Vitamin D metabolism in critically ill patients with acute kidney injury: a prospective observational study.

BACKGROUND: Vitamin D deficiency in critically ill patients is associated with poor outcomes, and vitamin D supplementation is recommended for patients with chronic kidney disease. Whether acute kidney injury (AKI) is associated with altered Vitamin D metabolism is unknown. We aimed to compare the longitudinal profiles of serum 25(OH)D and 1,25(OH)2D concentrations in critically ill patients with and without moderate to severe AKI and explore the impact of renal recovery and parathyroid hormone (PTH). METHODS: In this prospective, observational study in two centres in the UK, critically ill patients with and without AKI underwent serial measurement of serum 25(OH)D and 1,25(OH)2D and plasma PTH concentrations for 5 days. Linear mixed model analysis and sensitivity analyses were performed. RESULTS: Serial data of 137 patients were analysed. Seventy-one patients had AKI stage II/III of whom 23 recovered kidney function during the 5-day study period; 66 patients did not have AKI at enrolment of whom 14 developed new AKI. On day of enrolment, patients' serum 25(OH)D concentrations were low (median 18 nmol/L) but there was no significant difference between patients with and without AKI. Median serum 1,25(OH)2D levels were significantly lower in patients with AKI II/III (41 pmol/L [IQR 26, 58]) compared to similarly unwell patients without AKI (54 pmol/L [IQR 33, 69]) during the 5-day period. Recovery of kidney function in patients with AKI was associated with a rise in 1,25(OH)2D concentrations. Plasma PTH results were impacted by serum calcium and magnesium levels but not associated with 1,25(OH)2D levels. CONCLUSIONS: Critically ill patients with moderate-to-severe AKI have significantly lower serum 1,25(OH)2D concentrations than similarly sick patients without AKI but there was no difference in serum 25(OH)D concentrations. Recovery of AKI was associated with a rise in serum 1,25(OH)2D concentrations. More research is needed to investigate the health benefits and safety of supplementation with active vitamin D in critically ill patients with moderate-to-severe AKI. Trial registration Clinicaltrials.gov (NCT02869919), registered on 16 May 2016.

The additive effect of vitamin K supplementation and bisphosphonate on fracture risk in post-menopausal osteoporosis: a randomised placebo controlled trial.

This study assessed whether vitamin K, given with oral bisphosphonate, calcium and/or vitamin D has an additive effect on fracture risk in post-menopausal women with osteoporosis. No difference in bone density or bone turnover was observed although vitamin K1 supplementation led to a modest effect on parameters of hip geometry. PURPOSE: Some clinical studies have suggested that vitamin K prevents bone loss and may improve fracture risk. The aim was to assess whether vitamin K supplementation has an additive effect on bone mineral density (BMD), hip geometry and bone turnover markers (BTMs) in post-menopausal women with osteoporosis (PMO) and sub-optimum vitamin K status receiving bisphosphonate, calcium and/or vitamin D treatment. METHODS: We conducted a trial in 105 women aged 68.7[12.3] years with PMO and serum vitamin K1 ≤ 0.4 µg/L. They were randomised to 3 treatment arms; vitamin K1 (1 mg/day) arm, vitamin K2 arm (MK-4; 45 mg/day) or placebo for 18 months. They were on oral bisphosphonate and calcium and/or vitamin D. We measured BMD by DXA, hip geometry parameters using hip structural analysis (HSA) software and BTMs. Vitamin K1 or MK-4 supplementation was each compared to placebo. Intention to treat (ITT) and per protocol (PP) analyses were performed. RESULTS: Changes in BMD at the total hip, femoral neck and lumbar spine and BTMs; CTX and P1NP did not differ significantly following either K1 or MK-4 supplementation compared to placebo. Following PP analysis and correction for covariates, there were significant differences in some of the HSA parameters at the intertrochanter (IT) and femoral shaft (FS): IT endocortical diameter (ED) (% change placebo:1.5 [4.1], K1 arm: -1.02 [5.07], p = 0.04), FS subperiosteal/outer diameter (OD) (placebo: 1.78 [5.3], K1 arm: 0.46 [2.23] p = 0.04), FS cross sectional area (CSA) (placebo:1.47 [4.09],K1 arm: -1.02[5.07], p = 0.03). CONCLUSION: The addition of vitamin K1 to oral bisphosphonate with calcium and/or vitamin D treatment in PMO has a modest effect on parameters of hip geometry. Further confirmatory studies are needed. TRIAL REGISTRATION: The study was registered at Clinicaltrial.gov:NCT01232647.

Prevalence of elevated serum concentrations of biotin in patients from South East England, Korea, Singapore and Thailand and risk of immunoassay interference.

Biotin interference in immunoassays using biotin-streptavidin binding technology is well recognised by clinical laboratories, though the prevalence of elevated biotin in patient populations is largely unknown. We determined serum biotin concentrations in 4385 patient samples received sequentially by 6 laboratories for routine immunoassay analysis in England, and Korea, Singapore and Thailand (3 countries within the Asia Pacific region, APAC). Samples were initially analysed using a research use-only immunoassay, with those identified as having potentially elevated biotin concentrations referred for definitive analysis by LC-MS/MS. The prevalence of elevated serum biotin was 0.4% and 0.6% for England and APAC, respectively (range 10.0-129.0 µg/L). Our data adds to a report from a different region of England and is the first for APAC. Laboratories and clinicians benefit from an awareness of the prevalence of elevated serum biotin, which coupled with an understanding of the threshold at which interference occurs, reduces clinical impact of analytical error.

Age- and ethnicity-related reference intervals for serum vitamin B12.

BACKGROUND: Age and ethnicity are known to influence serum vitamin B12 (B12) concentration, yet universal reference intervals (RIs) are typically applied by laboratories. Both lower and upper RI limits for B12 are clinically relevant. Low values suggest deficiency leading to anemia and/or neurological impairment, while high values are not always an innocuous consequence of high B12 intake but are associated with some cancers, autoimmune, liver, and renal diseases. This work aimed to establish age- and ethnicity-related RIs for B12 using a modified indirect method based on Hoffmann's approach. METHODS: A total of 72,091 anonymized B12 results (Jan 2018-Nov 2019) were analyzed from an ethnically-diverse South-East London general practice patient population. Patients belonged to five ethnic groups: Asian, Black, White, Mixed, or Other. Multiple records for the same patient and results with missing ethnicity were excluded from the analysis of adult RIs. B12 analyses were performed using ARCHITECT® (Abbott Diagnostics). RESULTS: B12 was significantly higher in Black compared with Asian and White adults. There were no differences in B12 between Asian and White adults. Children (all ethnicities) between 2 and 5 years old had the highest B12. Because of the small number of children (up to the age of 13) in each ethnic-related age category, all ethnic groups were combined to obtain age-related RIs. The children's RIs ranged from 159 to 1025 pmol/L for 0-1-year-olds to 276-1102 pmol/L for 2-5-year-olds. The RIs for Black and White/Asian people >13 years of age were 166-805 pmol/L and 134-511 pmol/L respectively. CONCLUSIONS: The application of age- and ethnicity-appropriate RIs into diagnostic practice will provide a more accurate evaluation of B12 status when using the B12 test alone or in combination with other markers.

Exclusively breastmilk-fed preterm infants are at high risk of developing subclinical vitamin K deficiency despite intramuscular prophylaxis at birth.

BACKGROUND: There is near-global consensus that all newborns be given parenteral vitamin K1 (VK1 ) at birth as prophylaxis against VK deficiency bleeding (VKDB). Breastmilk has a low VK content and cases of late VKDB are reported in exclusively breastmilk-fed preterm infants despite VK prophylaxis at birth. OBJECTIVES: To assess the prevalence of functional VK insufficiency in preterm infants based on elevated under-γ-carboxylated (Glu) species of Gla proteins, factor II (PIVKA-II), and osteocalcin (GluOC), synthesized by liver and bone, respectively. PATIENTS/METHODS: Prospective, multicenter, observational study in preterm infants born <33 weeks' gestation. Blood samples and dietary history were collected before hospital discharge, and after discharge at 2-3 months' corrected age. Outcome measures were serum VK1 , PIVKA-II, and %GluOC (GluOC as a percentage of the sum of GluOC plus GlaOC) compared between exclusively breastmilk-fed and formula/mixed-fed infants after discharge. RESULTS: After discharge, breastmilk-fed babies had significantly lower serum VK1 (0.15 vs. 1.81 µg/L), higher PIVKA-II (0.10 vs. 0.02 AU/ml) and higher %GluOC (63.6% vs. 8.1%) than those receiving a formula/mixed-feed diet. Pre-discharge (based on elevated PIVKA-II), only one (2%) of 45 breastmilk-fed infants was VK insufficient. After discharge, eight (67%) of 12 exclusively breastmilk-fed babies were VK insufficient versus only one (4%) of 25 formula/mixed-fed babies. CONCLUSIONS: Preterm infants who remain exclusively or predominantly human breastmilk-fed after neonatal unit discharge are at high risk of developing subclinical VK deficiency in early infancy. Routine postdischarge VK1 supplementation of breastfed infants to provide intakes comparable to those from formula milks should prevent this deficiency.

Characterization and traceability of two generations of standard reference material for the measurement of vitamin K1 (phylloquinone) at endogenous concentrations in human plasma and serum.

Vitamin K is an essential micronutrient required for blood coagulation, regulation of vascular calcification and bone mineralization. Plasma and serum measurements of vitamin K1 (phylloquinone, K1 ) made using high-performance liquid chromatography with fluorescence detection, or tandem mass spectrometry are used clinically and in population studies to assess vitamin K status. Standard reference materials provide a validation tool for laboratories, helping assure clinical diagnosis and the comparability of data from different populations. We manufactured two K1 standard reference materials, in 2009 (KEQAS SRM-001) and in 2019 (KEQAS SRM-002). The target concentrations of K1 were assigned to each SRM using the All Laboratory Trimmed Mean of results reported by selected laboratories enrolled in the Vitamin K External Quality Assurance Scheme (KEQAS). The assigned concentrations of K1 for KEQAS SRM-001 and SRM-002 were 0.25 and 0.36 µg/L respectively. In 2019 KEQAS SRM-001 was re-analysed simultaneously with KEQAS SRM-002 to provide traceability between the two standards, therefore aiding comparability of analysis performed using these materials. Both standards were stored as aliquots at -80°C in the dark; annual re-analysis of the materials indicated that K1 is stable for at least 12 years in these conditions.

Macro-B12 masking B12 deficiency.

In clinical practice, the finding of an elevated serum B12 concentration is often the consequence of supplementation with B12 in either oral form or injections. Also, elevated serum B12 may be associated with underlying disorders, like liver diseases or a (haematologic) malignancy. Only a few studies have shown that it may also be the consequence of complex formation of B12-vitamin binding proteins with immunoglobulins, the so-called macro-B12 We describe a young woman who previously was diagnosed with B12 deficiency, and in whom, after cessation of B12 injection treatment, neurologic symptoms re-appeared, and despite this, repeatedly elevated serum B12 concentrations above the upper limit of the assay were found. We demonstrated that this was caused by the presence of macro-B12, which not only resulted in erroneous and longstanding elevated serum B12, but also masked her underlying B12 deficiency.

Vitamin B12 and Folate Markers Are Associated with Insulin Resistance During the Third Trimester of Pregnancy in South Asian Women, Living in the United Kingdom, with Gestational Diabetes and Normal Glucose Tolerance.

BACKGROUND: Gestational diabetes mellitus (GDM) can adversely affect the health of the developing fetus. Women of South Asian origin are particularly at risk of developing GDM. Insulin resistance (IR) contributes to the etiology of GDM, and although studies have shown associations of vitamin B12 (B12) and folate status with GDM and IR, only a limited number of B12 and folate markers have been used. OBJECTIVE: We used a comprehensive panel of B12 and folate markers to examine their association with IR in pregnant women with diet-controlled GDM and normal glucose tolerance (NGT). METHODS: In this cross-sectional study, 59 British-Bangladeshi women (24 GDM and 35 NGT) with a mean age of 29 y, BMI (in kg/m2) 26.7 and gestational age 33 wk were recruited. Serum total B12, holotranscobalamin, folate, methylmalonic acid, plasma homocysteine, 5-methyltetrahydrofolate, and red cell folate (RCF) were measured along with other parameters. The independent sample t-test and chi-squared test were used to assess differences in markers between GDM and NGT women. Spearman's test was used to look for correlations. A simple multiple regression analysis was used to investigate if markers of B12 and folate status predicted IR, using the HOMA-IR and adjusting for age, GDM status, and BMI. RESULTS: There were no differences in concentrations of B12 and folate markers between GDM and NGT women. In Spearman's analysis HOMA-IR correlated negatively with total serum B12 (P < 0.001) and holotranscobalamin (P < 0.05), and positively with BMI (P < 0.001), blood pressure (P < 0.05) and triglycerides (P < 0.05) in all women. MMA did not correlate with any of the B12 markers. In regression analysis, total B12 (ß = -0.622, P = 0.004), RCF (ß = 0.387, P = 0.018), and BMI (ß = 0.024, P < 0.001) were the significant predictors of HOMA-IR variance. CONCLUSIONS: Significant associations between markers of B12 and folate status with HOMA-IR were found during the third trimester in British-Bangladeshi women. B12 markers correlated poorly with each other.

Ethnicity influences total serum vitamin B12 concentration: a study of Black, Asian and White patients in a primary care setting.

AIMS: A growing body of evidence suggests that ethnicity and race influence vitamin B12 metabolism and status yet clinical awareness of this is poor, causing doubts regarding diagnosis and treatment. Moreover, deficiency and insufficiency cut-offs are universally applied for this test in most diagnostic settings. The objective of this study was to assess serum vitamin B12 concentrations in Black, Asian and White primary care patients in London, UK, particularly in patients of Black or Black British ethnic origin and establish if there is a need for specific reference ranges. METHODS: Serum B12 results from 49 414 patients were processed between January 2018 and November 2019 using the Architect assay (Abbott Diagnostics) at St. Thomas' Hospital, London, UK. Age, sex and ethnicity data were collected from the laboratory Health Informatics Team. RESULTS: Black patients (n=13 806) were found to have significantly higher serum vitamin B12 concentration across all age groups and both sexes, especially Nigerian patients (median B12 505 pmol/L,IQR: 362-727, n=891), compared with Asian and White ethnic groups (p<0.001). Binary logistic regression analysis revealed that the Black or Black British ethnic group had the strongest association with elevated serum B12 (>652 pmol/L) (adjusted OR 3.38, 95% CI 3.17 to 3.61, p<0.0001). CONCLUSIONS: It is likely that a combination of genetic and acquired/environmental factors are responsible for the ethnic differences in serum B12. This suggests that there is a need for ethnic-specific reference ranges with indications for the incorporation of age and sex too.

Vitamin B12 status in health and disease: a critical review. Diagnosis of deficiency and insufficiency - clinical and laboratory pitfalls.

Vitamin B12 (cobalamin) is an essential cofactor for two metabolic pathways. It is obtained principally from food of animal origin. Cobalamin becomes bioavailable through a series of steps pertaining to its release from dietary protein, intrinsic factor-mediated absorption, haptocorrin or transcobalamin-mediated transport, cellular uptake, and two enzymatic conversions (via methionine synthase and methylmalonyl-CoA-mutase) into cofactor forms: methylcobalamin and adenosylcobalamin. Vitamin B12 deficiency can masquerade as a multitude of illnesses, presenting different perspectives from the point of view of the hematologist, neurologist, gastroenterologist, general physician, or dietician. Increased physician vigilance and heightened patient awareness often account for its early presentation, and testing sometimes occurs during a phase of vitamin B12 insufficiency before the main onset of the disease. The chosen test often depends on its availability rather than on the diagnostic performance and sensitivity to irrelevant factors interfering with vitamin B12 markers. Although serum B12 is still the most commonly used and widely available test, diagnostics by holotranscobalamin, serum methylmalonic acid, and plasma homocysteine measurements have grown in the last several years in routine practice. The lack of a robust absorption test, coupled with compromised sensitivity and specificity of other tests (intrinsic factor and gastric parietal cell antibodies), hinders determination of the cause for depleted B12 status. This can lead to incorrect supplementation regimes and uncertainty regarding later treatment. This review discusses currently available knowledge on vitamin B12, informs the reader about the pitfalls of tests for assessing its deficiency, reviews B12 status in various populations at different disease stages, and provides recommendations for interpretation, treatment, and associated risks. Future directions for diagnostics of B12 status and health interventions are also discussed.

Evaluating the Diagnostic Value of a Combined Indicator of Vitamin B12 Status (cB12) Throughout Pregnancy.

BACKGROUND: Inadequate provision of vitamin B12 during pregnancy is associated with a number of adverse maternal and fetal outcomes. We set out to (1) suggest pregnancy-specific reference ranges for a range of biomarkers of vitamin B12; (2) assess the temporal behaviors of these markers over the course of pregnancy; and (3) test whether any biomarkers, including the genetic marker HIBCH rs291466 strongly associated with MMA measured early in pregnancy could reliably and significantly predict future B12 status within a healthy UK population of pregnant women. MATERIALS AND METHODS: We used existing biobank samples from the placebo arm of the UK Selenium in PRegnancy Intervention (SPRINT) study, to generate biochemical data for serum folate, B12, holotranscobalamin (HoloTC), total homocysteine (tHcy), and MMA, calculate cB12, and genotyped the polymorphism rs291466 in gene HIBCH on a total of n=114 women across trimesters 1-3 of their pregnancy. We performed a series of exploratory cross-sectional and longitudinal analyses to investigate levels at each trimester, suggest references ranges, evaluate changes and correlations between the B12 biomarkers, and assess the predictive capabilities of each biomarker from 12-weeks to 35-weeks of gestation. RESULTS: Significant changes in all vitamin B12 biomarker values were observed over the three trimesters (P < 0.05). Our study shows that cB12 values were largely constant and stable throughout trimester 1 (T1) and T2 (i.e., up to week 20), but declined significantly in T3 (-66% | P < 0.001). Yet, cB12 generally remained within the normal boundaries. We identified pregnancy and trimester-specific reference ranges for each biomarker at each trimester, notably for total serum B12. This marker fell below the recommended cut-offs in 1/3 of the cohort at the third trimester, contrasting other markers (mostly normal). Our multivariate analyses indicated that none of the biomarkers could reliably and accurately predict any other biomarkers than themselves later in pregnancy. Yet, HoloTC seems to be a promising predictor within the limitations of our cohort, constituted of B12-replete individuals. Most notably, cB12 did not significantly predict itself between trimesters. Finally, we show that the HIBCH variant has little predictive power for MMA or cB12 as it does not explain the significant increase in MMA concentrations nor the decline of cB12 throughout pregnancy. CONCLUSION: Trimester-specific reference ranges for biomarkers of vitamin B12 in normal pregnancy are suggested. However, these biomarkers have limited predictive value in identifying mothers at elevated risk of vitamin B12 insufficiency/deficiency during pregnancy.

Serum vitamin K1 (phylloquinone) is associated with fracture risk and hip strength in post-menopausal osteoporosis: A cross-sectional study.

PURPOSE: Vitamin K may play a potential role in bone metabolism, although further evidence is needed. The mechanisms behind its skeletal effects and optimum intake for maintaining bone health remain poorly defined. To elucidate these two issues, we investigated the association between circulating vitamin K1 (phylloquinone) concentrations with fracture risk, bone mineral density (BMD), hip geometry and plasma dephospho-uncarboxylated-Matrix Gla Protein (dp-ucMGP), an extra-hepatic vitamin K dependent protein (VKDP), in post-menopausal osteoporosis (PMO). METHODS: We studied 374 women aged (mean [SD]) 68.7[12.3] years with PMO. Information including demographics, lifestyle habits and previous fractures was captured through a questionnaire. Serum was analysed for vitamin K1. BMD at the lumbar spine (LS), total hip (TH) and femoral neck (FN) (n = 277) and hip structural analysis (HSA) parameters (n = 263) were derived from DXA scans. VKDPs including undercarboxylated prothrombin (PIVKA-II) and dp-ucMGP were measured in a sub-group (n = 130). RESULTS: Serum vitamin K1 was significantly lower in the group with fractures (prevalent fractures: 0.53 [0.41], no fractures; 0.65 [0.66] µg/L, p = 0.04) and independently associated with fracture risk. The adjusted odds ratio (95% CI) per µg/L increase in vitamin K1 was 0.550 (0.310-0.978, p = 0.042). Among the HSA parameters, serum vitamin K1 was positively associated with cross-sectional area (CSA) (p = 0.02), cross sectional moment of inertia (CSMI) (p = 0.028) and section modulus (Z) (p = 0.02) at the narrow neck (NN) of femur. Dp-ucMGP was detectable in 97 (75%) participants with serum vitamin K1 of 0.26 [0.15] µg/L, whilst PIVKA-II was above the clinical threshold in only 3.8%. CONCLUSIONS: Our data suggest that the positive effect of vitamin K on fracture risk may be related to its effects on bone strength. Higher concentrations of serum vitamin K1 may be required for vitamin K's skeletal effects compared to coagulation. Further prospective or interventional studies are needed for confirmation and should include measures of bone quality.

Micronutrients in critically ill patients with severe acute kidney injury - a prospective study.

Malnutrition is common in patients with acute kidney injury (AKI) and the risk of mortality is high, especially if renal replacement therapy is needed. Between April 2013 through April 2014, we recruited critically ill adult patients (≥18 years) with severe AKI in two University hospitals in London, UK, and measured serial plasma concentrations of vitamin B1, B6, B12, C and D, folate, selenium, zinc, copper, iron, carnitine and 22 amino acids for six consecutive days. In patients receiving continuous renal replacement therapy (CRRT), the concentrations of the same nutrients in the effluent were also determined. CRRT patients (n = 31) had lower plasma concentrations of citrulline, glutamic acid and carnitine at 24 hrs after enrolment and significantly lower plasma glutamic acid concentrations (74.4 versus 98.2 µmol/L) at day 6 compared to non-CRRT patients (n = 24). All amino acids, trace elements, vitamin C and folate were detectable in effluent fluid. In >30% of CRRT and non-CRRT patients, the plasma nutrient concentrations of zinc, iron, selenium, vitamin D3, vitamin C, trytophan, taurine, histidine and hydroxyproline were below the reference range throughout the 6-day period. In conclusion, altered micronutrient status is common in patients with severe AKI regardless of treatment with CRRT.

The Role of Matrix Gla Protein (MGP) in Vascular Calcification.

Matrix Gla protein (MGP) is a vitamin K-dependent protein, which is synthesized in bone and many other mesenchymal cells, which is also highly expressed by vascular smooth muscle cells (VSMCs) and chondrocytes. Numerous studies have confirmed that MGP acts as a calcification-inhibitor although the mechanism of action is still not fully understood. The modulation of tissue calcification by MGP is potentially regulated in several ways including direct inhibition of calcium-phosphate precipitation, the formation of matrix vesicles (MVs), the formation of apoptotic bodies (ABs), and trans-differentiation of VSMCs. MGP occurs as four species, i.e. fully carboxylated (cMGP), under-carboxylated, i.e. poorly carboxylated (ucMGP), phosphorylated (pMGP), and non-phosphorylated (desphospho, dpMGP). ELISA methods are currently available that can detect the different species of MGP. The expression of the MGP gene can be regulated via various mechanisms that have the potential to become genomic biomarkers for the prediction of vascular calcification (VC) progression. VC is an established risk factor for cardiovascular disease and is particularly prevalent in those with chronic kidney disease (CKD). The specific action of MGP is not yet clearly understood but could be involved with the functional inhibition of BMP-2 and BMP-4, by blocking calcium crystal deposition and shielding the nidus from calcification.

Laboratory assessment of vitamin K status.

Vitamin K is required for the É£-carboxylation of specific glutamic acid residues within the Gla domain of the 17 vitamin K-dependent proteins (VKDPs). The timely detection and correction of vitamin K deficiency can protect against bleeding. Vitamin K also plays a role in bone metabolism and vascular calcification. Patients at increased risk of vitamin K deficiency include those with a restricted diet or malnutrition, lipid malabsorption, cancer, renal disease, neonates and the elderly. Coagulation assays such as the prothrombin time have been used erroneously as indicators of vitamin K status, lacking sufficient sensitivity and specificity for this application. The measurement of phylloquinone (K1) in serum is the most commonly used marker of vitamin K status and reflects abundance of the vitamin. Concentrations <0.15 µg/L are indicative of deficiency. Disadvantages of this approach include exclusion of the other vitamin K homologues and interference from recent dietary intake. The cellular utilisation of vitamin K is determined through measurement of the prevalence of undercarboxylated VKDPs. Most commonly, undercarboxylated prothrombin (Protein Induced by Vitamin K Absence/antagonism, PIVKA-II) is used (reference range 17.4-50.9 mAU/mL (Abbott Architect), providing a retrospective indicator of hepatic vitamin K status. Current clinical applications of PIVKA-II include supporting the diagnosis of vitamin K deficiency bleeding of the newborn, monitoring exposure to vitamin K antagonists, and when used in combination with α-fetoprotein, as a diagnostic marker of hepatocellular carcinoma. Using K1 and PIVKA-II in tandem is an approach that can be used successfully for many patient cohorts, providing insight into both abundance and utilisation of the vitamin.

A genetic epidemiological study in British adults and older adults shows a high heritability of the combined indicator of vitamin B12 status (cB12) and connects B12 status with utilization of mitochondrial substrates and energy metabolism.

Vitamin B12 deficiency is common among older adults. However, the most commonly used marker of deficiency, total serum vitamin B12 (B12), is not sensitive enough to diagnose true deficiency in a significant proportion of the population. The combined indicator of B12 status (cB12), formulated as a composite score of various biomarkers of vitamin B12 status (which also accounts for low folate status and age) has been shown to offer a more robust and powerful test to diagnose B12 deficiency. There are no epidemiological studies of cB12 variability in older adults. We carried out a twin study to characterize the relative contribution of heritable (h2) and environmental factors to the observed variability in cB12 score in an adult and older adult population (n=378). Furthermore, we tested for association between variability in cB12 and candidate polymorphisms and genes previously associated with B12 biomarker levels characterized in silico the mechanism linking the genetic variants and cB12 variability. We found the variability in cB12 and its constituents to be highly heritable (h2=55%-64%). The single nucleotide polymorphism rs291466 in HIBCH, previously associated with variation in MMA, was significantly associated with cB12 (R2=5%, P=5E-04). Furthermore, variants in MTRR, MMAB and MUT, underlying inborn errors of B12 metabolism, were nominally associated with variation in cB12. Pathway accompanied by expression quantitative trait loci analysis revealed that HIBCH rs291466 influences the concentration of MMA via the valine degradation pathway. Our study provides etiological insight into how B12 deficiency can manifest into impaired mitochondrial function through perturbations in mitochondrial "fuel" usage.

Laboratory assessment of folate (vitamin B9) status.

Folate (vitamin B9) plays a crucial role in fundamental cellular processes, including nucleic acid biosynthesis, methyl group biogenesis and amino acid metabolism. The detection and correction of folate deficiency prevents megaloblastic anaemia and reduces the risk of neural tube defects. Coexisting deficiencies of folate and vitamin B12 are associated with cognitive decline, depression and neuropathy. Folate deficiency and excess has also been implicated in some cancers. Excessive exposure to folic acid, a synthetic compound used in supplements and fortified foods, has also been linked to adverse health effects. Of at least three distinct laboratory markers of folate status, it is the total abundance of folate in serum/plasma that is used by the majority of laboratories. The analysis of folate in red cells is also commonly performed. Since the folate content of red cells is fixed during erythropoiesis, this marker is indicative of folate status over the preceding ~4 months. Poor stability, variation in polyglutamate chain length and unreliable extraction from red cells are factors that make the analysis of folate challenging. The clinical use of measuring specific folate species has also been explored. 5-Methyltetrahydrofolate, the main form of folate found in blood, is essential for the vitamin B12-dependent methionine synthase mediated remethylation of homocysteine to methionine. As such, homocysteine measurement reflects cellular folate and vitamin B12 use. When interpreting homocysteine results, age, sex and pregnancy, specific reference ranges should be applied. The evaluation of folate status using combined markers of abundance and cellular use has been adopted by some laboratories. In the presence of discordance between laboratory results and strong clinical features of deficiency, treatment should not be delayed. High folate status should be followed up with the assessment of vitamin B12 status, a review of previous results and reassessment of folic acid supplementation regime.

The Effects of Parenteral K1 Administration in Pseudoxanthoma Elasticum Patients Versus Controls. A Pilot Study.

INTRODUCTION: Pseudoxanthoma elasticum (PXE) is a rare disease caused by mutations in the ABCC6 gene. Vitamin K1 is involved in the posttranslational carboxylation of some proteins related to inhibition of the calcification process. Our aim was to investigate, in patients affected by PXE, baseline levels of vitamin K1-dependent proteins and -metabolites and whether parenteral administration of phytomenadione was effective in modulating their levels. METHODS: We included eight PXE patients with typical clinical symptoms (skin, retina, and vascular calcification) and two ABCC6 causative mutations; 13 clinically unaffected first-degree patients' relatives (9 carrying one ABCC6 mutation and 4 non-carriers). We assessed urinary vitamin K1 metabolites and serum Glu- and Gla-OC, Gas6 and undercaboxylated prothrombin (PIVKA-II), at baseline and after 1 and 6 weeks after a single intramuscular injection of 10 mg vitamin K1. RESULTS: Comparison of PXE patients, heterozygous, and non-carriers revealed differences in baseline levels of serum MK-4 and of urinary vitamin K metabolites. The response to phytomenadione administration on vitamin K-dependent proteins was similar in all groups. CONCLUSION: The physiological axis between vitamin K1 and vitamin K-dependent proteins is preserved; however, differences in the concentration of vitamin K metabolites and of MK-4 suggest that vitamin K1 metabolism/catabolism could be altered in PXE patients.