ann clin biochem 2013 hughes 315 29

8/13/2019 Ann Clin Biochem 2013 Hughes 315 29

1/16

http://acb.sagepub.com/ journal of biochem istry and la boratory medicineAnnals of Clinical Biochemistry: An international

http://acb.sagepub.com/content/50/4/315The online version of this article can be found at:

DOI: 10.1177/0004563212473279

2013 50: 315 originally published onlin e 18 June 2013Ann Clin Biochem Catherine F Hughes, Mary Ward, Leane Hoey and Helene McNulty

and ageing: current issues and interaction with folate12Vitamin B

Published by:

http://www.sagepublications.com

On behalf of:

Association for Clinical Biochemistry and Laboratory Medicine

Netherlands Society for Clinical Ch emistry and Laboratory Japan Society of Clinical C hemistry

can be found at:Annals of Clinical Biochemistry: An international journal of biochemistry and laboratory medicine tional services and information for

http://acb.sagepub.com/cgi/alertsEmail Alerts:

http://acb.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

What is This?

- Jun 18, 2013OnlineFirst Version of Record

- Jul 1, 2013Version of Record>>

by guest on January 17, 2014acb.sagepub.comDownloaded from by guest on January 17, 2014acb.sagepub.comDownloaded from
http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/content/50/4/315http://acb.sagepub.com/content/50/4/315http://acb.sagepub.com/content/50/4/315http://acb.sagepub.com/cgi/alertshttp://www.sagepub.com/journalsReprints.navhttp://www.sagepublications.com/http://www.sagepublications.com/http://www.sagepub.com/journalsPermissions.navhttp://www.acb.org.uk/http://www.nvkc.nl/http://acb.sagepub.com/content/50/4/315.full.pdfhttp://www.nvkc.nl/http://acb.sagepub.com/content/early/2013/05/04/0004563212473279.full.pdfhttp://www.jscc-jp.gr.jp/index.htmlhttp://acb.sagepub.com/cgi/alertshttp://acb.sagepub.com/subscriptionshttp://acb.sagepub.com/subscriptionshttp://www.sagepub.com/journalsReprints.navhttp://www.sagepub.com/journalsReprints.navhttp://www.sagepub.com/journalsPermissions.navhttp://www.sagepub.com/journalsPermissions.navhttp://online.sagepub.com/site/sphelp/vorhelp.xhtmlhttp://online.sagepub.com/site/sphelp/vorhelp.xhtmlhttp://acb.sagepub.com/content/early/2013/05/04/0004563212473279.full.pdfhttp://acb.sagepub.com/content/early/2013/05/04/0004563212473279.full.pdfhttp://acb.sagepub.com/content/50/4/315.full.pdfhttp://acb.sagepub.com/content/50/4/315.full.pdfhttp://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/http://online.sagepub.com/site/sphelp/vorhelp.xhtmlhttp://online.sagepub.com/site/sphelp/vorhelp.xhtmlhttp://acb.sagepub.com/content/early/2013/05/04/0004563212473279.full.pdfhttp://acb.sagepub.com/content/early/2013/05/04/0004563212473279.full.pdfhttp://acb.sagepub.com/content/50/4/315.full.pdfhttp://acb.sagepub.com/content/50/4/315.full.pdfhttp://www.sagepub.com/journalsPermissions.navhttp://www.sagepub.com/journalsPermissions.navhttp://www.sagepub.com/journalsReprints.navhttp://www.sagepub.com/journalsReprints.navhttp://acb.sagepub.com/subscriptionshttp://acb.sagepub.com/subscriptionshttp://acb.sagepub.com/cgi/alertshttp://acb.sagepub.com/cgi/alertshttp://www.jscc-jp.gr.jp/index.htmlhttp://www.jscc-jp.gr.jp/index.htmlhttp://www.nvkc.nl/http://www.nvkc.nl/http://www.acb.org.uk/http://www.sagepublications.com/http://acb.sagepub.com/content/50/4/315http://acb.sagepub.com/


2/16

Review Article

Vitamin B 12 and ageing: current issues and interactionwith folate

Catherine F Hughes, Mary Ward, Leane Hoey and Helene McNulty

Abstract

A compromised vitamin B 12 status is common in older people despite dietary intakes that typically far exceed currentrecommendations. The maintenance of an optimal status of vitamin B 12 is not only dependent on adequate dietary intakebut more critically on effective absorption which diminishes with age. The measurement of vitamin B 12 is complicated bythe lack of a gold standard assay. There are a number of direct and functional indicators of vitamin B 12 status; however,none of these are without limitations and should be used in combination. Vitamin B 12 is of public health importance, notonly because deficiency leads to megaloblastic anaemia and irreversible nerve damage, but also because emergingevidence links low B12 to an increased risk of a number of age-related diseases, including cardiovascular disease, cognitivedysfunction, dementia and osteoporosis. Furthermore, there are concerns relating to potential adverse effects for olderadults with low vitamin B12 status of over-exposure to folic acid in countries where there is mandatory fortification of food with folic acid. The aim of this review is to examine the known and emerging issues related to vitamin B 12 in ageing,its assessment and inter-relationship with folate.

KeywordsVitamin B12 , ageing, B12 absorption, holotranscobalamin, folate

Accepted: 28th October 2012

IntroductionMajor advances in medicine over the last century haveresulted in a dramatic increase in life-expectancy. This,however, has been accompanied by a subsequentincrease in the prevalence of age-related chronic dis-eases; with morbidity, disability and poor quality of life frequently present in older age. Poor nutritionalstatus is recognized as a contributing factor and,recently, the role of vitamin B 12 in ageing has attractedconsiderable attention. Apart from the clinical featuresof B 12 deciency (i.e. megaloblastic anaemia and irre-versible neuropathy), emerging evidence indicates thatsubclinical deciency (i.e. low biomarker status) of B 12may be implicated in the development of severalchronic age-related diseases. The aim of this paper is

to consider this evidence and also review its inter-relationship with folate in ageing. The stages of vitaminB12 depletion and the analytical methods used to assessbiomarker status will be considered. The scientic evi-dence linking low/decient B

12 status to adverse health

outcomes will be reviewed, and consequences for rele-vant public health policies will be considered.

The Northern Ireland Centre for Food and Health, University of Ulster,Coleraine BT52 1SA, Northern Ireland, UK

Corresponding author:Prof. Helene McNulty, The Northern Ireland Centre for Food andHealth, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK.Email: [email protected]

This article was prepared at the invitation of the Clinical SciencesReviews Committee of the Association for Clinical Biochemistry.

Annals of Clinical Biochemistry50(4) 315329! The Author(s) 2013Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0004563212473279acb.sagepub.com

by guest on January 17, 2014acb.sagepub.comDownloaded from
http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/http://acb.sagepub.com/


3/16

Structure and physiological functions of vitamin B 12Vitamin B 12 is one of a group of cobalt containingcompounds known as cobalamin. There are a numberof different forms of the vitamin including the twometabolically active forms methylcobalamin and deox-yadenosylcobalamin and cyanocobalamin, the syn-thetic form of the vitamin typically used insupplements and fortied foods. Vitamin B 12 isrequired as a co-factor for only two mammalianenzymes, methionine synthase and methylmalonylCoA mutase. Adenosylcobalamin acts as a co-factorfor the latter enzyme which converts methylmalonylCoA to succinyl CoA, a metabolite in the tricarboxylicacid cycle (Figure 1). This is an important reaction inthe metabolism of branched-chain amino acids and

odd-chain length fatty acids.1

In vitamin B 12 depletion,the reduced activity of methylmalonyl-CoA mutasecauses the concentrations of its substrate methylmalo-nyl CoA to build up and form the by-product methyl-malonic acid (MMA), which is then excreted into thecirculation. Methylcobalamin acts as a co-factor forthe enzyme methionine synthase, which catalyses there-methylation of homocysteine to methionine, the pre-cursor of S -adenosylmethionine (Figure 2). S -adenosyl-methionine is a universal methyl donor essential for themethylation of phospholipids, neurotransmitters,amines, DNA, RNA and myelin basic protein. In vita-min B

12 depletion, inactivity of the methionine synthase

enzyme leads to a reduction in important methylationreactions and elevated homocysteine concentrations inthe circulation. 2

Vitamin B 12 and folate interrelationshipThe haematological sign of vitamin B 12 deciency ismegaloblastic anaemia characterized by megaloblastic(immature, enlarged) blood cells that occur as a resultof impaired DNA synthesis. An identical anaemiaarises with folate deciency because these two

vitamins are intrinsically linked via the enzymemethionine synthase. 2 Methionine synthase sits at the junction between two major biosynthetic pathways:the remethylation cycle and the folate cycle(Figure 2). Methionine synthase is the only knownenzyme to use 5-methyltetrahydrofolate, which inturn is converted to the metabolically active form,tetrahydrofolate, needed in one-carbon metabolism. 2

In vitamin B 12 depletion, methionine synthase activityis reduced and the formation of tetrahydrofolate isblocked, with folate essentially becoming trapped as5-methyltetrahydrofolate (i.e. the methyl-trap hypoth-esis). This is an unusable form of the vitamin as itcannot be converted back to its precursor 5,10-methy-lenetetrahydrofolate because the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate(by the enzyme methylenetetrahydrofolate reductase

[MTHFR]) is irreversible. S -adenosylmethionine is anallosteric inhibitor of MTHFR, thus reduced S -adeno-sylmethionine concentration (as seen in vitamin B 12depletion) removes the inhibition of MTHFR, pro-moting the reduction of 5,10-methylenetetrahydrofo-late to 5-methyltetrahydrofolate so that it can beused to re-synthesise methionine (the precursor of S -adenosylmethionine) thus further trapping folate as5-methyltetrahydrofolate. In this situation, cells sufferfrom a folate pseudo-deciency; folate is available butit is not in an active form that can be used by the cell,and consequently, the production of purines and pyr-imidines needed for DNA synthesis is disrupted. 3 Thisaffects the rapidly dividing cells of the bone marrow,leading to the production of megaloblastic blood cellsand subsequent megaloblastic anaemia. In addition,5-methyltetrahydrofolate is a poor substrate for folyl-polyglutamate synthase, the enzyme responsible forcellular retention of folate. In order for folate to beretained by the cell 5-methyltetrahydrofolate must rstbe converted to tetrahydrofolate via methionine syn-thase and thus, intracellular erythrocyte folate concen-tration has been reported to be lower in individualswith low vitamin B 12 .

35

Propionyl-CoA D -Methylmalonyl-CoA

Methylmalonic acid

AdenosylcobalaminSuccinyl-CoA

Tricarboxylicacid

cycle

L-Methylmalonyl-CoAmutase

L -Methylmalonyl-CoA

Figure 1. The vitamin B12 (i.e. adenosylcobalamin)-dependent conversion of methylmalonyl-CoA to succinyl-CoA.

316 Annals of Clinical Biochemistry 50(4)



4/16

Absorption, transport and storageThe absorption of vitamin B 12 is a highly complex pro-cess that is dependent on normal functioning of thegastrointestinal tract (Figure 3), consequently, it oftenbecomes less efficient with age. Vitamin B 12 is tightlybound to protein in food and must be released byhydrochloric acid and pepsin in the stomach in orderfor B 12 to be absorbed. The synthetic form of vitaminB12 , found in fortied foods and supplements, is how-ever already free and thus has no gastric acid require-ment. Free vitamin B 12 immediately binds withR-binding proteins and then with intrinsic factor (IF)in the duodenum forming the IF-B 12 complex whichtravels to the terminal ileum where it is absorbed viaspecic cubilin receptors by calcium-dependent endo-cytosis. There is a limit to the amount of vitamin B 12that can be absorbed at any given time via theIF-related pathway; receptors are reported to becomesaturated with B 12 intakes greater than 1.52.0 mg of vitamin B 12 .

6 In addition, an alternative, albeit ineffi-cient pathway exists whereby small amounts (1%) of vitamin B 12 are absorbed through passive diffusion(i.e. even in the absence of IF). 7 Once absorbed, vitamin

B12 then binds to one of two transport proteins, trans-cobalamin or haptocorrin. The majority of vitamin B 12binds to haptocorrin, a glycoprotein with an unknownfunction, while 2030% is bound to transcobalamin,forming holo-transcobalamin (holoTC), the metabolic-ally active fraction of the vitamin. 8 Total body stores of vitamin B 12 can reach 5 mg, with the majority stored inthe liver. 9 The liver excretes around 110 mg/d of vita-min B 12 into bile;

10 however, up to 90% of this is re-absorbed via a highly efficient enterohepatic cycle whilethe rest is excreted in faeces. The daily requirement forvitamin B 12 is so small in comparison to body storesthat it is considered to take years for vitamin B

12 de-

ciency to develop, even when there is a complete halt inB12 absorption.

11

Analytical measurement of vitamin B 12status

Serum total vitamin B12Measurement of serum/plasma total vitamin B 12 hasbeen the standard clinical test for vitamin B 12 deciencyfor many years and a variety of analytical approaches

Homocysteine

S -adenosyl-homocysteine

S -adenosyl-methionine

Methionine

Cysteine

Cystathionine

Methyl acceptor

5 Methyltetrahydrofolate

Sulphate + H 20

5, 10 Methylenetetrahydrofolate

Urine

Tetrahydrofolate

Methylatedacceptor MTHFR

Pyridoxal-5-phosphate

Methylcobalamin Methioninesynthase

Cystathionine-synthase

Folate cycle

Diet

NADPH

NADP*

FMN

Serine

Glycine

FAD

DMG

Homocysteinemetabolism

Choline Betaine

Figure 2. Metabolic pathway involving vitamin B12 of the remethylation of homocysteine to methionine and the recycling of folate.Shows co-factor forms for vitamin B 12 (methylcobalamin), vitamin B6 (pyridoxal-5-phosphate) and riboflavin (vitamin B2 ; flavinmononucleotide [FMN]; flavin adenine dinucleotide [FAD]). Also shows the metabolism of betaine and dimethylglycine (DMG).

Hughes et al. 317



5/16

are available such as the traditional microbiologicalassay, competitive binding assay, radioimmunoassayand chemiluminescence assay. Early versions of thenewer techniques lacked sensitivity and led to falselyelevated B 12 concentrations;

12 however, there is nowbetter agreement between the more recently developedprotein binding assays and the microbiological assay;generally considered to be the gold standard assay. 13

These newer assays have a number of advantagesover the microbiological assay, they are generally lesstime consuming, are not as sensitive to antibiotics orchemotherapy drugs, are widely available and have lowrunning costs. 13 It has become apparent, however, that

a low serum total vitamin B 12 concentration does notalways equate with B

12 deciency nor does a normal

value necessarily exclude it. 14 There is no establishedcut-off value used to dene vitamin B 12 deciency,with each laboratory recommended to determine itsown reference range depending on its assay method.There is a large variation between laboratories withone review reporting that the lower reference limit of values cited in the literature ranged from 110 to260 pmol/L. 15 Also, metabolic and clinical features of deciency have been reported in individuals with serumtotal B 12 within the normal range.

16 Furthermore, 5%of individuals with classical clinical signs of vitamin B 12

Free B 12

B12

B12 PDietary cobalaminbound to animal

protein

Gastricsecretion

HClPepsin

R-protein

Intrinsicfactor

B12 -intrinsic factorcomplex

B12

R R

IF

I FIF

IF B 1 2

B 1 2

B 1 2 T C

B 1 2 IF

IF

lleum

B12

Vitamin B 12 transported via transcobalamin (TC)or haptocorrin (HC)

B12B 1 2 HC HCT C

TC

B12

Adenosyl-

cobalamin

Methyl-cobalamin B 1 2

T C

T C

B 1 2

Ileal mucosal cell

Cubilin

I F

I F B 1 2

B 1 2

B 1 2

I F

B 1 2

B

1 2

B 1 2

R

B 1 2

P

Figure 3. The absorption and transport of vitamin B 12 (adapted from Andre s et al.127 ).




6/16

deciency were found to have serum total vitamin B 12concentrations within the normal range. 17

Serum total vitamin B 12 does not reect intracellularvitamin B 12 as it is a measure of all forms of the vita-min, the majority of which is bound to haptocorrin.Haptocorrin-bound vitamin B 12 is considered to be

metabolically inert and has been shown to respondslowly to changes in vitamin B 12 status comparedwith holoTC, which may explain its lack of sensitiv-ity. 12 Furthermore, liver disorders, myeloproliferativedisorders, renal failure, transcobalamin II deciency,bacterial overgrowth and haemolysis can all lead tofalsely elevated serum total B 12 concentrations. Falselow concentrations of serum total vitamin B 12 arelinked with severe folate or iron deciency, multiplemyeloma, pregnancy, HIV infection and oral contra-ceptive use. 18

Plasma total homocysteineThe re-methylation of homocysteine to methionine isdisrupted during vitamin B 12 depletion (Figure 2)because the necessary enzyme, methionine synthaserequires vitamin B 12 as cofactor. As a consequence,plasma homocysteine concentrations accumulate withB12 deciency thus providing a functional biomarkerof vitamin B 12 status. Although plasma homocysteineis highly sensitive to B 12 status, it lacks specicity. Thisis because plasma concentrations of homocysteine aredetermined not only by the status of vitamin B 12

19 butalso by folate, 20 and to a lesser extent vitamin B 6

21 and

riboavin.22

Once folate status is optimised (as in coun-tries with mandatory folic acid fortication) vitaminB12 emerges as the major nutritional determinant of homocysteine. 23 Apart from B vitamin status, plasmahomocysteine concentrations are also inuenced byother factors such as renal function, age and gender 24

and genetic polymorphisms, mainly homozygosity (TTgenotype) for the 677C ! T polymorphism in the geneencoding MTHFR, the enzyme required to generate5-methyltetrahydrofolate. 25 Blood sample collectionand handling can also impact considerably on homo-cysteine measurement. Homocysteine concentrationswill increase if the sample is kept at room temperature,or if separation of the sample is delayed following col-lection, as homocysteine is released from red bloodcells. However, this can be minimized if the sample isseparated immediately or placed on ice until centrifu-gation. 24,26 After separation, homocysteine is extremelystable and can be stored, once frozen, for many years. 24

Several analytical methods, e.g. high-performanceliquid chromatography, gas and liquid chromatogra-phy-mass spectrometry, stable-isotope dilution andimmunoassay techniques have been developed for mea-suring plasma homocysteine and there is generally good

agreement between the various techniques. 26 The assaysare reliable and have become less expensive in recentyears 5 but reference ranges may vary considerablybetween laboratories. 24 Although a plasma homocyst-eine concentration between 5 and 15 mmol/L is gener-ally considered normal, 26 this reference range may not

be appropriate for older adults. Despite its lack of spe-cicity, plasma homocysteine is a sensitive and usefulindicator of vitamin B 12 status in situations wherefolate is optimized.

Serum MMAVitamin B 12 acts as a co-factor for the enzyme methyl-malonyl-CoA mutase required for the conversion of methylmalonyl-CoA to succinyl CoA. In vitamin B 12deciency, the activity of this enzyme is reduced leadingto the accumulation of the by-product MMA, whichcan be detected in both blood and urine (Figure 1). 18

Plasma MMA is therefore a specic, functional markerof vitamin B 12 status that reects the availability of intracellular B 12 , although it becomes elevated in indi-viduals with renal impairment 27 and therefore may beless useful in older people where renal dysfunction is acommon problem. MMA is more concentrated in urineand methods for its measurement have been availablesince the 1950s. However, it lacks sensitivity andrequires a 24-hour urine collection as concentrationsare more sensitive to variation in food intake thanserum MMA concentrations. 28 More than 95% of patients with vitamin B 12 deciency have signicantly

elevated serum MMA.27

A variety of cut-off valueshave been used to dene vitamin B 12 status; however,serum MMA value < 0.30 mmol/L is generally con-sidered indicative of no deciency. 29 Although thedevelopment of a gas-chromatography-mass spectrom-etry method has increased sensitivity, the utility of MMA is compromised by high running costs, technicaldemands and dependence on normal renal function.

Serum holoTC Serum total vitamin B 12 and the functional assays(homocysteine; MMA) lack sensitivity and specicity,thus there has been an ongoing search for a more reli-able clinical measure of B 12 status. The major limitationof serum vitamin B 12 is that it measures the totalamount of the vitamin, while holoTC represents themetabolically active fraction of the vitamin that isinvolved in cellular reactions and thus may be a morereliable indicator of B 12 status. It is thought that adecrease in holoTC will indicate the earliest sign of B12 depletion as it has a much shorter half-life thanhaptocorrin. 30 Low holoTC concentrations have beendetected in populations at risk of deciency such as

Hughes et al. 319



7/16

vegetarians, vegans 31 and the elderly. 32 Preliminaryresults from an ongoing elderly Irish cohort indicatethat holoTC is a considerably better indicator of vita-min B 12 status than serum total vitamin B 12 or MMA inan elderly population. 33 In addition, several studieshave clearly demonstrated that holoTC is diagnostic-

ally superior to vitamin B 12 in the detection of vitaminB12 deciency31,34,35 and was better correlated with

symptoms of vitamin B 12 malabsorption than serumtotal vitamin B 12 .

36 There is also increasing evidencethat holoTC is more strongly correlated with diseasesassociated with low B 12 status, however, this has notbeen thoroughly evaluated. 37

Measurement of holoTC has been an attractive pro-spect for more than 20 years; however, until recently, itwas hampered by the lack of a good quality assay. 8

Currently, there are a number of different methodsavailable for the measurement of holoTC including amicrobiological assay, an enzyme-linked immunosorb-ent assay, a radioimmunoassay and a novel automatedholoTC specic monoclonal assay that is suitable forroutine use in clinical laboratories. 8 There is no generalconsensus as to what concentration of holoTC is suffi-cient, with the lower normal limit for holoTC varyingfrom 19 to 42 pmol/L depending on the laboratorymethod used. 8 Several studies have demonstrated thatholoTC increases dramatically after vitamin B 12 inges-tion, although the dose used in these studies was muchlarger than what would be typically consumed throughthe diet. 38,39 HoloTC is highly sensitive to altered renalfunction with concentrations increasing in renal impair-

ment31

and is inuenced by a common genetic poly-morphism of the transcobalamin gene (TC776C ! G),liver disease and female sex hormones. 40 The future of holoTC as a rst-line biomarker of vitamin B 12 is pro-mising, although further research is warranted toaddress the issues raised above before it can be usedroutinely.

Causes of low/deficient vitamin B 12status in older adultsVitamin B 12 absorption is a complex process and adefect at any step could lead to vitamin B

12 depletion.

Therefore, there are multiple causes of low B 12 in theolder population as shown in Table 1.

Inadequate dietary intakeRecommended daily intakes for vitamin B 12 vary fromcountry to country. 41 The UK National Diet andNutrition Survey found that dietary intakes of vitaminB12 in those aged over 65 were more than three timesgreater than current dietary recommendations. 42 Thus,inadequate intakes of vitamin B 12 are not a main

concern as diets of older people appear to provide vita-min B 12 well in excess of needs. Vitamin B 12 is onlyfound in foods of animal origin such as meats, poultry,sh, eggs and dairy products. It can also be obtainedfrom foods such as ready to eat breakfast cereals and

spreads fortied with the synthetic form of the vita-min. 10 In developed countries, inadequate dietaryintakes of vitamin B 12 are extremely rare, except forstrict vegetarians and vegans. 30

MalabsorptionIntrinsic factor deficiency pernicious anaemia. Perniciousanaemia is the classical form of vitamin B 12 deciencyand, although more prevalent in those over 60 years,only accounts for 12% of vitamin B 12 deciency foundin older populations. 11 It is an autoimmune diseasecharacterized by the destruction of the gastric mucosaand subsequent loss of IF, the essential transport pro-tein required for vitamin B 12 absorption. Loss of IFprevents not only the absorption of food-bound andfree vitamin B 12 but also the re-absorption of vitaminB12 secreted in bile. This creates a signicant negativevitamin B 12 balance with a progressive and permanentdepletion of body stores. 43 Although it may take anumber of years for clinical deciency to develop, per-nicious anaemia would ultimately lead to death if leftuntreated. 44 In order to bypass the gastrointestinaltract and the requirement for IF, patients are

Table 1. Causes of low/deficient vitamin B12status in older adults.

Causes of low vitamin B12 status

Inadequate dietary IntakeLow dietary Intake

VegetarianismVeganismMalabsorption

Pernicious anaemiaAtrophic gastritisBacterial overgrowth

Helicobacter pylori ZollingerEllison SyndromeGastric/intestinal resectionIntestinal diseases

Crohns diseaseCoeliac disease

Drugnutrient interactionsProton pump inhibitors

H2 antagonistsMetforminNitrous oxide

Pancreatic insufficiencyAlcohol abuse




8/16

traditionally treated with intramuscular B 12 injectionsfor life. However, there is evidence to suggest that oralvitamin B 12 administered at pharmacological doses of the vitamin can be as effective. 45

Food-bound malabsorption. Food-bound malabsorption of

vitamin B 12 is common in older people. It arises mainlyas a result of atrophic gastritis, an age-related disorderaffecting up to 30% of people 60 years. 46 Atrophicgastritis is a chronic inammation of the stomach lead-ing to atrophy of the mucosa which in turn results inreduced gastric acid secretion (hypochlorhydria), andtherefore diminished B 12 absorption as hydrochloricacid and pepsin are essential for the release of vitaminB12 from food proteins.

11 In addition, hypochlorhydriacan promote the overgrowth of bacteria in the stomachand small intestine which then utilize vitamin B 12 ,thereby further reducing the amount of the vitaminavailable for absorption. 47 In particular, Helicobacter pylori (H. pylori) infection has been associated with thedevelopment of atrophic gastritis and low vitamin B 12status. 48

In theory, individuals with food-bound malabsorp-tion of vitamin B 12 should still be able to absorb freeB12 because the secretion of IF remains intact and freevitamin B 12 has no gastric acid requirement (because itis not intrinsically bound to food proteins). Food-bound malabsorption leads to a more subtle less pro-found depletion of B 12 status and it remains unclearwhether this will ultimately progress to clinical vitaminB12 deciency.

44

Drug induced malabsorption proton pump inhibitors.Proton pump inhibitors (PPIs) are a group of acid sup-pressant drugs intended to treat gastric acid conditionssuch as gastro-oesophageal reux disorder, conditionsassociated with the use of non-steroidal anti-inamma-tory drugs, Barretts oesophagus and hypersecretory dis-orders such as ZollingerEllison syndrome. PPIs bind toand deactivate the gastric H /K adenosine triphospha-tase pump which reduces gastric acid secretion leadingto profound hypochlorhydria; 49 thus PPI therapy caninduce a state clinically identical to atrophic gastritis,the most common cause of vitamin B

12 deciency. This

has led to the concern that long-term acid-suppressionmedication may lead to food-bound malabsorption andultimately vitamin B 12 deciency especially in those vul-nerable to low status such as older people. 50 Chronicacid suppressant use in older people has been associatedwith the initiation of vitamin B 12 therapy.

51 Short-termstudies demonstrated that PPIs reduced the absorptionof food-bound vitamin B 12 .

52,53 Long-term omeprazoletreatment has been associated with a decrease in serumB12 status in both ZollingerEllison patients and olderadults; 54,55 however, results have not been entirely

consistent with two recent large studies reporting con-icting results. 55,56 Future studies, using a variety of vitamin B 12 biomarkers, are required to more fully inves-tigate the effect of prolonged PPI therapy on vitamin B 12status. The impact of PPIs on B 12 status is importantbecause they are frequently misprescribed to treat less

serious conditions and are in fact among the most fre-quently used medications, with millions of usersworldwide. 57

Gastric/intestinal resection and intestinal diseaseAs previously described vitamin B 12 absorption isdependent upon the normal functioning of the gastro-intestinal tract, a function that deteriorates with normalageing. Diseases that affect either the structure or func-tion of the stomach or part of the small intestine willhave a negative impact on the absorption of vitaminB12 . Gastric resection destroys the parietal and chief cells in the stomach so the secretion of hydrochloricacid, pepsin and intrinsic factor will be reduced andthus, the absorption of vitamin B 12 will be severely dis-rupted. 5 Furthermore, the subsequent hypochlorhydriapromotes bacterial overgrowth in the stomach andsmall intestine which further exacerbates the problemof vitamin B 12 malabsorption.

47 Ileal resection resultsin the loss of the IF-B 12 receptors in the terminal ileumwhich is one of the fundamental steps in vitamin B 12absorption. A variety of other intestinal conditionssuch as Crohns disease, coeliac disease, diverticulitis,intestinal blind loops and tapeworms have also been

associated with vitamin B 12 depletion.5

Genetic polymorphismsApart from the rare inborn errors of metabolism, anumber of polymorphisms in genes involved in B vita-min metabolism have been identied. The most import-ant of these is the 677C ! T variant in the gene codingfor the folate metabolizing MTHFR enzyme. This poly-morphism has been extensively reviewed elsewhere, butin brief, individuals homozygous for the 677C ! Tpolymorphism (TT genotype) in MTHFR haveimpaired folate metabolism, lower serum and red cellfolate and higher plasma homocysteine concentrationsand are at increased risk of certain age-related dis-eases. 58 Meta-analyses of genetic studies have alsoshown that those individuals with the TT genotypeare at a 1421% greater risk of cardiovascular disease(CVD) compared with those without the polymorph-ism. 59,60 A more recent meta-analysis reported thatindividuals with the TT genotype are at increased riskof stroke, however, this association was limited to thosewith low folate status. 61 There are also several knownpolymorphisms of the transcobalamin protein, the most

Hughes et al. 321



9/16

common being the 776C ! G polymorphism.Individuals homozygous for this polymorphism (GGgenotype) are reported to have lower concentrationsof holoTC compared with individuals with the wild-type genotype, hence, may impact the use of holoTCas a routine measurement for vitamin B 12 .

40,62 It is

thought that this polymorphism decreases the bindingaffinity and transport of vitamin B 12 .63

Vitamin B 12 diagnosis and treatmentVitamin B 12 depletion has been described in four stagesranging from mild depletion to clinical deciency. 30

Stage 1 is early vitamin B 12 depletion characterized bya decrease in holoTC concentration. Stage 2 is cellulardepletion characterized by a further decrease in holoTC.Serum total B 12 takes longer to respond to changes instatus and can remain within the normal range in stages1 and 2. Stage 3 is characterized by metabolic evidenceof deciency, with elevated concentrations of bothplasma homocysteine and serum MMA, low holoTCand normal or below normal serum total B 12 concentra-tions. Stage 4 is clinical deciency where both serumtotal B 12 and holoTC are low, the metabolic indicatorsare elevated and there are clinical signs of deciencyincluding haematological and neurological manifest-ations. Haematological features, which occur as aresult of impaired DNA synthesis, include megalo-blastic anaemia characterized by megaloblastic (largeimmature blood cells with low concentrations of haemo-globin) blood cells, hypersegmentation of neutrophils,

leukopaenia and thrombocytopaenia. Neurologicalsymptoms are diverse and irreversible if left untreated.The most severe form manifests as a sub-acute com-bined degeneration of the spinal cord and is character-ized by degeneration of the posterior and lateralcolumns of the spinal cord. 64

Currently, there is no consensus on how best to diag-nose vitamin B 12 deciency. Traditionally, diagnosis of vitamin B 12 deciency relied upon the symptoms asso-ciated with megaloblastic anaemia such as muscle weak-ness, fatigue and shortness of breath and the associatedhaematological signs. 65 However, the clinical signs of anaemia only occur in the late stages of depletion andfurthermore it is now clear that there is not always acontinuous progression from mild vitamin B 12 depletionto clinical deciency. 30,44 Clinical deciency is caused byIF deciency (i.e. pernicious anaemia) and leads to aprogressive and permanent depletion of vitamin B 12 ,but this accounts for only 12% of B 12 deciency. 11

Conversely, subclinical vitamin B 12 deciency is muchmore common and is usually as a result of food-boundmalabsorption of B 12 .

11 Diagnosis of pernicious anae-mia is relatively straightforward, patients have very lowvitamin B 12 concentrations and present with the typical

haematological and neurological symptoms. However,diagnosis of subclinical deciency is much more prob-lematic as it relies on metabolic evidence of deciency.Thus, it is recommended that the vitamin should bemeasured by more than one biomarker, both a direct(i.e. serum total vitamin B 12 or holoTC) and indirect

functional marker (i.e. plasma homocysteine or serumMMA) should be used in combination. 44

Treatment for IF deciency is well established withpatients receiving monthly intramuscular vitamin B 12injections for life. However, some studies demonstratethat oral supplementation with very high doses (i.e.1000 mg/day) B 12 may be equally effective because 1%of the vitamin can be passively absorbed without theneed for IF. 66,67 Although this is an attractive prospectfor clinicians, caution must be exercised as patients maynot be fully compliant in the long-term. 68 Conversely,there is no consensus on how to best treat (if at all)subclinical deciency as its clinical signicance remainsunclear. In the USA (but not in Europe or elsewhere),specic age-related dietary recommendations for thepopulation have been issued by the Institute of Medicine, 69 recommending that people aged 50years should consume most of their vitamin B 12 fromcrystalline sources, i.e. fortied food and supplements.A regular consumption of foods fortied with B 12 orlow-dose supplements will be more than sufficient foraddressing food-bound malabsorption. 70,71

Emerging roles in health of low/deficientvitamin B 12 status in the older populationThe potential role of homocysteine and the metabolic-ally related B-vitamins in the development of chronicdisease has attracted much interest within the scienticcommunity. This interest was initially sparked fromobservations that individuals with homocystinuria, aninborn error of metabolism characterized by excessiveconcentrations of homocysteine, had premature vascu-lar disease and mental retardation. 72 Since then, muchscientic literature has emerged focusing mainly onhomocysteine, but also on the metabolically related Bvitamins, most notably folate and vitamin B 12 , in rela-tion not only to CVD, 73,74 and stroke, 75 but also cog-nitive function, 76 fracture risk 77 and osteoporosis. 78

The possibility that B vitamin supplementation mighthelp delay or prevent chronic disease (via correction of suboptimal status and/or lowering homocysteine) is of major interest and could have important public healthimplications. 79

Cardiovascular diseaseCVD is the leading cause of death worldwide. Twomajor meta-analyses of epidemiological evidence




10/16

published in 2002 predicted that a lowering of plasmahomocysteine by 3 mmol/L (or by about 25% based ontypical homocysteine concentrations of 12 mmol/L)would reduce the risk of heart disease by 1116% andstroke by up to 1924%. 73,74 As recently reviewed, theevidence from the secondary prevention trials in at-risk

patients has failed to demonstrate a benet of homo-cysteine-lowering therapy with B vitamins (usuallyusing folic acid in combination with vitamin B 12 ) onCVD events. 58 Currently, the evidence is stronger forstroke, with meta-analyses of randomized controlledtrials (RCTs) showing that homocysteine loweringreduces the risk of stroke. 75,80 Few studies have inves-tigated the independent effects of vitamin B 12 on CVDrisk. Subgroup analysis of the Vitamin Intervention forStroke Prevention (VISP) Trial conned to those indi-viduals considered to benet from supplementation (i.e.non-supplement users and those without perniciousanaemia) suggests that higher doses of vitamin B 12may be required as they reported that individualswith higher baseline vitamin B 12 and taking the high-dose B vitamin had reduced risk of recurrent vascularevents while those with the lowest vitamin B 12 status onthe low-dose combination had the greatest risk of recurrent vascular events. 81 In addition, a number of other B vitamin RCTs have detected benecial effectson a number of cardiac endpoints. Combined B vitaminsupplementation including high-dose vitamin B 12 hasbeen associated with reduced re-stenosis and cardiovas-cular events in patients following coronary angio-plasty. 82,83 Combined vitamin B 12 and folic acid

supplementation was also associated with improvedcoronary blood ow in patients with coronary arterydisease. 84 Evidence from meta-analyses also supports arole for vitamin B 12 in CVD, with a greater effect onstroke reported when folic acid was used in combin-ation with vitamins B 12 and B 6 compared with folicacid alone. 80,85 Further research is warranted to morefully investigate the potential benets of B vitamin sup-plementation on CVD risk, particularly in those withpre-existing vascular disease.

Cognitive functionDementia and cognitive impairment disorders are amajor public health concern in ageing populations. 86

Observations from case-control studies, that patientswith Alzheimers disease (the most common form of dementia) had signicantly higher homocysteine con-centrations than age-matched controls, prompted thetheory that B vitamin status might be an importantmodiable risk factor for cognitive dysfunction inageing. 76,87 Cross-sectional and longitudinal studiesprovide a wealth of evidence to support an associationbetween homocysteine and/or related B vitamins with

cognitive function in both healthy older adults andpatients with dementia. 88 A recent meta-analysis of epi-demiological studies estimated that a 3- mmol/L reduc-tion in homocysteine would reduce the risk of dementiaby up to 22%. 89 Both folate and vitamin B 12 have beenextensively investigated in relation to cognitive per-

formance in ageing; however, the evidence is less con-sistent for vitamin B 12 .9093 Many of these studies relied

on serum total vitamin B 12 as the sole biomarker meas-ure of status; however, those that used MMA orholoTC are generally more supportive of a protectiverole for vitamin B 12 in maintaining cognitive functionin ageing. 79,94

A number of RCTs have investigated the potentialbenets of B vitamin supplementation on cognitive func-tion; however, many of these studies were of insufficientduration or sample size necessary to reach useful con-clusions. 95104 One large trial of long-term duration thatreported no benet was inherently awed as the cogni-tive arm of the investigation was initiated 1.2 years aftercommencement of B vitamin treatment; therefore, anypotential impact on cognitive function may have beenmissed. 96 Two a priori RCTs, both conducted in healthyolder people for a considerable duration, have been pub-lished with conicting results. 101,102 McMahon et al. 101

found no benet of combined B vitamin supplementa-tion (1000 mg folate, 500 mg vitamin B 12 , 10 mg vitaminB6 daily) on cognitive performance in 276 dementia-freeolder people (aged 65 years). Conversely, a supplemen-tation trial with folic acid (800 mg/day) alone in 818people aged 5070 years reported signicant improve-

ments in cognition over a-three year interventionperiod. 102 This discrepancy is intriguing given thatboth studies were similar in design, interestingly how-ever, the baseline B vitamin status between the two stu-dies was very different. The study by Durga wasconducted in the Netherlands, a country which doesnot permit folic acid fortication (even on a voluntarybasis), while the trial by McMahon showing a null effectwas conducted in New Zealand which has had an activevoluntary fortication programme since 1996. Hence,the participants in Durgas study had lower baselineconcentrations of folate and vitamin B 12 comparedwith McMahons study and therefore may have beenmore likely to benet from folic acid intervention tooptimize status. More recently,combined B vitamin sup-plementation was shown to improve cognitive perform-ance in community-dwelling older adults with depressivesymptoms. 103 The strongest evidence to date to providesupport for a cause and effect relationship between Bvitamin status and cognitive function is from thehomocysteine and B vitamins in cognitive impairment(VITACOG) study. B vitamin supplementation (0.8 mgfolic acid, 0.5 mg vitamin B 12 , 20 mg vitamin B6) sloweddown the rate of brain atrophy (as measured using

Hughes et al. 323



11/16

magnetic resonance imaging) 100 and improved cognitiveperformance 104 in patients with mild cognitive impair-ment. A number of RCTs conducted to investigatethe effect of B vitamin intervention on CVD also includea measurement of cognitive function. While not primar-ily designed to examine cognitive health, the evidence

from these trials should be helpful in contributing tothe evidence linking optimal B vitamin status to themaintenance of cognitive function in ageing, althoughthey will be confounded to some extent by the fact thatmany of the participants will have existing vasculardisease.

Bone healthOsteoporosis is characterized by low bone mass andmicro-architectural deterioration in bone tissue, result-ing in an increased risk of fracture and affects over 75million people in Europe, USA and Japan. Evidencefrom epidemiological studies has reported that elevatedplasma homocysteine concentrations and/or low B vita-min status are associated with lower bone mineral dens-ity (BMD) 78,105 and a higher risk of osteoporosis 106 andosteoporotic fracture. 107 Several studies have linkedlow vitamin B 12 status with osteoporosis and lowBMD. 106,108,109 A retrospective study of postmenopau-sal women found that those with pernicious anaemiahad a more than two-fold risk of fracture comparedwith those without pernicious anaemia. 110 Moreover,an increase in BMD and a reversal of osteoporosiswas demonstrated in a patient with pernicious anaemia,

severe osteoporosis and multiple vertebral compressionfractures after two years treatment with vitamin B 12 .

111

Low vitamin B 12 status has been associated withincreased risk of fracture 77,107 and more rapid boneloss in older women; 112 however, the evidence is notentirely consistent. 107

Many RCTs investigating the effect of B vitamin sup-plementation showed no evidence of a benecial effect;however, many of these interventions were conductedover too short a timeframe and typically measuredbone turnover biomarkers, the validity of which hasyet to be established. 113,114 One RCT of sufficient dur-ation and with robust bone outcomes did, however,show that combined folic acid and vitamin B 12 supple-mentation for two years was effective in reducing the riskof hip fractures in Japanese patients following stroke. 115

There is a need for further well-conducted RCTs beforeany rm conclusions can be made in relation to the roleof vitamin B 12 and related B vitamins in bone health.

Public health implicationsThe policy in North America and elsewhere of manda-tory fortication of staple foods with folic acid for the

prevention of neural tube defect (NTD) pregnancieshas been an indisputable success. 116 In addition to low-ering NTDs, this measure may also have had a benetin reducing stroke mortality. 117 The implementation of a similar policy has, however, been delayed in the UKbecause of concerns that high-dose folic acid could

have a detrimental effect on health.

Potential adverse effects of over exposure to folic acid in those with low vitamin B12Evidence from historic case reports of vitamin B 12 -de-cient patients mistakenly treated with folic acid have ledto the concern that high doses of folic acid may exacer-bate the neurological symptoms and mask anaemia inindividuals with low vitamin B 12 .

118 Analysis of postfolic acid fortication data from the USA andCanada has been useful in assessing the impact of high folic acid exposure on vitamin B 12 status. Sincethe introduction of mandatory fortication there hasbeen a dramatic increase in blood folate concentrationsby up to 40%, 119 with unmetabolized folic acid (i.e. notthe normal circulating co-factor form) now beingdetected in the circulation. 120 This in turn has led toan intriguing scenario where an increasing number of older adults have very high folate concentrationscoupled with low vitamin B 12 status. This is a relativelynew phenomenon and there are concerns over potentialadverse effects on health. The most widely documentedconcern is that folic acid at high doses may potentiallymask the anaemia of vitamin B 12 deciency, tradition-

ally diagnosed by macrocytosis (elevated mean corpus-cular volume [MCV] > 100fL). 5 This could arise as aresult of folic acid being converted to tetrahydrofolate(through the activity of dihydrofolate reductase),bypassing the vitamin B 12 -dependent enzyme methio-nine synthase, thus promoting DNA synthesis and cor-recting signs of anaemia (independent of whether it wascaused by folate or vitamin B 12 deciency) but not theneurological damage specically caused by vitamin B 12deciency. This is of concern in individuals with lowB12 status because correction of anaemia will alleviatethe symptoms associated with it and thus may delaydiagnosis allowing the irreversible neurologicaldamage to progress unabated. 5,118

An early report concluded, however, that the intro-duction of mandatory folic acid fortication in the USAhas not had an adverse effect on the diagnosis of vitaminB12 deciency.

121 Analysis from the National Healthand Nutrition Examination Survey (NHANES) in theUSA, however, observed another potential adverseinteraction between folate and vitamin B 12 . Low vitaminB12 was, as expected, associated with elevated homocyst-eine and MMA. 122 Unexpectedly, however, homocyst-eine and MMA were highest in individuals with low




12/16

vitamin B 12 coupled with high folate, and furthermore,these individuals were at greater risk of both anaemiaand cognitive impairment. 123 Two subsequent studiesconducted in the UK and Ireland failed to detect similaradverse interactions between high folate and low vita-min B 12 , an inconsistency that might be explained by

the absence of mandatory folic acid fortication inboth countries resulting in generally lower folate, andparticularly lower unmetabolized folic acidconcentrations. 124,125

Apart from concerns regarding potential adverseinteractions with vitamin B 12 , a new concern hasemerged in recent years that excessively high intakesof folic acid (the synthetic vitamin form) could have acancer-promoting effect in segments of ageing popula-tions, but this is rather controversial. On the onehand, there is consistent epidemiological evidencethat higher folate intake within the dietary rangeplays a protective role against cancer at varioussites, on the other hand there is evidence suggestingthat long-term exposure to high intakes of folic acid(greater than 1 mg/day) may promote colorectaltumorigenesis in patients with pre-existing lesions oreven increase cancer risk generally. 126 Thus policy-makers worldwide (including the UK government)have delayed decisions to implement population-based folic acid fortication policies similar to thoseintroduced in 1996 in North America.

Given the potentially serious consequences of over-exposure to folic acid some have suggested that vita-min B 12 should be included together with folic acid in

any new food fortication policy. Not only would thisprevent any adverse outcomes in individuals with lowvitamin B 12 status, but would also address the issue of food-bound malabsorption of vitamin B 12 , the mostcommon cause of low vitamin B 12 . Alternatively, aspecic age-related dietary recommendation similarto that issued by the Institute of Medicine (IOM) inthe USA where people aged 50 years are recom-mended to consume most of their vitamin B 12 fromcrystalline sources (i.e. fortied food and supplements)would also address these concerns. 69 If mandatoryfolic acid fortication proceeds in the UK, with orwithout the addition of vitamin B

12, it will be import-

ant to closely monitor any health effects in the olderpopulation.

ConclusionLow vitamin B 12 status is highly prevalent in older ageand is much more common than generally perceived.There is a wealth of epidemiological evidence to suggestthat optimal vitamin B 12 status is important for healthyageing and that low status is associated with anincreased risk of chronic diseases of ageing including

CVD, dementia, cognitive impairment and osteopor-osis. The evidence to date from RCTs examining theeffect of B vitamin supplementation on chronic diseasehas often failed to demonstrate benecial effectsalthough many of these trials were secondary preven-tion trials. Thus, there is a need for large scale,

well-designed intervention studies to investigate thepotential benets of vitamin B 12 , alone or in combin-ation with related B vitamins, in preventing disease inthose without existing disease pathology and particu-larly in those with lower vitamin B 12 status. Theimportance of detecting low vitamin B 12 status hasbeen highlighted in this review; however, conventionalvitamin B 12 assays may lack sensitivity and could meanthat low/decient B 12 status remains undetected in asignicant proportion of the older population.HoloTC shows promise as a sensitive biomarker of vitamin B 12 status; however, further investigations areneeded to determine its sensitivity (and response tovitamin B 12 intervention) before its usefulness as thefront-line tool in the diagnosis of vitamin B 12 deciencycan be conrmed. The issue of food-bound malabsorp-tion of vitamin B 12 and consequences of mild subclin-ical deciency have still not been fully explored andrequire further investigation. The possible adverse con-sequences of high folic acid exposure in older peoplewith low vitamin B 12 status will require close monitor-ing over the coming years. Optimization of vitamin B 12in older people should be an urgent public healthpriority.

AcknowledgementsNone.

Declaration of Conflicting InterestsNone.

FundingCFH was funded through a PhD studentship provided by theDepartment for Employment and Learning, NorthernIreland.

Ethical approvalNot applicable.

Guarantor HMcN.

ContributorshipCFH wrote the rst draft of the manuscript. All authorsreviewed and edited the manuscript and approved the nalversion.

Hughes et al. 325



13/16

References1. Scott JM. Folate (folic acid) and vitamin B12. In: Garrow

JS, James WPT and Ralph A (eds) Human Nutrition and Dietetics , 10th edn. London: Churchill Livingstone, 2003,pp.27180.

2. Molloy AM. Folate-vitamin B12 interrelationships; links

to disease risk. In: Bailey LB (ed.) Folate in Health and Disease , 2nd edn. Boca Raton: CRC Press, 2010,pp.381408.

3. Scott JM and Weir D. Folate/vitamin B12 inter-relation-ships. Essays Biochem 1994; 28: 6372.

4. Shane B. Folate chemistry and metabolism. In: Bailey LB(ed.) Folate in Health and Disease , 2nd edn. Boca Raton:CRC Press, 2010, pp.124.

5. Carmel R, Green R, Rosenblatt DS and Watkins D.Update on cobalamin, folate, and homocysteine.Hematology 2003; 1: 6281.

6. Scott JM. Bioavailability of vitamin B12. Eur J Clin Nutr1997; 51(Suppl. 1): S4953.

7. Berlin H, Berlin R and Brante G. Oral treatmentof pernicious anemia with high doses of vitamin B12without intrinsic factor. Acta Med Scand 1968; 184:24758.

8. Nexo E and Hoffmann-Lu cke E. Holotranscobalamin, amarker of vitamin B-12 status: analytical aspects andclinical utility. Am J Clin Nutr 2011; 94: 359S65S.

9. Chanarin I. The Megaloblastic Anaemias , 2nd edn.Oxford: Blackwell Scientific, 1979.

10. Carmel R. Cobalamin deficiency. In: Carmel R andJacobsen DW (eds) Homocysteine in Health and Disease . Cambridge: Cambridge University Press, 2001,pp.289305.

11. Carmel R. Cobalamin, the stomach, and aging. Am J Clin

Nutr 1997; 66: 7509.12. Green R. Indicators for assessing folate and vitamin B12status and for monitoring the efficacy of interventionstrategies. Food Nutr Bull 2008; 29: s5263.

13. Gibson RS. Principles of Nutritional Assessment , 2nd edn.New York: Oxford University Press, 2005.

14. Nilsson-Ehle H. Age related changes in cobalamin (vita-min B12) handling. Implications for therapy. Drugs Aging1998; 12: 27792.

15. Briddon A. Homocysteine in the context of cobalaminmetabolism and deficiency states. Amino Acids 2003; 24:112.

16. Lindenbaum J, Rosenberg IH, Wilson PW, Stabler SPand Allen RH. Prevalence of cobalamin deficiency inthe Framingham elderly population. Am J Clin Nutr1994; 60: 211.

17. Lindenbaum J, Savage DG, Stabler SP and Allen RH.Diagnosis of cobalamin deficiency: II. Relative sensitiv-ities of serum cobalamin, methylmalonic acid, and totalhomocysteine concentrations. Am J Hematol 1990; 34:99107.

18. Chatthanawaree W. Biomarkers of cobalamin (vitaminB12) deficiency and its application. J Nutr Health Aging2011; 15: 22731.

19. Eussen SJ, de Groot LC, Clarke R, et al. Oral cyano-cobalamin supplementation in older people with vitamin

B12 deficiency: a dose-finding trial. Arch Intern Med 2005; 165: 116772.

20. Homocysteine Lowering Trialists Collaboration. Dose-dependent effects of folic acid on blood concentrationsof homocysteine: a meta-analysis of the randomizedtrials. Am J Clin Nutr 2005; 82: 80612.

21. McKinley MC, McNulty H, McPartlin J, et al. Low-dosevitamin B-6 effectively lowers fasting plasma homocyst-eine in healthy elderly persons who are folate and ribo-flavin replete. Am J Clin Nutr 2001; 73: 75964.

22. McNulty H, Dowey le RC, Strain JJ, et al. Riboflavinlowers homocysteine in individuals homozygous for theMTHFR 677C- > T polymorphism. Circulation 2006; 113:7480.

23. Quinlivan EP, McPartlin J, McNulty H, et al. Importanceof both folic acid and vitamin B12 in reduction of risk of vascular disease. Lancet 2002; 359: 2278.

24. Refsum H, Smith AD, Ueland PM, et al. Facts and rec-ommendations about total homocysteine determinations:an expert opinion. Clin Chem 2004; 50: 332.

25. Frosst P, Blom HJ, Milos R, et al. A candidate geneticrisk factor for vascular disease: a common mutation inmethylenetetrahydrofolate reductase. Nat Genet 1995; 10:1113.

26. Ueland PM, Refsum H, Stabler SP, Malinow MR,Andersson A and Allen RH. Total homocysteine inplasma or serum: methods and clinical applications.Clin Chem 1993; 39: 176479.

27. Savage DG, Lindenbaum J, Stabler SP and Allen RH.Sensitivity of serum methylmalonic acid and total homo-cysteine determinations for diagnosing cobalamin andfolate deficiencies. Am J Med 1994; 96: 23946.

28. Rasmussen K. Studies of methylmalonic acid in humans.

I. Concentrations in serum and urinary excretion innormal subjects after feeding and during fasting, andafter loading with protein, fat, sugar, isoleucine, andvaline. Clin Chem 1989; 35: 22716.

29. Hvas AM and Nexo E. Diagnosis and treatment of vita-min B12 deficiency an update. Haematologica 2006; 91:150612.

30. Herbert V. Staging vitamin B-12 (cobalamin) status invegetarians. Am J Clin Nutr 1994; 59: 1213S22S.

31. Herrmann W, Schorr H, Obeid R and Geisel J. VitaminB-12 status, particularly holotranscobalamin II andmethylmalonic acid concentrations, and hyperhomocys-teinemia in vegetarians. Am J Clin Nutr 2003; 78: 1316.

32. Miller JW. Assessing the association between vitaminB-12 status and cognitive function in older adults. Am J Clin Nutr 2006; 84: 125960.

33. Valente E, Scott JM, Ueland PM, Cunningham C,Casey M and Molloy AM. Diagnostic accuracy of holo-transcobalamin, methylmalonic acid, serum cobalamin,and other indicators of tissue vitamin B12 status in theelderly. Clin Chem 2011; 57: 85663.

34. Hvas AM and Nexo E. Holotranscobalamin-a firstchoice assay for diagnosing early vitamin B deficiency?J Intern Med 2005; 257: 28998.

35. Miller JW, Garrod MG, Rockwood AL, et al.Measurement of total vitamin B12 and




14/16

holotranscobalamin, singly and in combination, inscreening for metabolic vitamin B12 deficiency. ClinChem 2006; 52: 27885.

36. Lindgren A, Kilander A, Bagge E and Nex E.Holotranscobalamin a sensitive marker of cobalaminmalabsorption. Eur J Clin Invest 1999; 29: 3219.

37. Refsum H, Johnston C, Guttormsen AB and Nexo E.Holotranscobalamin and total transcobalamin inhuman plasma: determination, determinants, and refer-ence values in healthy adults. Clin Chem 2006; 52: 12937.

38. Bor MV, Nexo E and Hvas AM. Holo-transcobalaminconcentration and transcobalamin saturation reflectrecent vitamin B12 absorption better than does serumvitamin B12. Clin Chem 2004; 50: 10439.

39. von Castel-Roberts KM, Morkbak AL, Nexo E, et al.Holo-transcobalamin is an indicator of vitamin B-12absorption in healthy adults with adequate vitaminB-12 status. Am J Clin Nutr 2007; 85: 105761.

40. Solomon LR. Disorders of cobalamin (Vitamin B12)metabolism: emerging concepts in pathophysiology, diag-

nosis and treatment. Blood Rev 2007; 21: 11330.41. Dhonukshe-Rutten RA, de Vries JH, de Bree A, van derPut N, van Staveren WA and de Groot LC. Dietaryintake and status of folate and vitamin B12 and theirassociation with homocysteine and cardiovascular diseasein European populations. Eur J Clin Nutr 2009; 63:1830.

42. Finch S, Doyle W, Lowe C, et al. National Diet and Nutrition Survey: People Aged 65 Years and Over.Volume 1: Report of the Diet and Nutrition Survey .London: TSO, 1998.

43. Scott JM. Folate and vitamin B12. Proc Nutr Soc 1999;58: 4418.

44. Carmel R. Biomarkers of cobalamin (vitamin B-12)

status in the epidemiologic setting: a critical overview of context, applications, and performance characteristics of cobalamin, methylmalonic acid, and holotranscobalaminII. Am J Clin Nutr 2011; 94: 348S58S.

45. Hathcock JN and Troendle GJ. Oral cobalamin for treat-ment of pernicious anemia? JAMA 1991; 265: 967.

46. van Asselt DZ, de Groot LC, van Staveren WA, et al.Role of cobalamin intake and atrophic gastritis in mildcobalamin deficiency in older Dutch subjects. Am J ClinNutr 1998; 68: 32834.

47. Fried M, Siegrist H, Frei R, et al. Duodenal bacterialovergrowth during treatment in outpatients with omepra-zole. Gut 1994; 35: 236.

48. Carmel R, Perez-Oerez G and Blaser M. Helicobacterpylori infection and food-cobalamin malabsorption. DigDis Sci 1994; 39: 30914.

49. Jain K, Shah A, Bariwal J, et al. Recent advances inproton pump inhibitors and management of acid-pepticdisorders. Bioorg Med Chem 2007; 15: 1181205.

50. Richter JE, Peura D, Benjamin SB, Joelsson B andWhipple J. Efficacy of omeprazole for the treatment of symptomatic acid reflux disease without esophagitis.Arch Intern Med 2000; 160: 18106.

51. Mitchell SL and Rockwood K. The association betweenantiulcer medication and initiation of cobalamin replace-ment in older persons. J Clin Epidemiol 2001; 54: 5314.

52. Marcuard SP, Albernaz L and Khazanie PG.Omeprazole therapy causes malabsorption of cyano-cobalamin (vitamin B12). Ann Intern Med 1994; 120:2115.

53. Saltzman J, Kemp J, Golner B, Pedrosa M, Dalial G andRussell R. Effect of hypochlorhydria due to omeprazoletreatment or atrophic gastritis on protein-bound vitaminB12 absorption. J Am Coll Nutr 1994; 13: 58491.

54. Termanini B, Gibril F, Sutliff V, Yu F, Venzon D andJensen R. Effect of long-term gastric acid supressive ther-apy on serum vitamin B12 levels in patients withZollinger-Ellison Syndrome. Am J Med 1998; 104:42230.

55. Dharmarajan TS, Kanagala MR, Murakonda P, LebeltAS and Norkus EP. Do acid-lowering agents affect vita-min B12 status in older adults? JAMDA 2008; 9: 1627.

56. den Elzen WP, Groeneveld Y, de Ruijter W, et al. Long-term use of proton pump inhibitors and vitamin B12status in elderly individuals. Aliment Pharmacol Ther2008; 27: 4917.

57. Forgacs I and Loganayagam A. Overprescribing protonpump inhibitors. BMJ 2008; 336: 23.58. McNulty H, Strain JJ, Pentieva K and Ward M. One-

carbon metabolism and cardiovascular disease outcomesin older adults. Proc Nutr Soc 2012; 71: 21321.

59. Klerk M, Verhoef P, Clarke R, et al. MTHFR 677C ! Tpolymorphism and risk of coronary heart disease: a meta-analysis. JAMA 2002; 288: 202331.

60. Lewis SJ, Ebrahim S and Smith GD. Meta-analysis of MTHFR 677C ! T polymorphism and coronary heartdisease: does totality of evidence support causal role forhomocysteine and preventive potential of folate? BMJ 2005; 331: 105358.

61. Holmes MV, Newcombe P, Hubacek JA, et al. Effect

modification by population dietary folate on the associ-ation between MTHFR genotype, homocysteine, andstroke risk: a meta-analysis of genetic studies and rando-mised trials. Lancet 2011; 378: 58494.

62. Castro R, Barroso M, Rocha M, et al. The TCN2 776C! G polymorphism correlates with vitamin B12 cellulardelivery in healthy adult populations. Clin Biochem 2010;43: 6459.

63. von Castel-Dunwoody KM, Kauwell GPA, Shelnutt KP,et al. Transcobalamin 776C ! G polymorphism nega-tively affects vitamin B-12 metabolism. Am J Clin Nutr2005; 81: 143641.

64. Healton EB, Brust JC, Garrett TJ and Lindenbaum J.Neurologic aspects of coabalmin deficiency. Medicine1991; 70: 22945.

65. Carmel R. How I treat cobalamin (vitamin B12) defi-ciency. Blood 2008; 112: 221421.

66. Kuzminski AM, Del Giacco EJ, Allen RH, Stabler SPand Lindenbaum J. Effective treatment of cobalamindeficiency with oral cobalamin. Blood 1998; 92: 11918.

67. Bor MV, C etin M, Aytac S, Altay C , Ueland PM andNexo E. Long term biweekly 1 mg oral vitamin B12ensures normal hematological parameters, but does notcorrect all other markers of vitamin B12 deficiency. Astudy in patients with inherited vitamin B12 deficiency.Haematologica 2008; 93: 17558.

Hughes et al. 327



15/16

68. Carmel R. Current concepts in cobalamin deficiency.Annu Rev Med 2000; 51: 35775.

69. Institute of Medicine Panel on folate, other B vitaminsand choline. Dietary Reference Intake: Thiamin,Riboflavin, Niacin, Vitamin B6, Vitamin B12,Pantothenic Acid, Biotin and Choline Washington DC:National Academy Press, 1998, pp.30656.

70. Bor MV, Lydeking-Olsen E, Mo ller J and Nexo E. Adaily intake of approximately 6 mg vitamin B12 appearsto saturate all the vitamin B12 related variables in Danishpostmenopausal women. Am J Clin Nutr 2006; 83: 528.

71. Vogiatzoglou A, Smith AD, Nurk E, et al. Dietarysources of vitamin B-12 and their association withplasma vitamin B-12 concentrations in the general popu-lation: the Hordaland Homocysteine Study. Am J ClinNutr 2009; 89: 107887.

72. McCully KS. Vascular pathology of homocysteinemia:implications for the pathogenesis of atherosclerosis. AmJ Pathol 1969; 56: 11128.

73. Homocysteine Studies Collaboration. Homocysteine and

risk of ischemic heart disease and stroke: a meta-analysis.JAMA 2002; 288: 201522.

74. Wald DS, Law M and Morris JK. Homocysteine andcardiovascular disease: evidence on causality from ameta-analysis. BMJ 2002; 325: 12028.

75. Wang X, Qin X, Demirtas H, et al. Efficacy of folic acidsupplementation in stroke prevention: a meta-analysis.Lancet 2007; 369: 187682.

76. Clarke R, Smith AD, Jobst KA, Refsum H, Sutton L andUeland PM. Folate, vitamin B12, and serum total homo-cysteine levels in confirmed Alzheimer Disease. ArchNeurol 1998; 55: 144955.

77. Dhonukshe-Rutten RA, Pluijm SM, de Groot LC, Lips P,

Smit JH and van Staveren WA. Homocysteine and vita-min B12 status relate to bone turnover markers, broad-band ultrasound attenuation, and fractures in healthyelderly people. J Bone Miner Res 2005; 20: 9219.

78. Gjesdal CG, Vollset SE, Ueland PM, et al. Plasma totalhomocysteine level and bone mineral density: theHordaland homocysteine study. Ann Intern Med 2006;166: 8894.

79. Clarke R, Birks J, Nexo E, et al. Low vitamin B-12 statusand risk of cognitive decline in older adults. Am J ClinNutr 2007; 86: 138491.

80. Lee M, Hong KS, Chang SC and Saver JL. Efficacy of homocysteine-lowering therapy with folic acid in strokeprevention: a meta-analysis. Stroke 2010; 41: 120512.

81. Spence JD, Bang H, Chambless LE and Stampfer MJ.Vitamin intervention for stroke prevention trial: an effi-cacy analysis. Stroke 2005; 36: 24049.

82. Schnyder G, Roffi M, Pin R, et al. Decreased rate of coronary restenosis after lowering of plasma homocyst-eine levels. N Engl J Med 2001; 345: 1593600.

83. Schnyder G, Roffi M, Flammer Y, Pin R and Hess OM.Effect of homocysteine-lowering therapy with folic acid,vitamin B12, and vitamin B6 on clinical outcome afterpercutaneous coronary intervention: the Swiss Heartstudy: a randomized controlled trial. JAMA 2002; 288:9739.

84. Bleie O, Strand E, Ueland PM, et al. Coronary bloodflow in patients with stable coronary artery disease trea-ted long term with folic acid and vitamin B12. CoronArtery Dis 2011; 22: 2708.

85. Rafnsson S, Saravanan P, Bhopal R and Yajnik C. Is alow blood level of vitamin B12 a cardiovascular and dia-betes risk factor? A systematic review of cohort studies.Eur J Nutr 2011; 50: 97106.

86. Smith AD. The worldwide challenge of the dementias: arole for B vitamins and homocysteine? Food Nutr Bull 2008; 29(2 suppl): s14372.

87. McCaddon A, Davies G, Hudson P, Tandy S and CattellH. Total serum homocysteine in senile dementia of Alzheimer type. Int J Geriatr Psychiatry 1998; 13: 2359.

88. Herrmann W and Obeid R. Biomarkers of neurodegen-erative diseases. Clin Chem Lab Med 2011; 49: 3434.

89. Wald DS, Kasturiratne A and Simmonds M. Serumhomocysteine and dementia: meta-analysis of eightcohort studies including 8669 participants. AlzheimersDement 2011; 7: 4127.

90. Nurk E, Refsum H, Tell GS, et al. Plasma total homo-cysteine and memory in the elderly: the HordalandHomocysteine study. Ann Neurol 2005; 58: 84757.

91. Campbell A, Jagust WJ, Mungas DM, et al. Low erythro-cyte folate, but not plasma vitamin B12 or homocysteine,is associated with dementia in elderly Latinos. J NutrHealth Aging 2005; 9: 3943.

92. Mooijaart SP, Gussekloo J, Frolich M, et al.Homocysteine, vitamin B-12, and folic acid and the riskof cognitive decline in old age: the Leiden 85-Plus Study.Am J Clin Nutr 2005; 82: 86671.

93. Tettamanti M, Garri MT, Nobili A, Riva E and Lucca U.Low folate and the risk of cognitive and functional def-

icits in the very old: the Monzino 80-plus Study. J AmColl Nutr 2006; 25: 5028.94. McCracken C, Hudson P, Ellis R and McCaddon A.

Medical Research Council Cognitive Function andAgeing Study. Methylmalonic acid and cognitive functionin the Medical Research Council Cognitive Function andAgeing Study. Am J Clin Nutr 2006; 84: 140611.

95. Wald DS, Kasturiratne A and Simmonds M. Effect of folic acid, with or without other B vitamins, on cognitivedecline: meta-analysis of randomized trials. Am J Med 2010; 123: 5227.

96. Kang JH, Cook N, Manson J, Buring JE, Albert CM andGrodstein F. A trial of B vitamins and cognitive functionamong women at high risk of cardiovascular disease. AmJ Clin Nutr 2008; 88: 160210.

97. Brady CB, Gaziano JM, Cxypoliski RA, et al.Homocysteine lowering and cognition in CKD: theVeterans Affairs Homocysteine study. Am J Kidney Dis2009; 54: 4409.

98. Ford A, Flicker L, McCaul K, et al. The B-VITAGEtrial: A randomized trial of homocysteine lowering treat-ment of depression in later life. Trials 2010; 11: 8.

99. Andreeva VA, Kesse-Guyot E, Barberger-Gateau P,Fezeu L, Hercberg S and Galan P. Cognitive functionafter supplementation with B vitamins and long-chainomega-3 fatty acids: ancillary findings from the




16/16

SU.FOL.OM3 randomized trial. Am J Clin Nutr 2011;94: 27886.

100. Smith AD, Smith SM, de Jager CA, et al.Homocysteine-lowering by B vitamins slows the rateof accelerated brain atrophy in mild cognitive impair-ment: a randomized controlled trial. PLoS ONE 2010; 5:e12244.

101. McMahon JA, Green TJ, Skeaff CM, Knight RG,Mann JI and Williams SM. A controlled trial of homo-cysteine lowering and cognitive performance. N Engl J Med 2006; 354: 276472.

102. Durga J, van Boxtel MPJ, Schouten EG, et al. Effect of 3-year folic acid supplementation on cognitive functionin older adults in the FACIT trial: a randomised, doubleblind, controlled trial. Lancet 2007; 369: 20816.

103. Walker JG, Batterham PJ, Mackinnon AJ, et al. Oralfolic acid and vitamin B-12 supplementation to preventcognitive decline in community-dwelling older adultswith depressive symptoms the Beyond AgeingProject: a randomized controlled trial. Am J Clin Nutr

2012; 95: 194203.104. de Jager CA, Oulhaj A, Jacoby R, Refsum H andSmith AD. Cognitive and clinical outcomes of homo-cysteine-lowering B-vitamin treatment in mild cogni-tive impairment: a randomized controlled trial. Int J Geriatr Psych 2012; 27: 592600.

105. Naharci I, Bozoglu E, Karadurmus N, et al. VitaminB12 and folic acid levels as therapeutic target in preser-ving bone mineral density (BMD) of older men. ArchGerontol Geriat 2012; 54: 46972.

106. Dhonukshe-Rutten RA, Lips M, de Jong N, et al.Vitamin B-12 status is associated with bone mineral con-tent and bone mineral density in frail elderly women butnot in men. J Nutr 2003; 133: 8017.

107. McLean RR, Jacques PF, Selhub J and Tucker KL.Homocysteine as a predictive factor for hip fracture inolder persons. New Engl J Med 2004; 350: 20429.

108. Morris MS, Jacques PF and Selhub J. Relation betweenhomocysteine and B-vitamin status indicators and bonemineral density in older Americans. Bone 2005; 37:23442.

109. Tucker KL, Hannan MT, Qiao N, et al. Low plasmavitamin B12 is associated with lower BMD: theFramingham Osteoporosis study. J Bone Miner Res2005; 20: 1528.

110. Goerss JB, Kim CH, Atkinson EJ, Eastell R, OFallonM and Melton LJ. Risk of fractures in patients withpernicious anemia. J Bone Miner Res 1992; 7: 5739.

111. Melton ME and Kochman ML. Reversal of severeosteoporosis with vitamin B12 and etidronate therapyin a patient with pernicious anemia. Metabolism 1994;43: 4689.

112. Stone KL, Bauer DC, Sellmeyer D and Cummings SR.Low serum vitamin B-12 levels are associated withincreased hip bone loss in older women: a prospectivestudy. Clin Endocrinol Metab 2004; 89: 121721.

113. Green TJ, McMahon JA, Skeaff CM, Williams SM andWhiting SJ. Lowering homocysteine with B vitamins hasno effect on biomarkers of bone turnover in older

persons: a 2-y randomized controlled trial. Am J ClinNutr 2007; 85: 4604.

114. Herrmann M, Umanskaya N, Traber L, et al. The effectof B-vitamins on biochemical bone turnover markersand bone mineral density in osteoporotic patients: a1-year double blind placebo controlled trial. Clin ChemLab Med 2007; 45: 178592.

115. Sato Y, Honda Y, Iwamoto J, Kanoko T and Satoh K.Effect of folate and mecobalamin on hip fractures inpatients with stroke: a randomized controlled trial.JAMA 2005; 293: 10828.

116. Honein MA, Paulozzi LJ, Mathews TJ, Erickson JDand Wong LY. Impact of folic acid fortification of theUS food supply on the occurrence of neural tube defects.JAMA 2001; 285: 29816.

117. Yang Q, Botto LD, Erickson JD, et al. Improvement instroke mortality in Canada and the United States, 1990to 2002. Circulation 2006; 113: 133543.

118. Reynolds E. Vitamin B12, folic acid, and the nervoussystem. Lancet Neuro 2006; 5: 94960.

119. Pfeiffer CM, Caudill SP, Gunter EW, Osterloh J andSampson EJ. Biochemical indicators of B vitaminstatus in the US population after folic acid fortification:results from the National Health and NutritionExamination Survey 19992000. Am J Clin Nutr 2005;82: 44250.

120. Troen AM, Mitchell B, Sorensen B, et al.Unmetabolized folic acid in plasma is associated withreduced natural killer cell cytotoxicity among postme-nopausal women. J Nutr 2006; 136: 18994.

121. Mills JL, Von Kohorn I, Conley MR, et al. Low vitaminB-12 concentrations in patients without anemia: theeffect of folic acid fortification of grain. Am J ClinNutr 2003; 77: 14747.

122. Selhub J, Morris MS and Jacques PF. In vitamin B12deficiency, higher serum folate is associated withincreased total homocysteine and methylmalonic acidconcentrations. Proc Natl Acad Sci USA 2007; 104:1999520000.

123. Morris MS, Jacques PF, Rosenberg IH and Selhub J.Folate and vitamin B-12 status in relation to anemia,macrocytosis, and cognitive impairment in olderAmericans in the age of folic acid fortification. Am J Clin Nutr 2007; 85: 193200.

124. Clarke R, Sherliker P, Hin H, et al. Folate and vitaminB12 status in relation to cognitive impairment and anae-mia in the setting of voluntary fortification in the UK.Br J Nutr 2008; 100: 10549.

125. Mills JL, Carter TC, Scott JM, et al. Do high bloodfolate concentrations exacerbate metabolic abnormal-ities in people with low vitamin B-12 status? Am J ClinNutr 2011; 94: 495500.

126. Mason JB. Folate consumption and cancer risk: a con-firmation and some reassurance, but were not out of thewoods quite yet. Am J Cin Nutr 2011; 94: 93664.

127. Andre s E, Loukili NH, Noel E, et al. Vitamin B 12(cobalamin) deficiency in elderly patients. CMAJ 2004;171: 2519.

Hughes et al. 329

ann clin biochem 2013 hughes 315 29

Documents