Severe acquired vitamin K deficiency: a hypothesis for rapid response to therapy.

The potential mechanism underlying the rapid response to vitamin K replacement in acquired deficiency states is incompletely understood. To examine vitamin K metabolism, a 10-year-old boy with autoimmune enteropathy on oral vitamin K supplementation, who presented with profuse nosebleeds and calf tenderness, was evaluated. Laboratory analyses were consistent with severe vitamin K deficiency: vitamin K dependent protein (VKDP) levels < 5%, normal vitamin K epoxide level and depressed total prothrombin antigen (carboxylated and undercarboxyated forms). Intramuscular vitamin K (10 mg) was administered. Nine hours following therapy, VKDP levels corrected completely. Total prothrombin antigen increased indicating new prothrombin synthesis. However, the increase in the prothrombin-clotting assay far exceeded the increase in total prothrombin, supporting storage of undercarboxylated prothrombin in vitamin K deficiency states, with carboxylation and secretion after vitamin K replacement. Although this mechanism is known to occur in rodents, it has not been reported in humans. Our findings suggest a new potential mechanism of prothrombin metabolism in humans.

Gender differences in hepatic phylloquinone and menaquinones in the vitamin K-deficient and -supplemented rat.

Gender differences in relation to vitamin K were investigated in the rat. Hepatic phylloquinone and menaquinone (MK-1 to MK-10) concentrations, gamma-carboxyglutamic acid (Gla) excretion, plasma phylloquinone and percent prothrombin were measured in male and female rats on a chow diet (24.5 ng phylloquinone and 8.8 microgram menadione), and on phylloquinone-deficient and -supplemented purified diets (0.38 and 1400 ng phylloquinone/g, respectively). Mean hepatic phylloquinone concentrations varied with dietary intake and ranged from 6.8+/-9.0 pmol/g in the deficient male, to 171. 1+/-56.9 pmol/g in the supplemented female. Menaquinones accounted for a large proportion of total vitamin K in the liver of males and females with MK-4, MK-6, and MK-10 present in highest concentrations. On the chow and supplemented diets, females had significantly higher MK-4, MK-6, and MK-10 concentrations in their livers (P<0.05). On the phylloquinone-deficient diet (-K1), hepatic phylloquinone, MK-4, and to a lesser extent MK-6 (but not MK-10) were significantly reduced (P<0.05). In the phylloquinone-supplemented male and female groups, which did not receive menadione during the experimental period, MK-4 increased above that in the chow groups suggesting synthesis of MK-4 from phylloquinone which was statistically significant in the female (P<0.01). A significant gender difference (P<0.05) was also observed for urinary Gla excretion with less Gla excreted by the females indicating that females may require less dietary phylloquinone than males of the same body weight.

Plasma lipoproteins as carriers of phylloquinone (vitamin K1) in humans.

The purpose of this study was to characterize the absorption and transport of phylloquinone (vitamin K1) by plasma lipoproteins. Twenty-six healthy subjects (11 men and 15 women) aged 20-78 y received phylloquinone in the amount of either 1.43 or 50 microg/kg body wt orally with a fat-rich meal containing 1.0 g/kg body wt of fat, carbohydrate, and protein and 7.0 mg cholesterol/kg body wt. Blood was obtained at baseline (0 h) and 3, 6, 9, and 12 h after the meal for the measurement of plasma lipid and phylloquinone concentrations in plasma and lipoprotein subfractions. In both groups of subjects, triacylglycerol concentrations peaked after 3 h in plasma and in the triacylglycerol-rich lipoprotein fraction, composed of chylomicrons and VLDLs. Plasma phylloquinone concentrations peaked at 6 h. At baseline and during the postprandial phase, > 53% of plasma phylloquinone was carried by the triacylglycerol-rich lipoprotein fraction. In 9 of the 11 subjects supplemented with 50 microg phylloquinone/kg, plasma lipoproteins were isolated by sequential ultracentrifugation. In these subjects the fraction of plasma phylloquinone carried by LDLs and by HDLs increased progressively from 3% and 4% at 3 h to 14% and 11% at 12 h, respectively. Our data indicate that whereas triacylglycerol-rich lipoproteins are the major carriers of phylloquinone, LDL and HDL may carry small fractions of this vitamin.

Diurnal variation in total and undercarboxylated osteocalcin: influence of increased dietary phylloquinone.

A diurnal variation exists in blood levels of the vitamin K-dependent bone protein osteocalcin. However, it is not known whether the carboxylated and undercarboxylated constituents of osteocalcin also vary. Therefore, osteocalcin and undercarboxylated osteocalcin were measured in specimens collected every 4 hours over a 24-hour period in nine healthy subjects (five males, four females) ages 20-33 years who were consuming a mixed diet containing 100 microg of phylloquinone. Osteocalcin and undercarboxylated osteocalcin were measured by radioimmunoassay (RIA) before and after treatment with barium sulfate. Although the percent undercarboxylated osteocalcin did not change, a diurnal variation was observed in total osteocalcin, carboxylated osteocalcin, and undercarboxylated osteocalcin, with peak concentrations at 4 a.m. and the lowest concentrations between 12 p.m. and 4 p.m. The difference between the total osteocalcin peak and trough concentrations averaged 28 +/- 7 (SEM)%. There were no gender differences in these rhythms. The effect of dietary phylloquinone as a modulator of these rhythms was evaluated in a randomized study by increasing phylloquinone intake to 420 microg/day with fortified corn oil, split between the lunch and dinner meals. Total and carboxylated osteocalcin fluctuations and concentrations were not affected by the dietary treatment. The diurnal variation in undercarboxylated osteocalcin was abolished with supplementation and concentrations at 8 a.m. (14 hours following supplementation) (2.3 +/- 0.2 ng/ml) were significantly lower than the unsupplemented levels (2.7 +/- 0.2 ng/mL, P = 0.006). The percentage of undercarboxylated osteocalcin was similarly decreased after supplementation (19.7 +/- 1.3%) in relation to the mixed diet cycle (24.2 +/- 1.6%, P = 0.006) at 8 a.m. on the second day. Dietary supplementation induced a fluctuation in percentage undercarboxylated osteocalcin with a decline in levels starting at approximately 12 a.m. Therefore, additional dietary phylloquinone does not appear to modulate the total osteocalcin diurnal rhythm, but can influence its undercarboxylated component.

Effect of vitamin K1 supplementation on vitamin K status in cystic fibrosis patients.

BACKGROUND: Patients with cystic fibrosis are at risk for impaired vitamin K status due to fat malabsorption from pancreatic insufficiency. This study was designed to assess vitamin K status and measure the effect of vitamin K1 supplementation in cystic fibrosis patients. METHODS: Eighteen outpatients participated in a crossover study to determine the effect of vitamin K1 (phylloquinone) supplementation. After obtaining initial data, each subject was randomly assigned to either a 4-week study treatment of 5 mg oral vitamin K1 supplementation per week, or no supplementation and then crossed over to the other treatment for a second 4 week period. Plasma, serum and urine samples were collected and analyzed pre-study and at the end of each study period. RESULTS: The mean concentration of plasma vitamin K1 for the supplemented group was significantly higher than the unsupplemented group, [0.34 nmol/L and 0.21 nmol/L, respectively (p < 0.05)]. The percent of undercarboxylated osteocalcin increased on supplementation from 17% to 31%, (p < 0.005). Prothrombin induced in vitamin K absence (PIVKA-II) increased on supplementation from 5 ng/mL to 22 ng/mL, (p < 0.005). The ratio of urinary gamma-carboxyglutamic acid/creatinine was similar for both study periods. CONCLUSIONS: In contrast to other studies in cystic fibrosis, this study demonstrated a need for vitamin K1 supplementation. The carboxylation state of osteocalcin and PIVKA-II were the most sensitive indices of changes in vitamin K1 status. Although the 5 mg vitamin K1/week dose improved these vitamin K parameters, normal levels were not achieved.

Changes in serum osteocalcin, plasma phylloquinone, and urinary gamma-carboxyglutamic acid in response to altered intakes of dietary phylloquinone in human subjects.

The response of osteocalcin and other biochemical markers of vitamin K status to diets formulated to contain different amounts of phylloquinone was assessed in nine healthy subjects aged 20-33 y. Subjects resided in a metabolic ward for two 15-d cycles with a minimum of 6 wk between cycles. A mixed diet containing 100 micrograms phylloquinone/d was fed throughout both cycles; however, the phylloquinone content of one of the cycles was increased to a total of 420 micrograms/d on days 6 through 10 by fortifying corn oil in the diet with phylloquinone (supplemented diet). Total serum osteocalcin concentrations were not affected by either of the dietary treatments. The percentage of undercarboxylated osteocalcin increased an average of 28% over the 15-d cycle with the mixed diet (P < 0.05) and declined significantly an average of 41% with 5 d of the supplemented diet (day 6: 21.9 +/- 1.3%, day 11: 12.8 +/- 1.1%; P = 0.0001) with a rise after the return to the mixed diet (16.7 +/- 1.3%, P < 0.001). Plasma phylloquinone concentrations increased significantly with supplementation (day 6: 0.95 +/- 0.16 nmol/L, day 11: 1.40 +/- 0.29 nmol/L; P < 0.001) and then rapidly returned to presupplementation concentrations on returning to the mixed diet. Twenty-four-hour ratios of urinary gamma-carboxyglutamic acid to creatinine were unchanged with the supplemented diet; however, excretion declined to 91 +/- 2% of baseline after 10 d on the mixed diet (P = 0.01). These results show that undercarboxylated osteocalcin, plasma phylloquinone, and urinary gamma-carboxyglutamic acid excretion appear to be sensitive measures of vitamin K nutritional status because all of these variables were responsive to changes in dietary intake.

Plasma concentrations of dihydro-vitamin K1 following dietary intake of a hydrogenated vitamin K1-rich vegetable oil.

Dihydro-vitamin K1 is a dietary form of vitamin K1 (phylloquinone) produced during the hydrogenation of vegetable oils. To determine if dihydro-vitamin K1 is present in plasma following dietary intake of a hydrogenated fat, eight healthy adults consumed each of two diets containing 30% of calories from fat, of which 20% was either soybean oil or a partially hydrogenated soybean oil-based stick margarine. Of the fats and oils analyzed, dihydro-vitamin K1 was only found in the hydrogenated products. The soybean oil diet contained 180 +/- 12 micrograms (mean +/- SD) of vitamin K1/day and nondetectable levels of dihydro-vitamin K1, whereas the stick margarine diet contained 199 +/- 7 micrograms of vitamin K1/day and 23 +/- 2 micrograms of dihydro-vitamin K1/day. After consuming each diet for five weeks, plasma dihydro-vitamin K1 concentrations were higher (P = 0.002) in all eight subjects when consuming the stick margarine diet (0.56 +/- 0.33 nmol/L) compared to the soybean oil diet (0.12 +/- 0.11 nmol/L). There was no significant change in plasma vitamin K1 concentrations when the two diets were compared. In conclusion, dihydro-vitamin K1 is detectable in plasma following dietary intake of a hydrogenated vitamin K1-rich vegetable oil.

Dihydro-vitamin K1: primary food sources and estimated dietary intakes in the American diet.

Dihydro-vitamin K1 was recently identified as a dietary form of vitamin K produced during the hydrogenation of vitamin K1-rich vegetable oils. Dihydro-vitamin K1 is absorbed, with measurable levels in human plasma following dietary intake. To determine the primary food sources of dihydro-vitamin K1 in the American diet, 261 foods from the U.S. Food and Drug Administration's (FDA) Total Diet Study (TDS) were analyzed by high-performance liquid chromatography. Of these foods, 36 contained dihydro-vitamin K1. Fast-food items that were otherwise poor sources of vitamin K1, such as french fries and fried chicken, contained appreciable amounts of dihydro-vitamin K1 (36 and 18 micrograms/100 g, respectively). These nutrient values were then applied to the FDA TDS consumption model to determine average dietary intake of dihydro-vitamin K1 in 14 age-gender groups. With the exception of infants, all age-gender groups had estimated mean daily dihydro-vitamin K1 intakes of 12-24 micrograms, compared to mean daily vitamin K1 intakes of 24-86 micrograms. The vitamin K1 and dihydro-vitamin K1 intakes were summed, and the dietary contribution of dihydro-vitamin K1 was expressed as a percentage of total vitamin K intake. Children reported the highest intakes of dihydro-vitamin K1 (30% of total vitamin K intake), followed by a progressive decrease in percentage contribution with age. There are currently no data on the relative bioavailability of dihydro-vitamin K1 but given its abundance in the American diet, this hydrogenated form of vitamin K warrants further investigation.

The pharmacokinetics and lipoprotein fraction distribution of intramuscular vs. oral vitamin K1 supplementation in women of childbearing age: effects on hemostasis.

Prenatal maternal vitamin K1 supplementation to improve the hemostatic status of the fetus may depend upon the route of administration and subsequent presentation at the placental barrier. We investigated intramuscular (IM) vs oral (PO) vitamin K1 supplementation in eight healthy, nonpregnant women of childbearing age. Pharmacokinetics were studied in each subject after a 5 mg IM dose and after a 5 mg oral dose of vitamin K1 approximately one month later. Plasma collected at the peak vitamin K level for each treatment was separated into very low density lipoproteins (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) and lipoprotein-free fractions by density gradient ultracentrifugation. Vitamin K1 was measured in the plasma and lipoprotein fractions using HPLC. The concentration of vitamin K1 in plasma reached a peak 2 h after an IM dose and remained high throughout the 30 h course of the study. In contrast, the oral dose of vitamin K1 peaked at 4 h and rapidly decreased to near baseline by 18 to 30 h. The distribution of vitamin K1 in the lipid fractions was different for IM compared to PO. The percentage of vitamin K1 in the VLDL fraction at the peak for an oral dose was significantly higher than for an IM dose (80.8% +/- 3.5 vs 10.8% +/- 6.5, p < 0.0001). After the oral absorption stage, the subjects took 5 mg of vitamin K1 orally, once a day, for 12 days. No significant differences were observed for the following coagulation proteins and hemostatic markers measured immediately before and after long-term oral vitamin K supplementation: factor II, factor VII, protein C, and thrombin-antithrombin III complex. In conclusion, physiological processing of supplemented vitamin K1 differs in the IM vs PO routes of administration and 12 days of oral vitamin K1 does not alter the concentration of selected vitamin K-dependent coagulation proteins or thrombin-antithrombin complex generation.

Assessment of dietary phylloquinone intake and vitamin K status in postmenopausal women.

OBJECTIVE: To examine the relationship between dietary phylloquinone intake and vitamin K status of postmenopausal Caucasian women. DESIGN: Cross-sectional study, in which dietary intake was estimated using weighed record techniques and vitamin K status was measured by a single plasma phylloquinone concentration and 24-h urinary gamma-carboxyglutamic acid (Gla) excretion. SETTING: The metabolic research unit at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA. SUBJECTS: 402 healthy postmenopausal Caucasian women who were participating in a randomized trial to determine the effect of calcium supplementation on bone loss. Of the original group, 362 had complete weighed diet records, 358 had corresponding plasma phylloquinone concentrations, and 346 had corresponding urinary Gla measurements. RESULTS: There was a significant correlation (r = 0.13, P = 0.01) between total dietary intake of phylloquinone (geometric mean = 89 micrograms/day) and plasma phylloquinone levels (mean = 1.12 nmol/l). Dietary intake was neither correlated with urinary Gla excretion (mean = 4.0 mumol/mmol creatinine) nor did it vary by season. The ratio of intra- to interindividual variance in phylloquinone intake was 2.6, from which it was estimated that 5 days of independent recording is necessary to estimate true usual dietary intake, assuming a correlation of 0.8. CONCLUSIONS: A weighed record has the potential to be a reliable method for estimating dietary intakes of vitamin K which relate to plasma phylloquinone levels used as an indicator of vitamin K status in postmenopausal Caucasian women.

The vitamin K content of intravenous lipid emulsions.

Commercially available intravenous lipid emulsions are largely derived from vegetable oils, a natural source of phylloquinone (vitamin K1). We therefore examined the concentration of vitamin K1 in two widely used intravenous lipid emulsions by using a previously validated high performance liquid chromatography technique. The vitamin K1 concentrations of 10% emulsions of Intralipid and Liposyn II were 30.8 and 13.2 micrograms/dL, respectively. The concentration of vitamin K1 in the 20% emulsions of these products was essentially double that in the 10% emulsions. The coefficients of variation between the vitamin K1 content in three different lots of each product were consistently less than 7.0%. The observed concentrations of the vitamin in these lipid emulsions paralleled the predicted content on the basis of the type of vegetable oil(s) used to make the product. The type of vegetable oil used for production therefore seems to be a major determinant of the final vitamin K1 content. The vitamin K1 contained in these intravenous lipid emulsions is substantial and may have great impact on the vitamin K status of the recipient.

Comparison of micronutrient intake measured by a dietary questionnaire and biochemical indicators of micronutrient status.

We compared the intake of 12 micronutrients as reported on a semiquantitative food frequency questionnaire with corresponding biochemical indicators of nutrient status in a sample of 57 males and 82 females aged 40-83 y. Age-, sex-and energy-adjusted correlation coefficients ranged from near zero for thiamin, vitamin A, and zinc to 0.63 for folate. Correlation coefficients between intake and the biochemical measures were > 0.30 for carotenoids, vitamin D, vitamin E, vitamin B-12, folate, and vitamin C. Differences of 50% or more were observed between extreme quartiles of intake for mean plasma concentrations of folate, vitamin B-12, and vitamin C. Excluding nutrient supplement users generally reduced the correlations. These data demonstrate that food frequency questionnaires can provide valid information on intake for a number of micronutrients.

The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits.

Although Freund's adjuvant has been used for decades as an immune enhancer in rabbits, adverse physiologic side effects have prompted the search for more suitable alternatives. We used osteocalcin, a bovine bone protein (M.W. 5,800), as the test antigen to evaluate four adjuvant regimens: a) primary inoculation with complete Freund's adjuvant (CFA) followed by three boosts with incomplete Freund's adjuvant (IFA), b) four serial inoculations with RIBI MPL+TDM+CWS adjuvant, c) four serial inoculations with TiterMax #R-1, and d) primary inoculation (only) with TiterMax #R-1. The antibody yield associated with the CFA/IFA regimen (mean OD = 2.152) was at least sixfold that of either TiterMax (mean OD = 0.358) or RIBI (mean OD = 0.239) multiple injection regimens. No antibody response was observed after the single injection of TiterMax antigen emulsion. Maximal antibody production occurred rapidly in response to Freund's adjuvant (day 31) as compared with TiterMax (day 74) and RIBI (day 66).

Surreptitious ingestion of a long-acting vitamin K antagonist/rodenticide, brodifacoum: clinical and metabolic studies of three cases.

The vitamin K metabolism of three patients with factitious purpura due to brodifacoum ingestion was studied. These patients, who presented with bleeding disorders due to deficiency of the vitamin K-dependent blood clotting proteins, were refractory to vitamin K1 at standard doses and required fresh frozen plasma to control bleeding until large doses of vitamin K1 were used. Metabolic studies demonstrated a blockade in vitamin K utilization, consistent with the presence of a vitamin K antagonist, but the patients denied use of anticoagulants. Warfarin assays were negative. We show that the factitious purpura in each patient was due to the surreptitious ingestion of brodifacoum, a potent second generation long-acting vitamin K antagonist used as a rodenticide. The coagulopathies responded to long-term therapy with large doses of vitamin K1. The serum elimination half-time for brodifacoum ranged from 16 to 36 days in these patients. The anticoagulant effect is of long duration, requiring chronic vitamin K treatment. With increasing availability of new rodenticides, factitious purpura due to surreptitious ingestion of these potent vitamin K antagonists is emerging as a new problem, previously associated with warfarin, with important implications for diagnosis and treatment.

Relationship of vitamin A and vitamin E intake to fasting plasma retinol, retinol-binding protein, retinyl esters, carotene, alpha-tocopherol, and cholesterol among elderly people and young adults: increased plasma retinyl esters among vitamin A-supplement users.

We studied the relationships of supplemental and total vitamin A and supplemental vitamin E intake with fasting plasma biochemical indicators of vitamin A and vitamin E nutritional status among 562 healthy elderly people (aged 60-98 y) and 194 healthy young adult (aged 19-59 y) volunteers. All subjects were nonsmokers. For the young adults, plasma retinol was significantly greater in males than in females (p less than 0.01); retinol was not related to supplemental vitamin A intake for either group. Fasting plasma retinyl esters demonstrated a significant increase with vitamin A supplement use. For supplemental vitamin A intakes of 5001-10,000 IU/d, a 2.5-fold increase over nonusers in fasting plasma retinyl esters was observed for elderly people (p less than 0.05) and a 1.5-fold increase for young adults (p greater than 0.20). For elderly people, greater fasting plasma retinyl esters were associated with long-term vitamin A supplement use (greater than 5 y) and biochemical evidence of liver damage. Elderly people who take vitamin A supplements may be at increased risk for vitamin A overload.

Vitamin C status and nutrient interactions in a healthy elderly population.

Vitamin C status and interactions with other nutrients were studied in 677 healthy, noninstitutionalized elderly people aged 60-98 y. Although 6% of the males and 3% of the females showed marginal vitamin C status (plasma ascorbic acid 11 to less than 23 mumol/L), only one person had a plasma ascorbic acid (AA) level less than 11 mumol/L. At all levels of total vitamin C intake, mean plasma AA levels were higher in females than males. Vitamin C supplement use was associated with generally higher blood levels of vitamins B-6, B-12, and E and folate in both sexes and with higher levels of retinol in females. However, after both age and the total dietary intake of the specific nutrient being examined were controlled for, plasma AA levels were significantly correlated only with plasma levels of vitamin E and folate in females.

Fat-soluble vitamin nutriture in primary biliary cirrhosis.

We measured serum levels of vitamins A, E, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D, as well as levels of abnormal (des-gamma-carboxy) prothrombin, in 52 patients with primary biliary cirrhosis. Decreased serum levels of retinol (vitamin A) and 25-hydroxyvitamin D and elevated levels of abnormal prothrombin were common in these patients and correlated with the histologic stage of the disease and with the clinical severity of disease as judged by elevated serum bilirubin levels and decreased serum albumin levels. The increased levels of abnormal prothrombin were due primarily to vitamin K deficiency but also, in part, to the severity of the liver disease itself. Vitamin E deficiency was rare. Only 1 patient had clinical manifestations of fat-soluble vitamin deficiency, night blindness, and gastrointestinal bleeding related to a marked prolongation of the prothrombin time. Deficiencies of fat-soluble vitamins are most likely to be present in jaundiced patients with long-standing, severe cholestasis. We suggest that fat-soluble vitamin status be determined in all patients with primary biliary cirrhosis by appropriate blood tests and that vitamin supplements be given only to those patients who require them.