Prevalence of vitamin K deficiency in older people with hip fracture.

Introduction: Vitamin K plays an important role in blood coagulation. Diet is the main source of vitamin K and body stores are depleted in days, hence deficiency is common in malnourished older people. A high proportion of people who sustain a hip fracture are already malnourished, compounded by fasting for surgery which might further increase deficiency. We wanted to explore the prevalence of vitamin K deficiency in hip fracture patients and the impact of a short period of fasting.Methods: In consecutive patients hospitalised with a hip fracture, we measured vitamin K and PIVKA-II (undercarboxylated factor II - a marker of subclinical vitamin K status) on admission and on first post-operative day. We excluded those on anticoagulants.Results: N = 62 participated; 4 had missing pre-op vitamin K samples and n = 3 had no surgery leaving n = 55 with paired samples. Mean age was 80.0 ± 9.6 years, 33% males. Prevalence of subclinical vitamin K deficiency on admission was 36% (20/55) based on reference range of > 0.15µg/L. The proportion with subclinical K deficiency after surgery rose to 64% (35/55), p < 0.05. 13% had detectable PIVKA-II concentrations pre-operatively, 15% did post-operatively. None had abnormal prothrombin time. Vitamin K status was not associated with post-operative haemoglobin drop or transfusion requirements.Conclusion: Prevalence of vitamin K deficiency in hip fracture patients is high and increases further following a short period of fasting. Though no significant impact was noted on peri-operative blood loss, larger studies are warranted to explore this, and the potential role of vitamin K supplements peri-operatively.

Undernutrition in childhood: Clinically based assessment tools and biological markers: Where are we and where should we go?

Despite its association with poor clinical outcomes and increased hospital costs, as of today undernutrition still goes undetected in paediatric hospitals. The reported prevalence of undernutrition in paediatric patients varies considerably. This disparity is partly due to the diversity of methods for its detection and assessment, as well as to the lack of consensus regarding its definition. Several methods, based on varied combinations of morphology characteristics, estimated nutritional intakes and medical conditions have been developed during the last 25 years. However, these tools suffer from poor sensitivity and selectivity particularly in acute conditions. Also while having their own merit, these tools mainly view malnutrition from the energy standpoint, disregarding assessment of specific micronutrients such as minerals and vitamins. In this position paper we make the point that in the era of personalized medicine, present technology offers the possibility of going beyond the traditional nutritional tools for assessing patients' status, and propose the measurement of selected micronutrients and allied metabolic markers in nutritional workup schemes adapted to each clinical condition.

Therapeutic warfarin use and the extrahepatic functions of vitamin K-dependent proteins.

The impact of warfarin therapy on the functions of extrahepatic vitamin K-dependent proteins (VKDP) is less clearly understood and less widely recognised in clinical practice than that on the hepatic counterparts (clotting factors II, VII, IX and X). Warfarin inhibits osteocalcin, an abundant extrahepatic VKDP involved in the mineralisation and maturation of bone and thus, primarily by this mechanism, may have an adverse effect on bone health. Whilst some studies do link warfarin use to an increase in osteoporosis and fracture risk others have not. Warfarin also inhibits the extrahepatic VKDP matrix gla protein (MGP) which acts to prevent ectopic calcification of the vasculature. Studies have consistently found a correlation between warfarin use and vascular calcification with inhibition of MGP believed to be the main cause. Inhibition of MGP also appears to explain warfarin's well established teratogenic effect. Further adverse effects may also arise from warfarin's inhibition of other known extrahepatic VKDPs. The available evidence is intriguing, and suggests that the impact of warfarin on the extrahepatic functions of vitamin K-dependent proteins warrants further careful consideration.

An infant and mother with severe B12 deficiency: vitamin B12 status assessment should be determined in pregnant women with anaemia.

The vitamin B12 status of infants depends on maternal B12 status during pregnancy, and during lactation if breastfed. We present a 9-month-old girl who was admitted to the metabolic unit for assessment of developmental delay. She was exclusively breastfed and the introduction of solids at 5 months was unsuccessful. Investigations revealed pancytopenia, undetectable B12 and highly elevated methylmalonic acid and homocysteine. Methylmalonic acid and homocysteine normalised following B12 injections. Marked catch-up of developmental milestones was noted after treatment with B12. Investigations of parents showed normal B12 in the father and combined B12 and iron deficiency in the mother. Maternal B12 deficiency, most likely masked by iron deficiency, led to severe B12 deficiency in the infant. Exclusive breastfeeding and a subsequent failure to wean exacerbated the infant's B12 deficiency leading to developmental delay. This case highlights the need for development of guidelines for better assessment of B12 status during pregnancy.

Safety of long-term restrictive diets for peroxisomal disorders: vitamin and trace element status of patients treated for Adult Refsum Disease.

BACKGROUND: Adult Refsum's Disease (ARD) is caused by defects in the pathway for alpha-oxidation of phytanic acid (PA). Treatment involves restricting the dietary intake of phytanic acid by reducing the intake of dairy-derived fat. The adequacy of micronutrient intake in patients with ARD is unknown. METHODS: Patients established on the Chelsea low-PA diet had general diet macronutrients, vitamins and trace elements assessed using 7-day-weighed intakes and serial 24-h recalls. Intakes were compared with biochemical assessments of nutritional status for haematinics (ferritin), trace elements (copper, zinc, iron, selenium), water- (vitamin B6 , B12 and folate) and fat-soluble vitamins (A, D, E and K). RESULTS: Eleven subjects (four women, seven men) were studied. Body mass index was 27 ± 5 kg/m(2) (range 19-38). All subjects had high sodium intakes (range 1873-4828 mg). Fat-soluble vitamin insufficiencies occurred in some individuals (vitamin A, n = 2; vitamin D, n = 6; vitamin E, n = 3; vitamin K, n = 10) but were not coincident. Vitamin B6 levels were normal or elevated (n = 6). Folate and 5-methyltetrahydrofolate concentrations were normal. Metabolic vitamin B12 insufficiency was suspected in four subjects based on elevated methylmalonic acid concentrations. Low copper and selenium intakes were noted in some subjects (n = 7, n = 2) but plasma levels were adequate. Iron, ferritin and zinc intakes and concentrations were normal. CONCLUSION: Subjects with ARD can be safely managed on the Chelsea low PA without routine micronutrient supplementation. Sodium intake should be monitored and reduced. Periodic nutritional screening may be necessary for fat-soluble vitamins, vitamin B12 , copper or selenium.

Circulating concentrations of vitamin E isomers: Association with bone turnover and arterial stiffness in post-menopausal women.

The effects of vitamin E on cardiovascular and bone health are conflicting with beneficial and detrimental findings reported. To investigate this further, we carried out a cross-sectional study to determine the relationship between circulating concentrations of the 2 vitamin E isomers, α- and γ-tocopherol (TP) with bone turnover and arterial stiffness. Two hundred and seventy eight post-menopausal women with mean age [SD] 60.9 [6.0] years were studied. Fasting serum α-TP and γ-TP, bone turnover markers; procollagen type 1 amino-terminal propeptide (P1NP) and C-terminal telopeptide of type 1 collagen (CTX), parathyroid hormone (PTH), total cholesterol (TC) and triglycerides (TG) were measured. Pulse wave velocity (PWV) and central augmentation index (AI) as markers of arterial stiffness were also determined. A positive correlation was observed between α-TP and γ-TP (r=0.14, p=0.022). A significant negative association between α-TP and P1NP only was seen in multiple linear regression analysis following adjustment for serum TC and TG (p=0.016). In a full multi-linear regression model, following correction for age, years since menopause, smoking habits, alcohol intake, use of calcium supplements, BMI, PTH, serum calcium, and estimated glomerular filtration rate (eGFR), the association between α-TP and P1NP remained significant (p=0.011). We did not observe any significant association between γ-TP or α-TP/γ-TP ratio with P1NP or CTX. P1NP was significantly lower in subjects with α-TP concentrations of >30 µmol/L (α-TP >30 µmol/L; P1NP: 57.5 [20.7], α-TP<30 µmol/L; P1NP: 65.7 [24.9] µg/L, p=0.005). PWV was significantly associated with α-TP/γ-TP ratio (p=0.04) but not with serum α-TP or γ-TP in a full multi-linear regression model adjusting for serum lipids, age, and blood pressure. The data suggest that high serum concentrations of α-TP may have a negative effect on bone formation. The balance of α-TP and γ-TP may be important in maintaining arterial compliance. Longitudinal studies are needed to investigate the impact of the vitamin E isomers on bone and cardiovascular health.

Vitamin K and other markers of micronutrient status in morbidly obese patients before bariatric surgery.

BACKGROUND: Micronutrient deficiencies occur in morbidly obese patients. The aim of this study was to assess vitamin deficiencies prior to bariatric surgery including vitamin K about which there is little data in this population. METHODS: A prospective assessment of 118 consecutive patients was performed. Clinical allied with haematological and biochemical variables were measured. Micronutrients measured included vitamins K1 , PIVKA-II (protein-induced in vitamin K absence factor II), vitamin D, vitamin B12 (holotranscobalamin), iron, transferrin and folate. RESULTS: Patients were aged 49 ± 11 [mean (SD, standard deviation)] years, body mass index (BMI) 50 ± 8 kg/m(2), 66% female and 78% Caucasian. Hypertension was present in 47% and type 2 diabetes in 32%. Vitamin D supplements had been prescribed in 8%. Micronutrient insufficiencies were found for vitamin K (40%), vitamin D (92%) and vitamin B12 (25%), and also iron (44%) and folate (18%). Normocalcaemic vitamin D insufficiency with secondary hyperparathyroidism was present in 18%. Iron and transferrin levels were associated with age, sex and estimated glomerular filtration rate. Vitamin K levels were associated with age, and inversely with BMI and diabetes mellitus; and PIVKA-II with smoking, triglycerides and liver function markers. Vitamin D levels were associated with statin use and prescription of supplements and inversely with BMI. Vitamin B12 levels were associated with ethnicity and HbA1c. CONCLUSION: Micronutrient status shows differing relationships with age, gender and BMI. Vitamin K insufficiency was present in 40% and not related to deficiencies in other vitamins or micronutrients. Vitamin D and vitamin K supplementation should be considered prebariatric surgery in patients with diabetes or severe insulin resistance.

Association of bone turnover markers and arterial stiffness in pre-dialysis chronic kidney disease (CKD).

Vascular calcification (VC) is highly prevalent in CKD and leads to increased vascular stiffness and cardiovascular disease (CVD). Non-traditional cardiovascular risk factors include abnormal bone turnover and/or dysregulation of the calcification inhibitors, although their relative contribution remains unclear. We investigated the association between bone turnover, the calcification inhibitors (matrix gla protein; MGP and Fetuin-A), and the phosphate regulating hormone; fibroblast growth factor-23 (FGF-23) and arterial stiffness in pre-dialysis CKD patients. One hundred and forty-five patients with CKD stages 1-4 (74 M, 71 F) aged (mean [SD]) 53 [14] years were studied. Bone turnover markers (bone-specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase (TRACP)) and MGP, Fetuin-A and FGF-23 were determined. BMD was measured at the lumbar spine (LS), femoral neck (FN), forearm (FARM) and total hip (TH). Arterial stiffness was assessed by contour analysis of digital volume pulse (SI(DVP)). There was a significant positive correlation between TRACP:BALP ratio and SI(DVP) ( r=0.19, p=0.023). Following multi-linear regression analysis, significant associations were seen between serum BALP (p=0.037), TRACP (p=0.009) and TRACP:BALP ratio (p=0.001) and SI(DVP) independently of traditional CVD risk factors. No significant relationship between SI(DVP) and MGP, Fetuin-A and FGF-23 was observed. A significant negative correlation was seen between BMD at the FARM and SI(DVP) in CKD stage 4 (r=-0.35, p=0.024). The association remained significant following correction for age, gender and cardiovascular risk factors (p=0.029). Our data suggest a link between imbalances in bone turnover and arterial stiffness in pre-dialysis CKD. Longitudinal studies are needed to evaluate the clinical usefulness of these bone turnover markers as predictors of CVD in CKD.

Pharmacodynamic resistance to warfarin is associated with nucleotide substitutions in VKORC1.

BACKGROUND: Vitamin K epoxide reductase subunit 1 (VKORC1) is the molecular target of coumarin anticoagulants and mutations in VKORC1 have been identified previously in individuals who required high warfarin doses. OBJECTIVE: Detailed characterization of the relationship between variation in VKORC1 and the warfarin resistance phenotype. PATIENTS AND METHODS: Serum warfarin concentration and coagulation parameters were determined in 289 subjects who required warfarin doses >20 mg day(-1). The VKORC1 sequence was studied in selected study subjects. RESULTS: Twenty-eight out of 289 (10%) subjects had serum warfarin >2.3 mg L(-1) during stable therapeutic anticoagulation indicating pharmacodynamic warfarin resistance. Detailed analysis of 15 subjects from this group showed that eight out of 15 (53%) had nucleotide substitutions in VKORC1 predictive of p.V66M, p.L128R, p.V54L or p.D36Y. VKORC1 was normal in the remaining seven out of 15 (47%) subjects and in nine out of nine (100%) subjects with high warfarin dose requirement not caused by pharmacodynamic resistance. At referral, subjects with VKORC1 mutations received a median warfarin dose of 32 mg day(-1) (range 22-55) and had a median serum warfarin concentration of 4.6 mg L(-1) (range 2.6-9.0). VKORC1 substitutions were associated with a requirement for high warfarin doses but not with adverse clinical events. Family members with VKORC1 nucleotide substitutions and not receiving warfarin had undetectable PIVKA-II and K(1) epoxide (K(1)O). CONCLUSIONS: Nucleotide variations in VKORC1 are a common cause of pharmacodynamic warfarin resistance but are not associated with adverse outcome during anticoagulation. Mutations associated with warfarin resistance do not cause a discernible defect in VKORC1 reductase function.

A study of the prevalence of vitamin K deficiency in patients with cancer referred to a hospital palliative care team and its association with abnormal haemostasis.

BACKGROUND: Many patients with advanced cancer are malnourished. Anorexia is common, as is the use of chemotherapy, which may cause nausea and poor appetite. Ten per cent of these patients experience haemorrhagic events. AIM: Since vitamin K deficiency (VKD) causes bleeding, to establish the prevalence of VKD in patients with advanced cancer receiving palliative care. METHODS: Serum concentrations of vitamin K(1) and undercarboxylated factor II (PIVKA-II) were determined in 46 (17 male/29 female) inpatients aged 26-85 (mean 58) years. INR and liver function tests (bilirubin, ALT, GGT and ALP) were also performed. RESULTS: Vitamin K(1) was below the lower limit of the reference range (0.33 nmol/l) in 22% of patients. 78% of patients had some degree of functional VKD indicated by raised (>0.2 AU/ml) PIVKA-II. Six patients (13%) had a prolonged INR, all of whom had raised PIVKA-II and GGT; 4 also had vitamin K(1) <0.33 nmol/l. Three patients (6.5%) had clinically significant VKD characterised by INR >1.5, PIVKA-II >10 AU/ml, and undetectable vitamin K(1). CONCLUSIONS: Patients with advanced cancer are prone to VKD which, while usually subclinical, may develop to a clinically relevant prolongation of the INR. Serum measurements of vitamin K(1) and PIVKA-II can be used to detect VKD and monitor vitamin K status before an increased risk of bleeding develops.

Does a first trimester dating scan using crown rump length measurement reduce the rate of induction of labour for prolonged pregnancy? An uncompleted randomised controlled trial of 463 women.

OBJECTIVE: To evaluate the effect of a first trimester ultrasound dating scan on the rate of induction of labour for prolonged pregnancy. DESIGN: Randomised controlled trial to include 400 women in each arm of the trial. SETTING: Participating general practices and a district general teaching hospital. POPULATION: Women attending their general practitioner in the first trimester to confirm pregnancy, in whom a first trimester ultrasound scan was not indicated. METHODS: Women randomised to the study group (scan group) underwent an ultrasound dating scan between 8 and 12 weeks, measuring crown-rump length. The estimated date of delivery (EDD) was changed if there was a discrepancy of more than 5 days from the gestation, calculated from the last menstrual period (LMP). For the remaining women (no-scan group), gestation was determined using the LMP. MAIN OUTCOME MEASURES: The rate of induction of labour for prolonged pregnancy. RESULTS: Due to circumstances beyond the researchers' control, recruitment was abandoned when 463 women had been enrolled. The EDD was adjusted in 13 (5.7%) women in the scan group and in 2 (0.9%) in the no-scan group. There was no difference in the rate of induction for prolonged pregnancy between the scan (19 [8.2%]) and the no-scan (17 [7.4%]) groups (relative risk 1.10; 95% CI 0.59-2.07). CONCLUSIONS: Acknowledging the reduced numbers recruited for study, it is concluded that there is no evidence that a first trimester ultrasound dating scan reduces the rate of induction of labour for prolonged pregnancy and may result in a more expensive healthcare strategy.

Ethnic differences in osteocalcin gamma-carboxylation, plasma phylloquinone (vitamin K1) and apolipoprotein E genotype.

OBJECTIVE: To investigate plasma osteocalcin gamma-carboxylation and its relationship to plasma phylloquinone concentration and apolipoprotein E (apoE) genotype in women from three ethnic groups with differing osteoporotic fracture risk. DESIGN AND SUBJECTS: Fasted blood samples were collected from postmenopausal Gambian (n=50), British (n=31) and Chinese women (n=23), and 11 premenopausal women in each group from three cross-sectional studies. RESULTS: After adjustment for total osteocalcin, plasma undercarboxylated osteocalcin (adjusted ucOC) was lowest in Chinese and highest in British women postmenopause (British vs Chinese 103% higher, P<0.0001; Gambian vs Chinese 66% higher, P<0.01). No differences were observed premenopause. Within each ethnic group, adjusted ucOC was similar pre- and postmenopause. Postmenopause, plasma phylloquinone was higher in Chinese women (1.0 ng/ml) than in British (0.31 ng/ml) and Gambian women (0.36 ng/ml) (P<0.0001). Premenopause, plasma phylloquinone was higher in Gambian and Chinese women (0.6 ng/ml) than in British women (0.3 ng/ml; P=0.01). Plasma phylloquinone and adjusted ucOC were inversely related in postmenopausal British women (R2=32.4%; P=0.0008). ApoE4 frequency was Gambian 32.6%, British 13.8% and Chinese 6%. A lower adjusted ucOC was associated with apoE2 genotype in British and Chinese women. Ethnic differences in adjusted ucOC persisted after adjustment for phylloquinone and apoE genotype. CONCLUSION: These preliminary data indicate suboptimal vitamin K status in postmenopausal British compared to Chinese and Gambian women. Ethnic differences in apoE genotype may also influence osteocalcin gamma-carboxylation status. The study highlights the need for larger epidemiological investigations of ethnic differences in vitamin K status and the possible implications to bone health.

Plasma phylloquinone (vitamin K1) concentration and its relationship to intake in a national sample of British elderly people.

Plasma phylloquinone (vitamin K1) concentration was examined according to season, socio-demographic and lifestyle factors and phylloquinone intake in a nationally representative sample of British people aged 65 years and over from the 1994-5 National Diet and Nutrition Survey. Values for both plasma phylloquinone concentration and phylloquinone intake were available from 1076 participants (561 men, 515 women). Eight hundred and thirty-four were living in private households, 242 in residential or nursing homes. Weighted geometric mean plasma phylloquinone concentrations were 0.36 (inner 95% range [corrected] 0.06, 2.01) and 0.24 (inner 94% range [corrected] 0.06, 0.96) nmol/l in free-living and institution samples respectively. Plasma phylloquinone concentrations did not generally differ between men and women, although values in free-living people were significantly lower during autumn and winter (October to March). Plasma phylloquinone concentration was not significantly associated with age. Plasma phylloquinone concentrations were positively correlated with phylloquinone intake in free-living men and women (r 0.18 and 0.30 respectively, both P<0.001). Stepwise multiple regression analysis found that 11 % of the variation in plasma phylloquinone concentration was explained by phylloquinone intake, season and plasma triacylglycerol concentration. After adjustment for age and corresponding nutrient intakes, plasma phylloquinone concentration was significantly associated (each P<0.01) with plasma concentrations of triacylglycerol, cholesterol, retinol and 25-hydroxyvitamin D in free-living women but not men, and with plasma concentrations of carotenes, alpha- and gamma-tocopherols and lutein in free-living men and women. The possibility of concurrent low fat-soluble vitamin status in elderly populations may be a cause for concern.

Structural basis of the enhanced stability of a mutant ribozyme domain and a detailed view of RNA--solvent interactions.

BACKGROUND: The structure of P4-P6, a 160 nucleotide domain of the self-splicing Tetrahymena thermophila intron, was solved previously. Mutants of the P4-P6 RNA that form a more stable tertiary structure in solution were recently isolated by successive rounds of in vitro selection and amplification. RESULTS: We show that a single-site mutant (Delta C209) possessing greater tertiary stability than wild-type P4-P6 also crystallizes much more rapidly and under a wider variety of conditions. The crystal structure provides a satisfying explanation for the increased stability of the mutant; the deletion of C209 allows the adjacent bulged adenine to enter the P4 helix and form an A-G base pair, presumably attenuating the conformational flexibility of the helix. The structure of another mutant (Delta A210) was also solved and supports this interpretation. The crystals of Delta C209 diffract to a higher resolution limit than those of wild-type RNA (2.25 A versus 2.8 A), allowing assignment of innersphere and outersphere coordination contacts for 27 magnesium ions. Structural analysis reveals an intricate solvent scaffold with a preponderance of ordered water molecules on the inside rather than the surface of the folded RNA domain. CONCLUSIONS: In vitro evolution facilitated the identification of a highly stable, structurally homogeneous mutant RNA that was readily crystallizable. Analysis of the structure suggests that improving RNA secondary structure can stabilize tertiary structure and perhaps promote crystallization. In addition, the higher resolution model provides new details of metal ion-RNA interactions and identifies a core of ordered water molecules that may be integral to RNA tertiary structure formation.

Human herpesvirus 6 and multiple sclerosis: systemic active infections in patients with early disease.

By means of immunohistochemical staining, cells actively infected with human herpesvirus 6 (HHV-6) were found in central nervous system tissues from 8 (73%) of 11 patients with definite multiple sclerosis (MS). Interestingly, 17 (90%) of 19 tissue sections showing active demyelination were positive for HHV-6-infected cells compared with only 3 (13%) of 23 tissue sections free of active disease (P<.0001). Central nervous system tissues from 2 of 28 normal persons and patients with other inflammatory demyelinative diseases were positive for HHV-6-infected cells (P<.0001), and the 2 positive cases were diagnosed as having HHV-6 leukoencephalitis. By use of a rapid culture assay, blood samples from 22 (54%) of 41 patients with definite MS were found to contain active HHV-6 infections, compared with 0 of 61 normal controls (P<.0001). No significant difference was found between HHV-6 viremia-positive and HHV-6 viremia-negative MS patients with respect to type of disease (relapsing/remitting or progressive). In contrast, patients with active HHV-6 viremia were significantly younger and had shorter durations of disease than did HHV-6 viremia-negative patients.

Identification of lipoprotein homologues of pneumococcal PsaA in the equine pathogens Streptococcus equi and Streptococcus zooepidemicus.

Streptococcus equi and Streptococcus zooepidemicus are major etiological agents of upper and lower airway disease in horses. Despite the considerable animal suffering and economic burden associated with these diseases, the factors that contribute to the virulence of these equine pathogens have not been extensively investigated. Here we demonstrate the presence of a homologue of the Streptococcus pneumoniae PsaA protein in both of these equine pathogens. Inhibition of signal peptide processing by the antibiotic globomycin confirmed the lipoprotein nature of the mature proteins, and surface exposure was confirmed by their release from intact cells by mild trypsinolysis.

Compilation of a provisional UK database for the phylloquinone (vitamin K1) content of foods.

This paper reports the compilation of a food composition database for phylloquinone (vitamin K1) derived from the direct analysis of foods, recipe calculation and the assignment of values based on food similarities. All the basic and other food items used in these calculations had been analysed by HPLC and about 170 of the items had been obtained and assayed in the UK. Recipe calculations took account of the cooking method and changes in water and fat content. Currently, approximately 1501 food items with Royal Society of Chemistry/Ministry of Agriculture, Fisheries and Food food codes have been allocated a vitamin K1 value, and a further 282 new recipe codes are included in the database. Representative values from each food group are reported together with an indication of the potential variation. Detailed examples of some recipe calculations are included, and also the impact of changing the type of fat in recipes. Vitamin K1 is associated with, and most abundant in, photosynthetic tissues of plants. Accordingly, the highest concentrations (3000-6000 micrograms/kg) are found in dark-green leafy vegetables and herbs, such as kale, parsley, spinach and green cabbage. Intermediate concentrations (1000-2000 micrograms/kg) are found in plants with paler leaves such as white cabbage and lettuce or in green, non-leafy vegetables such as broccoli and brussel sprouts. Fats and oils contain variable amounts of vitamin K1 with the highest concentrations (300-1300 micrograms/kg) in soyabean, rapeseed and olive oils and the margarines based on them. Other foods such as dairy products, meat dishes and cereal-based foods (bread, biscuits, cakes, desserts etc.), although not in themselves particularly rich in vitamin K1 (< 200 micrograms/kg), may contribute significantly to intakes when consumption of green vegetables is poor. Within the scope of this present study, it has not been possible to address issues such as inter-sample variability, losses during storage or the bioavailability from different foods and further work on these aspects is needed.

X-Ray crystal structure and molecular dynamics simulations of silver hake parvalbumin (Isoform B).

Parvalbumins constitute a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DF, Hartman KL, Laue TM, Nelson DJ, 1997, Protein Sci 7:2397-2408), we presented the sequence of the major parvalbumin isoform from the silver hake (Merluccius bilinearis) and presented spectroscopic and structural information on the excised "EF-hand" portion of the protein. In this study, the X-ray crystal structure of the silver hake major parvalbumin has been determined to high resolution, in the frozen state, using the molecular replacement method with the carp parvalbumin structure as a starting model. The crystals are orthorhombic, space group C2221, with a = 75.7 A, b = 80.7 A, and c = 42.1 A. Data were collected from a single crystal grown in 15% glycerol, which served as a cryoprotectant for flash freezing at -188 degrees C. The structure refined to a conventional R-value of 21% (free R 25%) for observed reflections in the range 8 to 1.65 A [1 > 2sigma(I)]. The refined model includes an acetylated amino terminus, 108 residues (characteristic of a beta parvalbumin lineage), 2 calcium ions, and 114 water molecules per protein molecule. The resulting structure was used in molecular dynamics (MD) simulations focused primarily on the dynamics of the ligands coordinating the Ca2+ ions in the CD and EF sites. MD simulations were performed on both the fully Ca2+ loaded protein and on a Ca2+ deficient variant, with Ca2+ only in the CD site. There was substantial agreement between the MD and X-ray results in addressing the issue of mobility of key residues in the calcium-binding sites, especially with regard to the side chain of Ser55 in the CD site and Asp92 in the EF site.