Mbs reviews vitamin b12 testing report february 2014 table of contents section Page

Conclusions

There has been a substantial increase in the number of claims for vitamin B12/folate testing over the past ten years. Analysis of MBS data indicates that the majority of vitamin B12 testing services are requested by GPs and OMPs for the purposes of screening or testing, rather than follow-up monitoring. There are no Australian clinical practice guidelines that either advocate or recommend against routine testing for vitamin B12. The international clinical practice guidelines vary widely in their recommendations. While some recommend vitamin B12 test as screening tools in commonly encountered illnesses such as dementia, others suggest restricting testing to patients who have already undergone pre-test investigations such as a full blood count or blood film examination. There are no recommendations on the frequency of vitamin B12 testing and there is no direct evidence regarding the clinical utility of vitamin B12 testing in any population.

1 BACKGROUND ON VITAMIN B12 TESTING

1.1 Description of current services

This section describes vitamin B12 and vitamin B12 testing, recommended vitamin B12 status, and the population groups and clinical conditions/risk factors in which vitamin B12 testing is recommended.

1.1.1 The mechanism of vitamin B12 absorption

Vitamin B12, also called cobalamin, is a water soluble vitamin that plays a fundamental role in the normal functioning of the brain and nervous system, and for the formation of blood. Dietary vitamin B12 initially binds a protein called haptocorrin (previously known as transcobalamin I or R-Factor), which is produced by the salivary glands of the oral cavity (as well as the parietal cells¹ of the stomach), and whose essential function is to protect vitamin B12 from degradation from the acidic environment of the stomach. Absorption of vitamin B12 occurs in the terminal ileum (i.e. most distal part of the small intestine) and is aided by the binding of intrinsic factor (IF) secreted by the parietal cells of the stomach to the vitamin⁽¹⁾ (Figure 1.1). In addition to this method of absorption, evidence supports the existence of an alternate pathway that is independent of the IF. This pathway is important in relation to oral supplementation (approximately 1% of a large oral dose of vitamin B12 is absorbed by this second mechanism).⁽²⁾ Once absorbed, vitamin B12 binds to a protein called transcobalamin II (holoTC or active B12 is the complex formed by the binding of vitamin B12 to transcobalamin II), and is transported throughout the body. The interruption of one or any combination of these steps places a person at risk of developing vitamin B12 deficiencies.⁽³⁾
Figure 1.1: Vitamin B12 absorption and transport⁽³⁾

1.1.2 The functions of vitamin B12 in the human body

In humans, vitamin B12 and folate are linked by two enzymatic reactions where they function as cofactors (i.e. a cofactor is a component, other than the protein portion, of many enzymes to facilitate the catalytic activity of the enzyme)⁽⁴⁾. Vitamin B12 is required as a cofactor in both reactions, whereas folate is required in only one of the reactions (see Figure 1.2).⁽³⁾
Figure 1.2: The enzymatic reactions that require vitamin B12 and folate (folic acid) as cofactors⁽⁵⁾

In the first reaction, vitamin B12 is required for the conversion of methylmalonic acid (MMA) to succinyl-CoA. MMA is a substance produced when proteins in the body are broken down.⁽⁶⁾ Folate does not play any role in this reaction. Deficiency in vitamin B12 can lead to increased levels of serum MMA.⁽³⁾
In the second reaction, both vitamin B12 (in the form of methylcobalamin) and folic acid act as cofactors in the conversion of the substrate homocysteine (a homologue of the amino acids cysteine and methionine) to methionine (an amino acid and one of the 20 building blocks of proteins) by the enzyme methionine synthase.^{(3, 7)} More importantly, this pathway is closely linked to the generation of thymidine which is vital for deoxyribonucleic acid (DNA, i.e. the building block of the human body which carries genetic information) synthesis. A deficiency in either vitamin B12 or folic acid or both can lead to increased homocysteine levels in plasma.⁽³⁾ In addition, deficiency of either vitamins can result in perturbation of these two key pathways with consequent disruption of DNA synthesis caused by thymidine lack and resulting in megaloblastic anaemia, as well as other adverse effects on the nervous system and other organs.⁽³⁾ It is this metabolic reaction that clearly links the two vitamins and is responsible for the common or shared neuropsychiatric and haematologic disorders discussed in the following sections.

1.1.3 Vitamin B12: dietary sources, fortification, and supplements

Vitamin B12 is present in animal products such as meat, poultry, fish (including shell fish), and to a lesser extent milk, cheese and eggs, and it is not present in plant products.⁽⁸⁾ The recommended dietary allowance for vitamin B12 is 2.4 µg/day⁽⁹⁾ and most individuals can meet this level through dietary intake.⁽¹⁰⁾ Table 1.1 lists some of the foods with substantial amounts of vitamin B12, along with their vitamin B12 content. Individuals over the age of 50 who have reduced protease secretions in the stomach (as well as strict vegetarians)⁽¹¹⁾ obtain their vitamin B12 from supplements or fortified foods (e.g. fortified cereal) because of the increased likelihood of food-bound vitamin B12 malabsorption.
Table 1.1: Examples of dietary sources of vitamin B12^{(8, 12)}

Type of food	Estimated vitamin B12 content (micrograms)
Clams, (85 grams)	84.0
Mussels, (85 grams)	20.4
Crab, (85 grams)	8.8
Salmon (85 grams)	2.4
Beef, (85 grams)	2.1
Chicken	0.3
Egg (whole)	0.6
Milk (85 grams), 1 glass)	0.9

Food fortification is defined as the process of adding micronutrients (such as vitamins and minerals) to food as permitted by the Australian and New Zealand Food Standards Code (ANZFSC).⁽¹³⁾ Regulations regarding the fortification of foods with vitamin B12 vary between countries. ANZFSC permits only a limited number of foods to be fortified with vitamin B12. This includes selected soy milks, yeast spread, and vegetarian meat analogues.⁽¹⁴⁾

The risk of toxicity from vitamin B12 intake from supplements and/or fortified foods is low .⁽¹⁵⁾ Vitamin B12 is a water soluble vitamin, and therefore any excess intake is usually excreted in the urine.

1.1.4 Causes of vitamin B12 deficiency

Table 1.2 describes causes of vitamin B12 deficiency which can be divided into four categories: nutritional deficiency, increased requirements, impaired absorption, and other gastrointestinal causes.^{(7, 16)}
Table 1.2: Causes of vitamin B12 deficiency

Nutritional deficiency	Increased requirements	Impaired absorption	Other gastrointestinal causes
Poor intake of meats and dairy products in the elderly population (aged 65 and above)^(17) Chronic alcoholism^(18, 19) Strict vegan diets^(17) Malnutrition^(20)	Due to pregnancy and lactation^(21-24)	Autoimmune disease with autoantibodies against the intrinsic factor (pernicious anaemia)^(25) ^(26) Atrophic body (corpus) gastritis (due to autoantibodies to gastric parietal cells)^(27) Prolonged use of acid-suppression therapy or drugs^(28) Gastrectomy^(29) or any intestinal surgery which involves gastric resection, sleeve or banding surgery^(30)	Chronic gastrointestinal symptoms e.g. dyspepsia, recurrent peptic ulcer, diarrhoea^(3) Coeliac disease^(31) Crohn’s disease^(32) Fish tapeworms and other intestinal parasites^(29) Ileocystoplasty (i.e. a surgical reconstruction of the bladder involving the use of an isolated segment of ileum to augment bladder capacity)^(33) Pancreatic failure

Vitamin B12 deficiency is usually the result of dietary insufficiency and is common in individuals who are strict vegetarians because vitamin B12 is only present in foods from animal origin. Because of the complex mechanism of vitamin B12 absorption, causes of malabsorption may also arise at several levels in the gastrointestinal tract.⁽²⁶⁾ At the gastric level, the most frequent cause of significant vitamin B12 malabsorption leading to deficiency is pernicious anaemia (PA), which is an autoimmune disorder caused by the frequent presence of gastric autoantibodies directed against IF and the parietal cells.⁽³⁴⁾ PA can affect both the elderly and young individuals.^{(35, 36)}

1.1.5 Diseases caused by vitamin B12 deficiency

Vitamin B12 plays an important role in DNA synthesis and neurologic function.⁽³⁷⁾ Deficiency in vitamin B12 is associated with a wide spectrum of haematologic, neurologic and psychiatric disorders (Table 1.3) that can often be reversed by early diagnosis and prompt treatment.⁽³⁾
Table 1.3: Clinical manifestations of vitamin B12 deficiency

Haematologic^(3)	Neurologic^(38)	Psychiatric^(38-41)	Cardiovascular ^(42, 43)
Megaloblastic anaemia Panycytopenia (leukopenia, thrombocytopenia) Pernicious anaemia (i.e. large immature RBCs)	Paresthesias (i.e. a skin sensation such as burning or itching with no apparent physical cause) Peripheral neuropathy Combined systems disease (demyelination of peripheral nerves, spinal cord, cranial nerves and the brain)	Irritability, personality change Mild memory impairment, dementia Depression Psychosis Alzheimer’s Disease^(41)	Possible increased risk of myocardial infarction and stroke

1.1.6 Vitamin B12 testing

Reliable and accurate assessment of vitamin B12 status is required to determine the prevalence of deficiencies of this vitamin in the Australian population and is necessary for developing suitable strategies to prevent these nutritional problems. The haematologic complications of vitamin B12 and folate deficiencies are identical. Therefore, detecting the presence of vitamin B12 or folate deficiency, and distinguishing one from the other, depends critically on laboratory testing. These tests may be used singularly or in combination to establish the nutritional status and prevalence of deficiencies of the vitamins.
The methods used to assess vitamin B12 and folate status can either measure the:⁽⁴⁴⁾

concentrations of the vitamins in the blood (e.g. serum vitamin B12 levels, serum or plasma folate levels); and/or

increased levels of metabolites such as MMA and/or homocysteine.
The diagnosis of vitamin B12 deficiency has traditionally been based on measuring the total serum levels of vitamin B12. There is currently no internationally agreed definition for vitamin B12 deficiency based on clinical manifestations or on the ‘cut-off’ values that are used to define vitamin B12 deficiency, which vary between 120-200 pmol/L. However, vitamin B12 is carried on two distinct binding proteins in plasma:^{(43, 44)}

Transcobalamin II: binds vitamin B12 to form a complex called holotranscobalamin (holoTC). HoloTC binds only 20–30% of vitamin B12 circulating in the blood, but is responsible for delivery of vitamin B12 to cells and is considered to be the functionally important fraction, thus its name active-B12. HoloTC levels fall in vitamin B12 deficiency. Therefore, testing for this carrier protein can identify low vitamin B12 status before total serum vitamin B12 levels drop.^{(45, 46)}

Haptocorrin: binds the major portion of plasma vitamin B12 which is essentially inert as far as vitamin B12 delivery to cells is concerned, although it may reflect the general underlying state of vitamin B12 stores. The complex formed by the binding of haptocorrin to vitamin B12 is called HoloHC ⁽⁴⁷⁾. Haptocorrin deficiency is associated with low serum vitamin B12 concentrations.⁽⁴⁸⁾
Research has shown that assays that measure holoTC-associated fraction of vitamin B12 (e.g. Axis-Shield ASA)⁽⁴⁹⁾ are a more reliable indicator for identifying vitamin B12 deficiency, when used in conjunction with other available tests, such as serum MMA or homocysteine measurements.^(50-53) Currently available assays to measure holoTC are developed by Axis-Shield. This company recently launched a new active-B12 assay (Abbott ARCHITECT) for use in high throughout laboratories.⁽⁵⁴⁾ Furthermore, elevated levels of metabolites such as MMA have been shown to be more sensitive in the diagnosis of vitamin B12 deficiency than measurement of serum B12 levels alone.^{(5, 16, 55, 56)} Urinary MMA can be measured using high performance liquid chromatography (HPLC).⁽⁵⁷⁾ Both biomarkers, holoTC and MMA, show a stronger association between low vitamin B12 concentrations and increased risk of cognitive decline and dementia in the elderly than total vitamin B12 measurements.^(58-60) Table 1.4 compares the three tests that can be used to assess vitamin B12 status.
Table 1.4: Comparison of the three tests used to measure vitamin B12⁽⁴⁴⁾

Biomarkers	Serum/plasma B12	Serum holoTC	Serum/plasma MMA
Assessing intake	+	++	++
Sensitivity	+	+	++
Specificity	--	-	+
Assessing long term and short term status of vitamin B12	+ Long term status	++ Long term and short term status	++ Long term and short term
Accepted cut-offs indicating deficient states	See Table 1.5	TC <35 pmol/L	>260 nmol/L deficient

Table 1.4 shows that sensitivity of serum vitamin B12 measurement for detection of vitamin B12 depletion or deficiency is good overall, but specificity is poor. The predictive value is improved when this test is combined with measurement of MMA. One study has shown that the use of a low serum vitamin B12 level as the sole means of diagnosis of vitamin B12 deficiency may miss up from 10% to 26% of patients with actual tissue B12 deficiency.⁽⁵⁾ The holoTC assay used on its own is also not very predictive of vitamin B12 deficiency unless it is used in conjunction with plasma MMA or with the total plasma vitamin B12.⁽⁵¹⁾ Therefore, for an accurate measure of vitamin B12 status and reserves, it is recommended that serum vitamin B12 levels are combined with a measure of a metabolic marker of vitamin B12 reserves such as MMA, holoTC or homocysteine.⁽⁶¹⁾

1.1.7 Serum vitamin B12 target values

The cut-off value for vitamin B12 deficiency varies markedly between laboratories worldwide. Table 1.5 presents the “usual or approximate” reference intervals for vitamin B12 deficiency.
Table 1.5: Vitamin B12⁽⁶²⁾ reference intervals

Status	Vitamin B12 levels
Normal range	200-900 pg/ml† (130-850 pmol/L)
Deficient	< 200* pg/ml (< 130 pmol/L)
* This is an unsafe range as many in the population exhibit neurological symptoms of deficiency at much higher concentrations. The lowest concentration to be considered normal is 221 pmol/L.^(63) † pmol/L = 0.738 x pg/ml

As discussed earlier, elevated homocysteine levels can be a useful indicator for vitamin B12 deficiency, because serum homocysteine levels increase as vitamin B12 stores fall. Serum homocysteine levels greater than 9 µmol/L suggest the beginning of depleted vitamin B12 reserves and levels greater than 15 µmol/L is indicative of depleted vitamin B12 reserves.⁽⁶⁴⁾ However, caution should be taken with this test as homocysteine levels may also increase with folate deficiency.⁽⁶⁵⁾

It is important to distinguish between low vitamin B12 status (defined as subclinical cobalamin deficiency (SCCD)) and outright vitamin B12 deficiency. Low vitamin B12 status denotes a condition in which laboratory tests indicate depletion of vitamin B12 stores as judged by being outside of the normal reference range. In the case of direct measures of vitamin B12 [serum vitamin B12 or holotranscobalamin (holoTC)], low vitamin B12 status is indicated by being below the lower limit of the reference range (for vitamin B12 < 200 pg/mL or <148 pmol/L; for holoTC <35 pmol/L), whereas for indirect measures of metabolites (MMA or homocysteine), low vitamin B12 status would be indicated by a level above the upper limit of the reference range (for MMA >260 nmol/L; for homocysteine, > 12 µmol/L).⁽⁴⁴⁾

There are large numbers of individuals with low vitamin B12 status who do not progress to outright deficiency. This may be attributed to the degree of impairment of the process of assimilation and absorption of vitamin B12 in relation to the daily requirement for the vitamin. Complete abrogation of physiologic vitamin B12 absorption, such as occurs after total gastrectomy, ileal resection, or advanced autoimmune pernicious anaemia, will inexorably lead to a degree of depletion of the vitamin that can no longer sustain cellular requirements and that would, with time, lead to both functional and structural abnormalities. However, in the food vitamin B12 malabsorption states, the basic mechanism of intrinsic factor-dependent vitamin B12 absorption remains intact, but some aspect of the assimilative process is impaired, as in non-immune atrophic gastritis or with the chronic use of proton pump inhibitors. There is uncertainty and ongoing debate as to whether low vitamin B12 status per se may be associated with subtle degrees of deficiency that have consequences of public health significance.⁽⁴⁴⁾

1.1.8 Prevalence of vitamin B12 deficiency in Australia

The true prevalence of vitamin B12 deficiency in the general Australian population remains unknown. The incidence appears to increase with age (>65 years) and with the ubiquitous use of gastric acid–blocking agents.⁽⁶⁶⁾ An Australian study published in 2012 found 14% of 130 patients living in residential aged care facilities in southern Tasmania were vitamin B12 deficient, defined as serum vitamin B12 levels less than 150 pmol/L.⁽⁶⁷⁾ Another study published in 2006 examined the prevalence of low serum vitamin B12 in a representative sample of 3,508 persons aged 50+ years between 1997 and 2000.⁽⁶⁸⁾ Low serum vitamin B12 (defined as < 185 pmol/L) was found in 22.9% of participants.

1.1.9 Service providers claiming MBS benefits for vitamin B12 testing

Most pathology in Australia is provided in comprehensive laboratories that provide a wide range of testing services at a single location. Only approved pathology practitioners are eligible to claim vitamin B12 testing.