sign in sign up
<<
Home , Adenosylcobalamin, Methylcobalamin, Vitamin K

NUTRISTASIS Are supplements necessary for all? A role for vitamin status assessment

disease, coupled with media attention, has Nutristasis covers the development and application of led to the impression by some people that taking in supplement form is analytical tools to support the assessment of vitamin status. necessary. Although high exposure to most vitamins is generally thought to cause no Here, Agata Sobczynska-Malefora and Dominic J Harrington harm, and vitamin toxicity is normally ´ associated with a very high intake (ie vitamin look at the laboratory’s role in vitamin status assessment. B6 leading to sensory neuropathy, causing a rise in intracranial pressure or and joint pain), the adverse effects of The word ’vitamine‘ was first used in 1911 by while controversies exist regarding others long-term supplementation have not been the Polish scientist Dr Kazimierz Funk who, (ie folic acid fortification leading to the studied for most vitamins. while working in London, referred to an presence of unmetabolised folic acid in It should also be noted that some vitamins amine-containing compound which was , or supplementation in impede the action of others. The excessive found to be vital for life – (now also smokers).1 intake of one vitamin may lead to an known as vitamin B1). It was subsequently Although a balanced provides a increased utilisation or masking deficiency found that vitamins do not share any sufficient daily intake of all vitamins needed of the other vitamin, resulting in the depletion commonality in their structure, nor do they for most people, sustaining an adequate of this vitamin or exacerbating clinical all contain amines, hence the word ‘vitamin’ status can be challenging for some (ie the symptoms. Examples include the interaction was coined. Today, although the role that elderly because of impaired absorption or of (vitamin B9) with , or the 13 recognised vitamins now play in during pregnancy because of increased negative impact of high-dose vitamin E numerous developmental and metabolic requirement). In many cases optimal supplementation on status.2 processes has been well defined, many other treatment regimes have yet to be established. functions need to be better understood. Self-administered supplementation and Nutristasis Unit Similarly, the clinical utility of vitamin adverse effects due to an excessive intake of The Nutristasis Unit was established in 2002 supplementation in the treatment and vitamins from pharmaceutical preparations to develop and apply analytical tools to prevention of disease has been well (ie or support the assessment of vitamin status. established for some disorders (ie for vitamin B12 deficiency) is also a concern. The unit currently performs 300,000 tests to prevent scurvy, for rickets and Research in the past few decades related to annually, using various analytical techniques vitamin K prophylaxis to prevent ) the importance of vitamins in health and to estimate the abundance of a particular

The Nutistasis Unit team, with Dr Dominic Harrington (far left) and Dr Agata Sobczynska-Malefora (fifth from the right) ´ THE BIOMEDICAL SCIENTIST JANUARY 2016 23 NUTRISTASIS

THF (tetrahydrofolate), Diet/folic acid Diet 5-MTHF (5-methyltetrahydrofolate), MTHFR (methylene tetrahydrofolate reductase), MS (), THF CBS (cystathionine beta-synthase), Methionine SAM (S-Adenosyl methionine), SAM Cbl (cobalamin),

TC II (transcobalamin), MS CH3 holoTC (holotranscobalamin), 5, 10 methylene THF Vitamin B HC (haptocorrin), 2 Me-Cbl S-Adenosyl homocysteine OH-Cbl (), MTHFR cytoplasm MMA-CoA (methylmalonyl-CoA), Homocysteine MMA (methylmalonic acid, 5-MTHF Me-Cbl (methylcobalamin), CBS Ado-Cbl (adenocylcobalamin).

Vitamin B6 Cbl-TCII Cbl-TCII Cystathionine Cbl-food Cbl-HC Cbl-IF Cbl-HC (holo TC) Cbl-OH d s stomach intestine bloo cell cytoplasm

Cbl-HC Cysteine er liv Ado-Cbl ia Amino acids mitochondr Propionate MMA–CoA Succinyl–CoA odd-chain fatty acids

MMA

Fig 1. Homocysteine, folate and vitamin B12 . vitamin, as well as functional assays that Folate, vitamin B12 and homocysteine holohaptocorrin (Cbl-HC) and ‘active’ B12 establish utilisation within cells. Functional Vitamin B9 (folate) and vitamin B12 (Cbl-TC II) (Fig 1). If the holoTC result falls assays include total plasma homocysteine to (cobalamin) are essential for metabolism, within 25–70 pmol/L, we perform MMA 3 evaluate folate and B12 status, methylmalonic DNA synthesis and regulation of gene analysis. acid (MMA) for vitamin B12 and PIVKA-II for expression. Deficiencies of folate and/or Methylmalonic acid is a very sensitive the evaluation of vitamin K. Values that fall vitamin B12, resulting in megaloblastic test for identifying patients with suboptimal/ within the laboratory reference ranges are anaemia and neurological impairment, deficient vitamin B12 status. To assist our suggestive of adequate vitamin status are one of the most common vitamin users with the diagnosis of folate deficiency, respective to the metabolic process where deficiencies found in patients and the in addition to commonly used serum and the vitamin is being utilised. general population. In our recent study of red cell folate tests, we provide analysis of Whenever possible and applicable, we 17,875 patients from primary care, we 5-methyltetrahydrofolate (plasma, whole combine functional assays with assays found serum folate below our deficiency blood and CSF)6 and homocysteine. which assess the abundance of vitamins in cut-off of 7 nmol/L in 14% and serum B12 5-methyltetrahydrofolate (5-MTHF) is the blood/plasma, referred to as ‘static’ assays to <138 pmol/L in 4.5% of patients.3 main form of folate and an essential methyl reflect storage and transport. However, the Deficiency of folate and B12 is also the most donor for methionine synthase and vitamin interpretation of the combined results is not frequent reason for elevated plasma B12-mediated conversion of homocysteine always straightforward, since discrepancies homocysteine (hyperhomocysteinaemia to methionine (Fig 1). Low 5-MTHF most do occur as a result of test limitations in [HHcy]), a risk factor for cardiovascular commonly suggests folate deficiency, but certain clinical and analytical conditions. disease and dementia. As an example, the can also indicate vitamin B2 deficiency or Our scientists assist clinicians with the prevalence of HHcy in our cohort of patients methylene-tetrahydrofolate reductase interpretation of such results. We also with a history of thromboembolic disease (MTHFR) polymorphism (TT genotype). provide regular teaching sessions to doctors, was 27%. Hyperhomocysteinemia due to Patients with this polymorphism often have scientists in training and students from folate and/or vitamin B12 insufficiency could raised homocysteine, and hence are at medical schools and universities. The be explained in about 56% of patients of greater cardiovascular risk.7,8 Our Molecular Nutristasis Team also actively leads or these patients.4,5 Unit performs analysis of this polymorphism participates in research projects and studies, The diagnosis of folate and B12 and other common polymorphisms as well aiming to better understand the role of deficiency remains problematic and there is as full sequencing of MTHFR and CBS vitamins and improve our services for better no ‘gold standard’ test. There is no genes (Fig 1). patient care. consensus on cut-off points used by High vitamin B12 and folate Currently, the Nutristasis Unit routinely laboratories, given the variety of concentrations are also prevalent. In our assesses vitamin status of the following - methodologies and diagnostic approaches hospital and primary care cohort,3 we soluble vitamins: B1, B2, B6, B9, B12 and C, used. Our approach to the diagnosis of found 20% of patients with holoTC results and -soluble vitamins: A, E, K and D. It also vitamin B12 deficiency for hospital patients >128 pmol/L (upper limit of the linear provides analysis of vitamin K antagonists includes the use of the holotranscobalamin range of the method) and 9% of total B12 such as (oral ) and (holoTC) assay as a first-line marker, >652 pmol/L (upper limit of our reference (). Although followed by confirmatory testing with MMA if range), respectively. Elevated folate services are primarily provided for clinical indicated. HoloTC measures only the concentrations (>45.3 nmol/L) were found specimens, the unit can also perform biologically active fraction of vitamin B12, as in 3.8% of hospital and 10.3% of primary analyses for veterinary surgeries and the opposed to the widely used serum B12 test care patients, respectively. We also found food industry. which quantifies both ‘inactive’ that of those with high B12 status, 6% of

24 JANUARY 2016 THEBIOMEDICALSCIENTIST NUTRISTASIS

hospital and 10% of primary care patients Vitamin B6 vitamins K2) predominantly of bacterial origin. had serum folate within our deficiency Vitamin B6 is involved in over 100 enzymatic In Western diets, K1 and MK-n account for range (<7 nmol/L). Conversely, within reactions, including the metabolism of amino 90% and 10% of the vitamin K intake, patients with very high folate status, there acids, carbohydrates, neurotransmitters and respectively. Vitamin K is required for the were 4% of hospital patients with holoTC . Low vitamin B6 status can lead to HHcy conversion of -bound glutamate to <25 pmol/L and 2.3% of primary care (Fig 1) and has been associated with severe γ-carboxyglutamate (Gla), essential for the patients with total B12 <138 pmol/L (our cut- malnutrition and venous thromboembolism, synthesis of seven vitamin K-dependent offs for deficiency). while very high doses of (B6) that have a crucial role in blood It is possible that over-supplementation supplementation lead to toxicity presenting as (factors II, VII, IX and X; proteins of one vitamin exacerbated the deficiency of sensory neuropathy. Pyridoxine is used in the C, S, and Z). Other vitamin K-dependent the other. Some of the evidence which treatment of many conditions (ie cystinuria, proteins (Gla proteins), with a widespread supports this hypothesis comes from the homocystinuria, seizures or peripheral distribution, have now been shown to be analysis of data from the National Health neuropathy associated with isoniazid and essential for functions such as the regulation and Examination Survey hydralazine therapy). The prevalence of of bone turnover and , inhibition of

(NHANES), which found associations of vitamin B6 deficiency is low due to its vascular calcification, and roles in vascular high folate status with impaired activity of availability in various foods. repair processes, cell cycle regulation, cell-cell the two vitamin B12 methionine We only found pyridoxal 5’-phosphate adhesion and signal transduction. synthase and MMA-coenzyme mutase (Fig (PLP), the active coenzyme form of vitamin B6, Green leafy are a rich source of 1), as well as anaemia and cognitive below the lower cut-off of the reference range, vitamin K1, while dietary MKs are present in impairment in elderly subjects.9 Recently, it suggesting deficiency, in 2% of patients animal tissues. Like other fat-soluble vitamins, 13 has been also suggested that high folic acid referred for B6 assessment. However, 38% vitamin K is absorbed from the duodenum, consumption reduces MTHFR and of our patient cohort had PLP values above the where it is dependent on and pancreatic activity, creating pseudo-MTHFR deficiency upper limit of the reference range, including secretions for solubilisation. Any condition and leading to hepatocyte degeneration.10 7% of patients with concentrations >540 causing the of fat leads to a The potential adverse effects of high nmol/L (five times the upper limit). Of these secondary deficiency of fat-soluble vitamins. maternal folic acid have also been linked 19 patients, sensory neuropathy was present Poor absorption of vitamin K quickly leads with higher adiposity, and low vitamin B12 in three cases. Vitamin B6 toxicity should have to the depletion of its tissue stores, which is and high folate have predicted high insulin been considered as a contributing factor to indicated by a decrease in circulating levels of resistance in children.11,12 There is little this neuropathy. the vitamin long before pathological changes known about the adverse effects of develop. Cases of are excessive intake of vitamin B12 in general. Vitamin K often missed, or detected late, due to the use Some hypotheses include the formation of The major naturally occurring K vitamins are of inappropriate laboratory markers of vitamin antibodies to transcobalamin, leading to a the plant form phylloquinone (vitamin K1) and K status, commonly the international high plasma concentration of the vitamin. multiple forms of menaquinones (MK-n or normalised ratio (INR). The INR, which is

A rapid, economical and easy to use system offering full AST traceability and standardisation AST MICRO-READER Pro-Lab Diagnostics are The SIRscan™ Micro is a compact and easy to use pleased to announce the rapid micro-reader for disc susceptibility testing. Offering full traceability of images and launch of the SIRscan™ result management with minimal capital Micro to the UK Market. investment and running costs, the system is a safe and reliable way to move from manual AST to automated reading. Inoculum standardisation is available using the Inoclic™ system and a fully customisable expert system is available as part of the SIRweb™ Bacterio software package. Read time on the analyser is less than 90 seconds and results are available immediately. Reading may be performed for all and all media, including blood, , HTM as well as weak growth and double zone detection.

THE BIOMEDICAL SCIENTIST JANUARY 2016 25 NUTRISTASIS

based on the , is designed a b to detect bleeding tendencies and is a very Diet Diet insensitive and non-specific marker of vitamin K status. Vitamin K status is evaluated by the NAD(P)H measurement of K in serum. This is NAD(P)H 1 Vitamin K reductase Vitamin K reductase supported by the analysis of PIVKA-II (under- VKOR VKOR VKOR carboxylated prothrombin, an abnormal VKOR species of factor II that is only detectable in Warfarin Warfarin the circulation of patients with suboptimal Vitamin KO Vitamin KO vitamin K status or those taking vitamin K Vitamin KH2 Vitamin KH2 antagonists (ie warfarin; Fig 2). By running these two assays in tandem we are able to monitor the two most important determinants of vitamin K status, availability and utilisation.

A vitamin K1 concentration within the laboratory reference range (suggestive of γ-glutamyl carboxylase adequate vitamin K status) does not preclude an elevated PIVKA-II concentration, which is CO +O suggestive of hepatic deficiency. Gla 2 2 Glu Gla PIVKA-II Glu In the UK, vitamin K supplementation is commonly used for the prophylaxis of vitamin K deficiency bleeding (VKDB) of the newborn Fig 2. a) The vitamin K cycle and associated enzymes. b) Warfarin action on the vitamin K cycle. and to reverse anticoagulation by vitamin K Warfarin exerts its anticoagulant effect by inhibiting the vitamin K reductase (VKOR) complex antagonists (ie warfarin). The role of vitamin K that recycles vitamin K 2,3-epoxide (KO) to vitamin K quinol (KH2). supplements and their potential benefits for bone and vascular health and treatment of holotranscobalamin and methylmalonic indicators of vitamin B-12 deficiency. Am J 14 certain cancers is still being evaluated. acid in hospital patients and total vitamin B12 Clin Nutr 2009; 89 (2): 702S–6S. Toxicity related to vitamin K ingestion has in primary care patients to assess low vitamin 10 Christensen KE, Mikael LG, Leung KY et al. been mainly associated with B12 status. J Hematol Thromb 2015; 1 (2): 8. High folic acid consumption leads to pseudo-

(vitamin K3) formulations leading to 4 Sobczynska-Malefora A, Harrington DJ, MTHFR deficiency, altered metabolism, haemolytic anaemia. It is important to note Gorska´ R, Shearer MJ, Lomer MC. and injury in mice. Am J Clin Nutr 2015; that vitamin K3 is only indicated for the Hyperhomocysteinaemia and B vitamin 101 (3): 646–58. prophylaxis and treatment of vitamin K intakes in patients with a history of 11 Knight BA, Shields BM, Brook A et al. Lower deficiency in malabsorption syndromes.15 cardiovascular disease (oral communication). circulating B12 is associated with higher 10th International Conference: One carbon obesity and insulin resistance during Conclusions metabolism, vitamins B and homocysteine. pregnancy in a non-diabetic white British Deficiencies of some vitamins are prevalent Medical School, University of Lorraine, population. PLoS One 2015; 10 (8): in patients and general populations, and it is Nancy, France. July 2015. e0135268. important that these are diagnosed and 5 Sobczynska-Malefora A, Harrington DJ, 12 Yajnik CS, Deshpande SS, Jackson AA et al. treated early. However, the prevalence of Lomer MC´ et al. Erythrocyte folate and Vitamin B12 and folate concentrations during elevated vitamin concentrations is much 5-methyltetrahydrofolate levels decline pregnancy and insulin resistance in the more common, often resulting from during 6 months of oral anticoagulation with offspring: the Pune Maternal Nutrition Study. excessive intake. The adverse effects of over- warfarin. Blood Coagul Fibrinolysis 2009; Diabetologia 2008; 51 (1): 29–38. supplementation for many vitamins have not 20 (4): 297–302. 13 Critcher MS, Sobczynska-Malefora A. Vitamin yet been very well studied. These need to be 6 Sobczynska-Malefora A, Harrington DJ, B6: low and very high´ concentrations in taken into consideration when treatment is Voong K,´ Shearer MJ. Plasma and red cell hospital patients. The Biomedical Scientist reviewed/prescribed or self-administration reference intervals of 5-methyltetrahydrofolate 2015 Sept; 59 (9): 504–5. takes place. Assessment of vitamin status of healthy adults in whom biochemical 14 Shearer MJ, Gorska R, Harrington DJ, coupled with investigations of causes of functional deficiencies of folate and vitamin Schurgers LJ, Newman P. Vitamin K. In: vitamin status should precede any future B 12 had been excluded. Adv Hematol 2014; Herrmann W, Obeid R eds. Vitamins in the treatment. ɀ 2014: 465623. prevention of human diseases. Berlin/New 7 Shmeleva VM, Kapustin SI, Papayan LP, York: Walter de Gruyter, 2011: 515–60. The authors are grateful to the whole Sobczynska-Malefora A, Harrington DJ, 15 Card DJ, Shearer MJ, Schurgers LJ, Gomez K, Nutristasis Team for ongoing dedication to Savidge´ GF. Prevalence of Harrington DJ. What’s in a name? The provision and improvements of the hyperhomocysteinemia and the MTHFR pharmacy of vitamin K. Br J Haematol 2015 Nutristasis Unit laboratory services. C677T polymorphism in patients with arterial Oct 22. doi: 10.1111/bjh (Epub ahead of and venous thrombosis from North Western print). References Russia. Thromb Res 2003; 111 (6): 351–6. 1 Hemila H, Kaprio J. Modification of the 8 Sobczynska-Malefora A, Harrington DJ, effect of vitamin E supplementation on the Rangarajan´ S, Kovacs JA, Shearer MJ, Dr Agata Sobczynska-Malefora is a lead scientist mortality of male smokers by age and dietary Savidge GF. Hyperhomocysteinemia and and Dr Dominic ´J Harrington is head of the vitamin C. Am J Epidemiol 2009; 169 (8): B-vitamin status after discontinuation of oral Nutristasis Unit. The service is based at 946–53. anticoagulation therapy in patients with a St Thomas’ Hospital and is part of Viapath, the partnership between Guy’s and St Thomas’, 2 Card DJ, Gorska R, Cutler J, Harrington DJ. history of venous thromboembolism. Clin King’s College and Bedford NHS Trusts, Vitamin K metabolism: current knowledge Chem Lab Med 2003; 41 (11): 1493–7. and Serco. For more information and future research. Mol Nutr Food Res 9 Selhub J, Morris MS, Jacques PF, please visit www.viapath.co.uk 2014; 58 (8): 1590–600. Rosenberg IH. Folate-vitamin B-12 or email [email protected] 3 Sobczynska-Malefora A, Critcher MS, interaction in relation to cognitive or [email protected] Harrington´ DJ. The application of impairment, anemia, and biochemical

26 JANUARY 2016 THEBIOMEDICALSCIENTIST