Biomarkers of cobalamin (vitamin B12) deficiency and its application

Abstract

Cobalamin (vitamin B12) deficiency in the elderly is an under recognized problem in daily clinical practice. It seems to be important because the deficiency of this vitamin can lead to irreversible neurological damage, anemia, osteoporosis, and cerebrovascular and cardiovascular diseases. Some clinical abnormalities that we thought were related to the normal aging changes may actually be caused by cobalamin deficiency, such as lack of ankle jerk reflex. The prevalence of cobalamin deficiency increases with age (ranges from 0.6% to 46% depending on the population studies and criteria for diagnosis). Other than clinical manifestations, there are some biomarkers for detection of cobalamin deficiency: the red blood cell mean corpuscular volume (MCV); serum cobalamin level; plasma holotranscobalamin; serum methylmalonic acid (MMA) levels and serum homocysteine levels. The interpretation and the application of these biomarkers are here presented.

Introduction

Cobalamin (vitamin B12) deficiency seems to be an obscure problem in the elderly. Its deficiency can take 3-6 years to develop after absorption of dietary B12 had ceased and the initial clinical manifestations are subtle and non-specific or are attributed to the normal aging process. This problem was also underestimated in the past because of the belief that deficiency is unlikely except in strict vegetarian and pernicious anemia patients are known to be cobalamin deficient and that it usually takes about 20 years for stores of the vitamin to become depleted. Since a deficiency in this vitamin can lead to irreversible neurological damage (1) (sub-acute combined degeneration of the spinal cord and cognitive impairment in the elderly), megaloblastic anemia, osteoporosis (2), cerebrovascular and cardiovascular diseases (3), early diagnosis is essential. In recent years, new and sensitive diagnostic markers to determine a person's cobalamin status have become available. This article reviews the prevalence of cobalamin deficiency, metabolism of cobalamin and biomarkers for diagnosing cobalamin deficiency disease.

Prevalence

The prevalence of cobalamin deficiency in the elderly varies widely due to population samples and the use of different criteria for this deficiency. The reported prevalence of low plasma vitamin B12 concentrations among the elderly ranges widely from 0.6 to 46% (4, 5, 6, 7, 8, 9, 10) (table 1) with a higher prevalence of deficient concentrations among whites and the Latino population compared with Afro Americans or Asian Americans (11). These finding may be from genetic polymorphism of TC gene or the prevalence of pernicious anemia is higher in Caucasians. The prevalence increases with age in all reports.

Table 1.

Prevalence of cobalamin deficiency in the elderly

Study	Population	Criteria	Prevalence
Assantachai P, 2005 (4)	2,336 rural community Thai elderly (F:M=889:1,447) age 68.94 yr. (60-97 yr.)	Plasma B12<200 pg/mL	0.6%
Campbell AK et al, 2003 (5)	1,546 community of Latino ancestry in Sacramento, CA age 70.5 yr. M=43%	Plasma B12<200 pg/mL (<148 pmol/L)	6.5%
Clarke R et al, 2007 (6)	2,403 community elderly in Oxford city and Oxfordshire age 79.2 yr. M=41%	1. Plasma B12 <200 pmol/L2. Holotranscobalamin <45 pmol/L3. MMA > 0.45 nmol/L	30%25%49%
Hin H et al, 2006 (7)	1,000 community elderly in Oxfordshire age 81.4 yr. M=40%	Plasma B12<133 pmol/L	13%
Stone KL et al, 2004 (8)	83 community elderly in USA age 71.1 yr.	Plasma B12 =280 pg/mL (207.2 pmol/L)	19.3%
Clarke R et al, 2004 (9)	3,511 community elderly in UK age =65 yr.	Plasma B12<150 pmol/L	5% of age 65-74yr.10% of age =75 yr.
Dhonukshe-Rutten RA et al, 2005 (10)	1,267 community elderly in Netherlands age76 yr. M:F=615:652	1. Plasma B12 <200 pmol/L2. Homocysteine >15 μmol/L3. Combine 1 & 2	22%M, 16%F46%M, 29%F14%M, 9%F

Metabolism of cobalamin

Cobalamin is a complex organometallic compound in which a cobalt atom is situated within a corrin ring. It cannot be synthesized in the human body. Meat and dairy products provide the only dietary sources of cobalamin for humans. The usual western diet contains 3 to 30 μg of cobalamin per day, while the recommended dietary allowance (RDA) is 2.4 μg per day for adults (12, 13); pregnant women up to 6 μg per day (13). Total body stores of cobalamin are 2 to 5 mg, approximately one-half of which is in the liver. About 0.1% of this cobalamin reserve is lost per day by secretions into the gut and not all these secretions are reabsorbed. Thus it takes from 3 to 6 years to become deficient if absorption ceases abruptly.

Protein bound cobalamin from food is digested by pepsin and gastric acid in the stomach. Released free cobalamin then quickly binds to the R factor (haptocorrin), one of a group of closely related glycoproteins, found in saliva, milk, gastric juice, bile, phagocytes and plasma. Haptocorrin bound cobalamin itself is not absorbed but emptied into the duodenum. In the alkaline pancreatic enzyme milieu of the duodenum, cobalamin is freed from haptocorrin by pancreatic proteases and then binds specifically and rapidly to the gastric-derived intrinsic factor (IF). The intrinsic factor, a 45 kDa glycoprotein with very high affinity for cobalamin, is generated by gastric parietal cells in response to histamine, gastrin and pentagastrin, as well as the presence of food. The generation of IF-cobalamin complex will allow vitamin absorption and it protects against catabolism by intestinal bacteria and/or proteolytic enzymes. The IF-cobalamin complex binds to specific ileal enterocyte receptors (cubilin) at the terminal ileum, from which it is absorbed in an energy requiring process. In the enterocytes, cobalamin is subsequently released and transferred to transcobalamin II (TC II). The cobalamin-TC II complex, known as holotranscobalamin (holoTC), arrives in the blood circulation, from which it is rapidly taken up by the liver, bone marrow, and other cells (14). A maximum of 30% of circulating cobalamin is bound to TC II, which represents metabolically active vitamin B12. The half-life of holotranscobalamin is about 1 hour. Most circulating cobalamin is bound to transcobalamin I (TC I), a glycoprotein closely related to haptocorrin, which appears to be derived in part from leucocytes. Cobalamin-TC I is thought to transport the surplus of cobalamin to the liver and has a longer half-life of several days. An alternative route of cobalamin absorption occurs when large doses are taken orally (1,000-2,000 μg) (15). This relatively inefficient route of absorption probably occurs by means of passive diffusion throughout the length of the small intestine (16). It is estimated that approximately only 1% of the administered dose is absorbed by this route (15).

Cobalamin in the cells is metabolized into two coenzymes, adenosyl-cobalamin and methyl-cobalamin. Adenosyl-cobalamin is a cofactor of L-methylmalonyl-CoA-mutase. It is involved in the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Methyl-cobalamin is a cofactor for methionine synthase. This enzyme transfers a methyl group of 5-methyltetrahydrofolate to homocysteine during the synthesis of methionine. Methionine is converted to S-adenosylmethionine (SAM) by ATP. This product is the methyl donor of multiple methylated products such as methylated lipids, myelin basic protein, DOPA, DNA (figure 1). Methylation defects are postulated to be the likely cause of myelin damage and disturbed neurotransmitter metabolism (17). In case of intracellular deficiency of cobalamin, plasma concentrations of methylmalonic acid (MMA) and homocysteine will arise due to the fact that it cannot be synthesized to succinyl-CoA and methionine respectively. Large increases in the tissue levels of methylmalonyl-CoA and its precursor, propionyl-CoA, can produce nonphysiological fatty acids containing an odd number of carbon atoms and incorporated into neuronal lipids. This biochemical abnormality may contribute to the neurological complications of cobalamin deficiency.

Figure 1.

Metabolic pathways of cobalamin(Cbl), folate, homocysteine, methylmalonyl-CoA and fatty acid

Cbl is converted to either methylcobalamin (Met-Cbl) in the cytosol or to adenosylcobalamin (Ado-Cbl) in the mitochondria. Met-Cbl is an essential cofactor for methionine synthase (MS), an enzyme catalyzing the cytosolic remethylation of homocysteine to methionine, whereas Ado-Cbl is a cofactor for methylmalonyl-CoA mutase (MCM), which catalyzes the intramitochondrial isomerization of methylmalonyl-CoA to succinyl-CoA. ATP, adenosine triphosphate; DMG, dimethylglycine; BHMT, betaine homocysteine methyltransferase; CBS, cystathionine- -syntase; vit B6, pyridoxal-5-phosphate; CL, cystathionine- lase; DHFR, Dihydrofolate reductase; FPGS, folylpolyglutamate syntase; THFn, polyglutamate form of THF; FTHFS, formyl THF syntase; SGMT, serine-glycine methyltransferase; 5,10 MTHFR, 5, 10 methylene THF reductase; vit B2, riboflavin; LCAS, long chain acyl-CoA synthase; FAS, fatty acid synthase; NADPH, nicotinamide-adenine dinucleotide phosphate; CPT-1, carnitine palmitoyltransferase-1; CPT-2, carnitine palmitoyltransferase-2; CACT, carnitine acylcarnitine translocase; PCC, propionyl-CoA carboxylase; LMC, L-malyl-CoA lyase; ACC, acetyl-CoA carboxylase. (adapted from Cuskelly GJ et al (32), Thupari JN et al (33), Rossi A et al (34).

Biomarkers of cobalamin deficiency

Cobalamin deficiency causes abnormal metabolic processes in this vitamin, such as DNA and RNA biosynthesis defects, and Methylation cycle defects as already mentioned. There are some biomarkers for detection of cobalamin deficiency: red blood cell mean corpuscular volume (MCV), serum cobalamin level, plasma holotranscobalamin, serum methylmalonic acid (MMA) levels and serum homocysteine levels.

Red blood cell means corpuscular volume (MCV)

Methyl-cobalamin deficiency causes accumulation of 5-methyltetrahydrofolate, an inactive form of folate, and decreases tetrahydrofolate, its active form. As a result, the cell will suffer a form of folate deficiency, in that it will have adequate folate but it will be trapped in a form that cannot be used for DNA biosynthesis (18) (figure 1). The consequence will be an anemia identical to that seen in true folate deficiency. Andrès et al (19) reported the hematological manifestations or abnormalities in 201 patients (median age range 61 - 73 years) with well-documented cobalamin deficiency (mean serum vitamin B12 levels 125 ± 47 pg/mL). Hematological abnormalities were reported in at least two-thirds of the patients: anemia (37%), leucopenia (14%), thrombocytopenia (10%), macrocytosis (54%) and hypersegmented neutrophils (32%). About 10% of the patients displayed life-threatening hematological manifestations with documented symptomatic pancytopenia (5%), 'pseudo' thrombotic microangiopathy (Moschkowitz) (2.5%), severe anemia (defined as Hb levels < 6 g/dl) (2.5%) and hemolytic anemia (1.5%). Savage et al (20) reported the probability of a deficiency of folate and/or cobalamin when the MCV is normal (80 to 100 fL), 115 to 129 fL, or > 130 fL to be estimated at < 25, 50, and 100 percent respectively. Other causes of macrocytosis are immunosuppressive drugs, myelodysplastic syndromes, multiple myeloma, pure red cell aplasia, aplastic anemia, liver disease, alcohol abuse and hypothyroidism.

Serum cobalamin level

There are two common laboratory methods for measuring serum cobalamin level; radioimmunoassay and chemiluminescence. Thus, the normal ranges of cobalamin level depend on the methods but there is no gold standard. Clarke et al (21) had used tertiles of vitamin B12 level to show the association between the bottom tertile of vitamin B12 level and cognitive impairment. Hin et al (7) had used quartiles of vitamin B12 level to show the association between the bottom quartile of vitamin B12 level and cognitive impairment. Stone et al (8) had used quintiles of vitamin B12 level to show the association between the bottom quintile of vitamin B12 level (114-280 pg/mL) and increased hip bone loss in elderly women. Dhonukshe-Rutten et al (10) studied relationship between homocysteine or cobalamin concentration and fracture risk in the elderly, found that low serum cobalamin people (< 200 pmol/L) had elevated serum homocysteine level (> 15 μmol/L) about 63 percent. Normal serum cobalamin people (= 200 pmol/L) had elevated serum homocysteine level (> 15 μmol/L) about 31.57 percent. As a result, cobalamin level may not be a good marker for cobalamin deficiency or tissue deficiency. Clarke et al (6) reported the sensitivity and specificity when using the cobalamin level cut-off point at < 200 pmol/L which were 75.7% and 72.4%, respectively. In general, however, serum cobalamin level can be interpreted as follows:

• –> 300 pg/mL - cobalamin deficiency is unlikely (ie, 1 to 5 percent (22))
• –200 to 300 pg/mL - borderline result: cobalamin deficiency possible
• –<200 pg/mL - consistent with cobalamin deficiency (specificity of 95 to 100 percent (23))

In patients with a high degree of suspicion of cobalamin deficiency (borderline level), metabolite testing (serum methylmalonic acid levels, serum homocysteine levels) should be performed. Falsely-elevated serum cobalamin may be caused by liver disease, myeloproliferative disease, transcobalamin II deficiency, small intestinal bacterial overgrowth and hemolysis. Falsely-reduced serum cobalamin may be caused by severe folate deficiency, severe Fe deficiency, oral contraceptives, mild or severe haptocorrin deficiency and myeloma (24).

Plasma holotranscobalamin (holoTC)

Holotranscobalamin or cobalamin bound transcobalamin II is the active fraction of vitamin B12 that delivers vitamin B12 to all cells in the body. If cobalamin absorption ceases, holotranscobalamin is the first metabolite to decrease. Clarke et al (6) reported that using plasma holotranscobalamin cut-off point at 45 pmol/L had a better diagnostic accuracy than serum cobalamin (sensitivity 77% & 73% and specificity 76% & 72.4%). As we know, neurological damage in cobalamin deficiency is irreversible. The important point of plasma holotranscobalamin is that it is the earliest marker of a negative cobalamin balance. The question that should be answered is this: is neurological impairment reversible in patients with decreased holotranscobalamin levels but normal levels of both MMA and homocysteine. Smith et al (11) reported the association between cognitive impairment and the bottom 3 quartiles of holotranscobalamin levels, and estimated that a doubling of the holotranscobalamin concentration from 50 to 100 pmol/L would lead to a 30% reduction in the rate of cognitive decline. Other factors that can affect plasma holotranscobalamin include a common genetic polymorphism 776 (C776G) of the TC gene, TC Codon 259 Genetic Polymorphism, impaired renal function and liver disease (25, 26, 27) as well as female sex hormones (28).

Serum methylmalonic acid (MMA) levels

Adenosyl-cobalamin, one of the two metabolically active forms of cobalamin, is a cofactor of enzyme L-methylmalonyl-CoA-mutase that converts L-methylmalonyl-CoA to succinyl-CoA (figure 1). Deficiency of Adenosyl-cobalamin causes an accumulation of both L-methylmalonyl-CoA and its precursor D-methylmalonyl-CoA. Excess D-methylmalonyl-CoA is then converted into methylmalonic acid (measurable in both serum and urine). This marker is the hallmark only of cobalamin deficiency, but it may be increased in chronic kidney disease, bacterial overgrowth and hypovolemic states (20). The cut-off point of serum methylmalonic acid level varies from study to study. Campbell et al (5) assessed predictors of vitamin B12 status in an elderly Latino population, reported that high serum MMA (= 350 nmol/L) was found in 46% of deficient plasma B12 (< 200 pg/mL), 20.4% of marginal plasma B12 (200-300 pg/mL), and 2.3% of normal plasma B12 (> 300 pg/mL). Herrmann et al (29) reviewed the causes and early diagnosis of vitamin B12 deficiency, and used the cut-off point of serum MMA level > 271 nmol/L to be vitamin B12 deficiency. Hin et al (7) reported that the top quartile of serum MMA was related to cognitive impairment in the elderly. The National Health and Nutrition Examination Survey in the United States used serum MMA > 210 nmol/L ie, the 95th percentile for vitamin B-12–replete participants with normal renal function to be the gold standard indicator of vitamin B12 deficiency (30). Clarke et al (6) defined metabolic vitamin B12 deficiency as a definite diagnosis if serum MMA was > 0.75 μmol/L and only probable if serum MMA was > 0.45 μmol/L. Savage et al (20) defined normal range of serum MMA (ie, 70 to 270 nmol/L). In conclusion, serum MMA has a higher sensitivity and specificity for molecular detection (94% and 99% respectively (20)) than the serum cobalamin level. Therefore, measurement of the serum MMA is helpful in clarifying the diagnosis when serum cobalamin concentrations are equivocal or low normal level. The limitations of serum MMA as an indicator include the high cost of analysis and special equipment required such as mass spectrometers.

Serum homocysteine levels

Methyl-cobalamin, one of the two metabolically active forms of cobalamin, is a cofactor of enzyme methionine synthase that converts homocysteine to methionine. Deficiency of Methyl-cobalamin causes an accumulation of both homocysteine and 5-methyl tetrahydrofolate (the inactive form of folate). Thus serum homocysteine can be used to identify cobalamin deficiency. Serum homocysteine is also increased in the folate, vitamin B2 and vitamin B6 deficiencies (figure 1). Rare genetic defects in the enzymes involved in homocysteine metabolism (cystathionine synthase activity deficiency – homozygous and heterozygous mutations, methylene tetrahydrofolate reductase (MTHFR) deficiency – homozygous and heterozygous severe mutations and thermolabile MTHFR) can cause hyperhomocysteinemia. Other causes of nongenetic hyperhomocysteinemia are renal failure, liver disease, drugs (methotrexate, trimethoprim, cholestyramine, colestipol, phenytoin, carbamazepine, niacin, theophylline, androgen, cyclosporine and fibric acid derivatives). Normal range of serum homocysteine is 5 to 14 μmol/L (20). Campbell et al (5) reported that high serum homocysteine (= 13 μmol/L) was found in 50.9% of deficient plasma B12 (< 200 pg/mL), 25.7% of marginal plasma B12 (200-300 pg/mL), and 20.4% of normal plasma B12 (> 300 pg/mL). Hin et al (7) reported that the top 2 quartiles of serum homocysteine and Clark et al (21) reported that the top 2 tertiles of serum homocysteine were also related to cognitive impairment in the elderly. Dhonukshe-Rutten et al (10) reported the association of high serum homocysteine (> 15 μmol/L) to low broadband ultrasound attenuation of the calcaneus, increased fracture risk and high bone turnover markers in the elderly. In conclusion, serum homocysteine has a higher sensitivity and specificity than serum cobalamin level for detection of cobalamin deficiency. Increased serum homocysteine is specific for cobalamin and folate deficiencies, so folate deficiency must be excluded before diagnosis of cobalamin deficiency is made and vice versa.

Strategy for diagnosis of cobalamin deficiency

If cobalamin deficiency is suspected in patients with macrocytosis, pancytopenia, unexplained neurologic signs and symptoms (especially dementia), or special populations (such as the elderly, alcoholics, and people suffering from malnutrition), the first step should be to measure serum cobalamin and erythrocyte folate concentration (31).

If serum cobalamin and erythrocyte folate concentration are > 300 pg/mL and > 221 ng/mL respectively, deficiencies of the two vitamins are unlikely, and additional testing is not required.

If serum cobalamin is < 300 pg/mL and erythrocyte folate concentration is > 221 ng/mL, cobalamin deficiency is likely. The next step should be the evaluation of the serum homocysteine or methylmalonic acid (MMA). If both test results are normal (ie, MMA 70 to 270 nmol/L and serum homocysteine 5 to 14 μmol/L), cobalamin deficiency is effectively ruled out. If concentrations of both metabolites have increased, cobalamin deficiency is confirmed.

If serum cobalamin is < 300 pg/mL and erythrocyte folate concentration is < 221 ng/mL, combined vitamin deficiency is likely. The next step should be the evaluation of the serum homocysteine or methylmalonic acid (MMA). If both test results are normal, combined vitamin deficiency is effectively ruled out. If concentrations of both metabolites have increased, combined vitamin deficiency is confirmed. If MMA is normal and serum homocysteine has increased, folate deficiency is likely.

If serum cobalamin is > 300 pg/mL and erythrocyte folate concentration is < 221 ng/mL, folate deficiency is likely. The next step should be the evaluation of the serum homocysteine. If serum homocysteine is normal, folate deficiency is effectively ruled out. If concentration of homocysteine is increased, folate deficiency is confirmed.

Renal dysfunction should be excluded in patients with elevated serum MMA and/or serum homocysteine. If serum creatinine is increased, the reduction of serum MMA and/or serum homocysteine after vitamin B12 treatment should be determined.

Acknowledgements: I am grateful to: Professor JOHN BULGER and Mrs. Chommanard Sumngern to help me to prepare the manuscripts.

Author Disclosure Statement: There is no competing financial interests exist.

Financial disclosure: None of the authors had any financial interest or support for this paper.