K in liver. We measured hepatic concentrations of vitamin K1 (phylloquinone) and K2 (menaquinones) in 11 human livers (eight infants and three adults). Relatively small amounts of vitamin K were found in the liver at any age compared to other fat soluble vitamins. Vitamin K1 was the predominant form with much smaller concentrations of vitamin K2. Long-chain menaquinones (vitamin K2) were readily identified in most liver specimens. Hepatic vitamin K2 concentrations also increased with increasing age. These observations have implications for vitamin K supplementation in infants.

Vitamin K deficiency from dietary vitamin K restriction in humans.

Vitamin K is required for the maintenance of normal hemostatic function. Ten college-aged male subjects chose diets restricted in vitamin K content for 40 d. Median phylloquinone intakes based on analysis of food composites dropped from 82 micrograms/d during the prestudy period to 40 and 32 micrograms/d at d 9 and 27 of dietary restriction, respectively. Serum phylloquinone concentrations fell from a mean of 0.87 to 0.46 ng/mL during a 21-d period of vitamin K restriction. Supplementation with 50 micrograms phylloquinone/d for 12 d increased serum phylloquinone to 0.56 ng/mL, and supplementation with 500 micrograms phylloquinone/d increased serum phylloquinone to 1.66 ng/mL. Vitamin K restriction resulted in alterations in a functional clotting assay that detects undercarboxylated prothrombin species in plasma and in a decrease in urinary gamma-carboxyglutamic acid. Supplementation with either 50 or 500 micrograms of phylloquinone restored both these indices to near normal values. These data are consistent with a human dietary vitamin K requirement of approximately 1 microgram/kg body wt/d.

The availability of fluoride from NaF and phosphorus supplements.

The availability of fluoride (F) from dicalcium phosphate (DCP), defluorinated phosphate (DFP), and raw rock phosphate (RRP) has been assessed and compared to the availability of F as NaF. Diets containing 50-55 ppm F were fed to lambs and rats and skeletal F retention determined. The relative availability of F in the different phosphate sources measured by this technique compared to that of NaF was about 50% (DCP), 20% (DFP), and 65% (RRP). Digestibility of F in goats fed similar diets was determined in a 4 x 4 Latin Square design experiment and found to be 38% (DCP), 34% (DFP), 65% (RRP), and 75% (NaF).

Evidence for a lack of an effect of dietary fluoride level on reproduction in mice.

The possible essentiality of fluoride for reproduction was studied in female mice fed a low-fluoride ( less than 0.5 ppm F) basal diet for three generations. Reproduction of mice fed this basal diet was the same as when mice were fed the basal diet supplemented with 2 or 100 ppm F. A previous study [Messer et. al. (1973) J. Nutr. 103, 1319-1326], utilizing the same diet did result in an impairment of reproduction and the development of an anemic condition. The results of the present study suggest that the apparent essentiality of fluoride previously observed was due to a pharmacological effect of fluoride in improving iron utilization in a diet marginally sufficient in iron. The data do not support the previous claims of an essential role of fluoride in reproduction.