Vitamin B12 Deficiency Cascade

Serum

3 mL

2 mL (Note: This volume does not allow for repeat testing).

Red-top tube or gel-barrier tube

Diagnosis of Vitamin B12 Deficiency. Although often used as the first-line screening test for B12 deficiency, serum B12 measurement used in isolation has a generally poor sensitivity and specificity for detection of B12 deficiency.^1,6,19,32 The National Health and Nutrition Examination Survey (NHANES) opted to use the combination of serum total vitamin B12 and methylmalonic acid (MMA) to monitor B12 status in the United States population.³⁵ In the interest of economy, a number of groups have suggested the use of a sequential selection algorithm for the detection of B12 deficiency.^5,14,33,34 In this approach, a second-line assay (in this case MMA) is performed only when the outcome of the first-line assay (vitamin B12 level) falls in an "equivocal" range.^1,7 It has been suggested that borderline B12 levels (200-400 ng/L) should be followed up with measuring MMA levels.¹ MMA levels below the upper limit of the reference interval (0-378 nmol/L) are strongly suggestive of normal B12 status.

Ninety per cent of patients with pernicious anemia have gastric parietal cell antibodies, but specificity of this test is poor since they are also found in 15% of elderly subjects.

If IFA results are negative but suspicion for pernicious anemia remains, an elevated serum gastrin level is consistent with the diagnosis.⁷

Mutations in the gene encoding intrinsic factor, can also lead to an inherited form of B12 malabsorption and deficiency, which resembles pernicious anemia, but without autoantibody involvement.³⁶

In the presence of discordance between laboratory test result and strong clinical features of B12 deficiency, it remains important to proceed with treatment to avoid neurological impairment.¹⁴

MMA can increase (300-700 nmol/L) in renal failure and its refractory to B12 administration.¹

Some patients with gastric atrophy and diminished parietal cell function are not positive for IFA or PCA. Diminished acid secretion caused by gastric atrophy regardless of the etiology can cause increased secretion of gastrin. Elevated gastrin levels can support the diagnosis of PA in antibody negative patients.^24,29 It is important to diagnose hypergastrinaemia arising from loss gastric parietal cells drives development of antral enterochromaffin cell hyperplasia that can further develop into neoplasia and carcinoid syndrome.^1,3,24,30,31

B12 is essential for certain enzymatic reactions that are required for numerous physiologic functions including erythropoiesis and myelin synthesis.^1,2 Impaired DNA synthesis caused by B12 deficiency impacts nuclear maturation of rapidly dividing cells. This affects hematopoiesis and results in the presence of immature and ineffective red cells that are larger than normal (megaloblasts) in a context of severe anemia and pancytopenia. This megaloblastic anemia is characterized by the hypersegmented neutrophils that can be seen on peripheral smears and giant bands in bone marrow. Other rapidly dividing cells of the small-bowel epithelium can be affected resulting in malabsorption and diarrhea.³ Glossitis is a frequent hallmark of megaloblastic anemia, with the patient experiencing a painful, smooth, red tongue. Ineffective erythropoiesis and associated increased red cell turnover can result in elevation in bilirubin levels, manifesting as jaundice.³

B12 deficiency can also produce neurological manifestations including sensory and motor disturbances (symmetric paresthesias, numbness and gait problems), ataxia, cognitive decline leading to dementia and psychiatric disorders. These neurological symptoms often predominate and can frequently occur in the absence of hematological complications.^3,7 In fact, the majority of patients with suspected B12 deficiency do not have anemia.^5-8

Emerging evidence indicates that low (though not necessarily deficient) B12 is associated with increased risk of various chronic diseases of ageing including cognitive dysfunction, cardiovascular disease and osteoporosis.^5,6 Dietary vitamin B12 is normally bound to proteins in food and requires release by gastric acid and pepsin in the stomach.⁷ In the small intestine, vitamin B12 binds to intrinsic factor (IF) produced by gastric parietal cells. In the ileum, the B12-IF complex binds to specific receptors, which facilitates absorption into the blood. Large amounts of absorbed vitamin B12 are stored in the liver such that any reduction in vitamin B12 intake/absorption may take many years to manifest clinically.⁸ Low B12 status, especially in older adults, is rarely attributable to dietary insufficiency⁹ and is more typically the result of malabsorption related to atrophic gastritis, inflammatory bowel disease or use of proton pump inhibitors or other gastric acid suppressant drugs.^2,6,7,10-13

The diagnosis of vitamin B12 deficiency requires consideration of both the clinical state of the patient and the results of laboratory tests. Screening average-risk adults for vitamin B12 deficiency is not recommended.² However, testing should be considered in patients with risk factors and/or clinical blood count and serum vitamin B12 level.^2,5,7,14,15 The World Health Organization¹⁶ and the British Committee for Standards in Haematology¹⁴ suggested using 200 pg/mL as a cut-off to define B12 deficiency. In practice, detectable disturbances in metabolic networks consistent with possible deficiency occur at B12 levels as high as 400 pg/mL.¹⁷

A significant number of B12-deficient patients may be overlooked when serum B12 measurement is used in isolation.^5,17 Further investigation using a second-line test can be useful for serum B12 results that fall within the indeterminate range. The enzyme, methylmalonyl-CoA mutase requires vitamin B12 as a cofactor for the conversion of methylmalonyl-CoA to succinyl-CoA.⁵ In vitamin B12 depletion, reduced activity of this enzyme leads to an accumulation of methylmalonyl-CoA which is, in turn, hydrolyzed to methylmalonic acid. Measurement of serum methylmalonic acid provides biochemical evidence of metabolic abnormalities consistent with B12 insufficiency.^{2,5,7,10,14,18,19}

In the United States and the United Kingdom, the prevalence of vitamin B12 deficiency has been estimated to be approximately 6% of persons younger than 60 years, and nearly 20% in those older than 60 years.¹⁰ B12 status in the United States has been assessed in the National Health and Nutrition Examination Survey (NHANES).²⁰ Using NHANES data from 1999 to 2004, the prevalence of B12 status defined as low was estimated to be 2.9%, 10.6% or 25.7% based on serum B12 cut-off values of 200, 300 and 400 pg/mL, respectively.²⁰ Using these cut-off values, the prevalence of low B12 status increased with age from young adults (19-39 years of age) to older adults (greater than or equal to 60 years of age), and was generally higher in women than than in men (prevalence of 3.3% versus 2.4% with a serum B12 level of <200 pg/mL, respectively).²⁰ Using increased levels of MMA as a functional indicator of B12 status, the prevalence of low B12 status was 2.3% or 5.8% based on cut-off values of >376 and >271 nmol/L, respectively.²⁰ The prevalence of increased levels of MMA increased with age and was not different between men and women.²⁰ Notably, only 50-75% of participants in NHANES with low levels of serum B12 had increased levels of MMA.²⁰ It should also be noted that modest increases occur with renal failure.⁷

Pernicious Anemia (PA) caused by autoimmune destruction of gastric parietal cells and atrophy of the gastric mucosa is the most common cause of vitamin B12 deficiency.^3,6,7,21 Asymptomatic autoimmune gastritis, a chronic inflammatory disease of the gastric mucosa, precedes the onset of mucosal atrophy by 10-20 years.²²

With disease progression, an increasing number of the parietal cells that produce hydrochloric acid and intrinisic factor are destroyed.²² This may present initially as iron deficiency anemia due to loss of gastric acid, which is required for iron absorption.^1,23 Ultimately, diminished production of intrinsic factor together with development of neutralizing antibody against intrinsic factor itself leads to B12 malabsorption.^2,3,10,24 The autoimmune nature of PA is reflected by the presence of autoantibodies against the parietal cell proton pump protein (H/K ATPase) and to intrinsic factor.^3,20,24,25 This condition frequently coexists with other autoimmune disorders including Hashimoto's thyroiditis and type 1 diabetes mellitus.^3,24,29

Parietal Cell Antibodies (PCA) are present at a high frequency in PA (80%-90%), especially in early stages of the disease and are considered a predictive marker of subsequent gastric mucosa atrophy and its hematologic manifestations.^3,24 In the later stages of the disease, the incidence of PCA decreases due to the progression of autoimmune gastritis and a loss of gastric parietal cell mass, as a result of the decrease in antigenic rate.²⁶ PCA can precede the clinical symptoms of the gastric disease by several years.³ PCA are found in 90% of patients with PA, but have low specificity and are seen in various autoimmune disorders.¹ Intrinsic Factor Antibodies (IFA) are less sensitive, but are considered highly specific for PA.³ Studies have reported positivity for IFA in 40%-60% of patients with PA, which rises to 60%-80% with increasing duration of disease.^3,27 The combined assessment of both PCA and IFA increases diagnostic performance, with 73% sensitivity and 100% specificity.²⁸

Diminished acid secretion caused by gastric atrophy resulting from autoimmune disease or some other etiology elevates secretion of gastrin. Elevated gastrin levels can support the diagnosis of PA.^24,29 Hypergastrinaemia arising from loss gastric parietal cells drives development of antral enterochromaffin cell hyperplasia that can further develop into neoplasia and carcinoid syndrome.^1,3,24,30,31