Concept:Vitamin B12 Cobalamin and TrPs

Vitamin B₁₂ (cobalamin) is one of the most structurally complex of all vitamins and the only one whose only primary food source is bacteria. It is considered together with folic acid because their metabolism and function are intimately linked — the two independently essential enzyme cofactors share critical pathways, and deficiency of one can mask or precipitate deficiency of the other. Both are required for DNA synthesis, and inadequacy of either aggravates myofascial trigger points (TrPs) through mechanisms that include impaired red cell production, reduced oxygen-carrying capacity of muscle, neuropathy, and increased TrP irritability.

In one study of chronic myofascial pain subjects, 16% of 57 patients had serum vitamin B₁₂ levels below 261 pg/ml, and 10% had low serum or erythrocyte folate levels — proportions more convincing than in the fibromyalgia syndrome (FMS) comparison group. Two of the three FMS subjects with vitamin B₁₂ deficiency cleared completely with cobalamin replacement (Gerwin, unpublished data).

Discovery and History

In 1926, Minot and Murphy successfully treated pernicious anaemia by feeding patients liver. Previously, the disease had been invariably fatal. In 1948, the responsible agent, a cobalamin, was finally discovered and crystallised. Hodgkin won the 1964 Nobel Prize in Chemistry for delineating the structure of this complex molecule.

Understanding the overlapping contributions of folic acid and vitamin B₁₂ to the aetiology of macrocytic anaemia evolved slowly. Pteroylglutamic (folic) acid was purified in 1943 by Stokstad and was crystallised from liver in the same year by Pfiffner and associates. By 1948, Angier and his coworkers synthesised it and identified its structure. It then became clear that folic acid was the Wills factor, the vitamin M previously found in dry brewers' yeast, and the vitamin B_c of yeast identified in chick experiments.

The Cobalamin Molecule

Its central cobalt atom is linked to a variable anionic group:

• -CN in cyanocobalamin (the common synthetic form)
• -OH in hydroxocobalamin (the major form in plasma)
• -CH₃ in methylcobalamin

At least three other forms are known. It has been officially recommended that the term vitamin B₁₂ be reserved specifically for the cyanocobalamin form; "cobalamin" may apply to any of its forms. Methylcobalamin and 5'-deoxyadenosinecobalamin are the only two forms of the vitamin known to be physiologically active. Cyanocobalamin is physiologically inactive and must be converted to other forms — first to be absorbed, and then to be metabolically useful.

Biochemical Functions

Cobalamins serve numerous essential metabolic functions:

1. DNA synthesis — deoxyribonucleic acid (DNA) synthesis through the regeneration of intrinsic folate
2. Regeneration of intrinsic folate — critical to the synthesis of DNA
3. Transport of folate to, and its storage in, cells
4. Fat and carbohydrate metabolism — the conversion of methylalanate to succinate is cobalamin-dependent
5. Protein metabolism
6. Reduction of sulfhydryl groups

Since cobalamin and folic acid are required for the synthesis of DNA, both are necessary for normal growth and tissue repair.

The Methylation Cycle and Homocysteine

Cobalamin is essential for the methylation of homocysteine to methionine through a reaction involving methionine synthase, for which methylcobalamin (Me-Cbl) is the cofactor. The conversion of homocysteine to methionine is a key reaction in the synthesis of DNA, and requires both Me-Cbl and tetrahydrofolate (THF). The methyl donor is Me-THF (methyltetrahydrofolate).

Folic acid is stored intracellularly as a polyglutamate, which is the form that is also necessary for its enzyme cofactor function. When cobalamin is lacking, Me-THF cannot be demethylated, and an essential conversion prior to polyglutamation cannot proceed. Hence, the polyglutamated form of THF is decreased in serum and intracellularly when cobalamin is inadequate — the so-called methyl-folate trap. When cobalamin reserves are already depleted, large doses of folic acid increase the utilisation of cobalamin and can precipitate a serious cobalamin deficiency.

Fat Metabolism and Myelin

The cobalamins are involved in fat and carbohydrate metabolism since the conversion of methylalanate to succinate is cobalamin-dependent. It has been proposed, but not proved, that the neurological deficits characteristic of cobalamin deficiency are due to compromise of the lipid portion of the lipoprotein myelin sheath surrounding the affected nerve fibres.

In both the central and peripheral nervous systems, cobalamin deficiency is associated with inadequate myelin synthesis that leads to: 1. First, demyelination 2. Then, axonal degeneration 3. Finally, neuronal death

Comparable neurologic disease is less frequently caused by folate deficiency. Lesions of the myelinated peripheral nerves due to cobalamin deficiency occur more frequently and earlier than the central nervous system lesions of the myelinated posterior and lateral cords of the spinal column. The latter advanced deficiency is known as subacute combined degeneration — combined system disease, posterior lateral sclerosis, or funicular degeneration.

Nerve Function and TrPs

Vitamin B₁₂ inadequacy or deficiency causes a myelopathy — this has long been known. It is now known that there is also a peripheral neuropathy associated with vitamin B₁₂ deficiency. Folic acid deficiency has also been reported to cause a peripheral neuropathy that is less common than that seen with vitamin B₁₂ deficiency.

Neuropathy is associated with increased TrP irritability. The role of both vitamin B₁₂ and folic acid on nerve function raises the possibility that these vitamins produce central or peripheral nerve dysfunction that predisposes to altered nerve/muscle junction or motor endplate dysfunction. The mechanism in MPS patients is not clear.

Persons with acute lumbar or cervical radiculopathy can present with an acute MPS before there is any clinical sign of radiculopathy. Likewise, post-lumbar laminectomy scarring with nerve root entrapment can present with MPS in the distribution of the entrapped nerve root. These observations, made by Dr. Gerwin, support the concept that at least some cases of MPS are the result of nerve injury — and that metabolic nerve dysfunction (injury) can also result in the formation or the persistence of the myofascial trigger point.

Insufficiency and Deficiency

Cobalamin Insufficiency

The symptomatology of a marginal amount of cobalamin in the body may be highly variable and difficult to interpret:

• Nonspecific depression
• Fatiguability
• Increased susceptibility to myofascial TrPs
• An exaggerated startle reaction to unexpected noise or touch is occasionally a helpful guide

In Dr. Gerwin's experience, several cases presented only with fatigue, disturbed sleep, and diffuse muscle pain, all of which improved with cobalamin replacement.

Folate Insufficiency

Insufficient folate is the most common vitamin inadequacy and among those most likely to perpetuate myofascial TrPs. Patients with myofascial pain who have marginally low serum folate levels have symptoms similar in kind to, but less intense than, many of the symptoms reported by patients with obvious neurologic disorders responsive to folic acid therapy:

• Increased muscular irritability and susceptibility to TrPs
• They tire easily, sleep poorly, and feel discouraged and depressed
• These patients also frequently feel cold and have a reduced basal temperature (as do patients with thyroid hypofunction; their symptoms are often relieved by multivitamin therapy including folic acid)

Established Cobalamin Deficiency

The clinical presentations of megaloblastic anaemia (pernicious anaemia) and the neurological dysfunction caused by vitamin B₁₂ deficiency occur as two distinct syndromes, although there is considerable overlap in that 67% of persons with pernicious anaemia with pancytopenia will have some neurologic disorder. Neurologic dysfunction can occur in the absence of megaloblastic anaemia, and progress independently.

Symptoms are those of combined degeneration of the spinal cord:

• Loss of vibratory and position sense (posterior spinal cord column functions)
• Weakness and spasticity (lateral spinal cord column motor functions)
• Peripheral neuropathy — both an axonal and a demyelinating neuropathy; tends to be predominantly, but not exclusively, sensory
• Gait ataxia and spasticity produce neuromuscular stress in addition to that of the nerve disorder itself
• Constipation occurs when bowel motility is impaired
• Fatigue, personality change, memory loss are less specific symptoms
• Dementia, visual loss, and psychosis are seen in more severe cases, not likely to present as muscle pain syndromes

Established Folate Deficiency

Folic acid deficiency is associated with:

• Fatigue, diffuse muscular pain, restless legs
• In addition: megaloblastic anaemia, depression, peripheral sensory loss, and diarrhoea
• A subnormal serum folate level in time causes megaloblastic hematopoiesis and anaemia
• Evidence of peripheral neuropathy was found in 21% of one group of folate-deficient patients
• Similar findings in another group responded to folic acid therapy

Experimental deprivation of folate for 6 months produced the following documented effects:

• In 3 weeks: low serum folate
• In 7 weeks: hypersegmentation of polymorphonuclear leukocytes
• In 14 weeks: increased urinary excretion of formiminoglutamic acid
• In 18 weeks: low erythrocyte folate and macroovalocytosis
• In 19 weeks: megaloblastic bone marrow and anaemia
• During the fourth month: sleeplessness and forgetfulness appeared and gradually increased through the fifth month
• Mental symptoms disappeared within 48 hours after starting oral folic acid therapy

Epidemiology of Deficiency

Pernicious anaemia due to cobalamin deficiency occurs in 1–3% of persons of European ancestry over age 60, and is more common in younger persons, especially women, of Hispanic and African ancestry. Deficiency of both vitamin B₁₂ and folic acid is much more prevalent in the elderly — vitamin B₁₂ deficiency occurring in as many as 40% of subjects as determined by measuring homocysteine and methylmalonic acid levels.

Five percent of healthy elderly and 19% of hospitalised elderly were deficient in folic acid. In the cases of both vitamin B₁₂ and folic acid, metabolic deficiency was found in subjects whose serum vitamin levels were within the accepted range of normal.

The four commonest causes of folate deficiency are advanced age, pregnancy or lactation, dietary indiscretion, and drug abuse, most commonly alcohol.

One-third of all pregnant women in the world develop a folate deficiency so severe that they have megaloblastic anaemia. The prevalence of folate deficiency is so high, especially in vulnerable groups, that many more individuals must have insufficient folate nutrition. Among 269 pregnant low-income patients in Gainesville, FL, 15% were deficient in serum folate (< 3 ng/ml), and 48% were low (insufficient) in serum folate (3–6 ng/ml), on their first maternity visit.

Laboratory Tests and Diagnosis

Cobalamin

The diagnosis of cobalamin deficiency cannot be made reliably only by measuring serum vitamin B₁₂ levels. The conventional reference range is 200–835 pg/ml (IU: 148–616 pmol/l). More sensitive assessment uses serum levels of methylmalonic acid and homocysteine — both are elevated early in vitamin B₁₂ deficiency; in folate deficiency, homocysteine levels are elevated but methylmalonic acid levels are normal.

• When the serum B₁₂ level is 350 pg/ml or lower, serum and urine homocysteine and methylmalonic acid levels should be obtained — if these are elevated, supplementation should be given
• When serum B₁₂ levels are between 300–400 pg/ml, homocysteine and methylmalonic acid levels are obtained; if any are elevated, supplementation should be given
• If the situation is still unclear, cystathionine and HTC II levels are obtained
• The diagnosis of cobalamin deficiency cannot be made reliably only by measuring serum vitamin B₁₂ levels — several cases presented only with fatigue, disturbed sleep, and diffuse muscle pain, all of which improved with cobalamin replacement. Serum B₁₂ was below 300 pg/ml in these individuals
• Note: The use of the Schilling test has been largely supplanted by serologic testing for anti-parietal cell and anti-intrinsic factor antibodies as the preferred diagnostic approach for pernicious anaemia.

Assay pitfalls:

• Assay kits containing R binding proteins that bind other cobalamin analogues will result in falsely higher values of vitamin B₁₂
• Non-cobalamin corrinoids that are inactive analogues of vitamin B₁₂ can falsely elevate serum B₁₂ levels if the assay method does not use pure intrinsic factor
• Large amounts of vitamin C or other reducing agents can destroy vitamin B₁₂, giving falsely low values
• Acquired immunodeficiency syndrome can also give falsely low serum values of cobalamin
• Studies have shown that persons with vitamin B₁₂ levels in the normal range can have other laboratory or clinical evidence of vitamin B₁₂ deficiency

The Schilling test assesses absorption of an oral dose of radiolabelled vitamin B₁₂ by measuring the fraction excreted in the urine over 24 hours. The stage I test without intrinsic factor should always be abnormal in pernicious anaemia, and should be corrected by the concurrent administration of intrinsic factor in the stage II test. However, the test has serious limitations — the crystalline form of vitamin B₁₂ is not the same as food-bound vitamin B₁₂, and is absorbed more readily; hence the stage I Schilling test can be normal even in the presence of pernicious anaemia, particularly since only about 10% of the normal level of intrinsic factor is needed to absorb vitamin B₁₂.

Folate

Routine laboratory testing of folate levels in blood serum and in blood cells (tissue level) is now available. Normal human serum contains approximately 7–16 ng/ml of folate in the serum. Contrary to expectation, among hospitalised patients, a high mean corpuscular volume (MCV) of 95 cu mm or more had only a 0.18 correlation with folate deficiency, and therefore would not have been useful to screen for it — the absence of macrocytosis does not exclude folate deficiency.

Requirements

• Vitamin B₁₂: The daily requirement needed to maintain body stores is between 1–6 μg. The enterohepatic circulation is so frugal in conserving vitamin B₁₂ that little is lost each day — it can take nearly a year to deplete body stores. Recommended daily allowances (RDA) by age:
  • Infants 0–12 months: 0.4–0.5 mcg/day
  • Children 1–8 years: 0.9–1.2 mcg/day
  • Children 9–13 years: 1.8 mcg/day
  • ≥14 years: 2.4 mcg/day
  • Pregnant women: 2.6 mcg/day; lactating women: 2.8 mcg/day
• Folacin: The total folacin activity recommended as a daily dietary allowance is 400 μg/day for adults and adolescents; 800 μg/day during pregnancy; 500 μg/day during lactation. Evidence of depleted body stores of folacin appear in 2 months and symptoms become severe after 4 months of folic acid deprivation

Sources

Cobalamin

Cobalamins are unique because the only primary food source is bacteria. The cobalamins are synthesised by certain microorganisms found in soil, sewage, water, intestines, or rumen. Herbivorous animals depend entirely on microbial sources for their cobalamin. The vitamin is not found in vegetable food sources, and is available to man only from animal food products or supplements. Brewers' yeast, still used by some as a source of B vitamins, does not contain vitamin B₁₂ unless the yeast is grown on a special cobalamin-containing media.

Folate

The dietary sources of folate are leafy vegetables (foliage), as the name indicates. Sources also include yeast, liver and other organ meat, as well as fresh or fresh-frozen uncooked fruit or fruit juice, and lightly cooked fresh green vegetables, such as broccoli and asparagus.

Although folates are ubiquitous in nature, being present in nearly all natural foods, they are highly susceptible to oxidative destruction: 50–95% of the folate content of foods may be destroyed in processing and preparation. All folate is lost from refined foods, such as hard liquor and hard candies.

Causes of Insufficiency and Deficiency

Cobalamin

The complicated chain of events required for absorption of cobalamin presents many links that can fail:

• Freeing of ingested cobalamins from their polypeptide linkages in food by gastric acid and by gastric and intestinal enzymes
• Formation of complexes with the intrinsic factor produced by normal gastric parietal cells
• On reaching a protein receptor on the microvillar membrane of the terminal ileum, in the presence of ionic calcium and at pH about 6, the cobalamin passes through the mucous membrane into the portal venous blood
• There it must join the transport protein, transcobalamin II, which carries it to the liver

Several drug interactions reduce serum cobalamin levels: neomycin, colchicine, p-aminosalicylic acid, biguanide therapy (e.g., metformin), and ethanol have been associated with malabsorption of cobalamin.

Folate

Tissue deficiency in folate is common even in high-income states, in 15% of the white population and in over 30% of the black and Spanish-American groups. The commonest causes are:

• Advanced age (an increasing segment of the population)
• Pregnancy or lactation
• Dietary indiscretion
• Drug abuse, most commonly alcohol

Therapy

Cobalamin

Vitamin B₁₂ is only derived from animal products, whereas folic acid is available from both animal and vegetable foods. Treatment means replenishing body stores and then maintaining them at optimal levels.

• In pernicious anaemia, treatment is lifelong; it is associated with autoimmune endocrinopathy and may co-occur with vitiligo and alopecia areata
• In dietary deficiency, alteration of the diet may suffice once body stores have been replenished
• Initial replacement: cyanocobalamin (CNCbl) 1 mg intramuscularly twice weekly for 2 weeks, then weekly for 2 months, then monthly — weekly injections generally restore the body pool to normal levels
• Sublingual route is considered appropriate even in pernicious anaemia or states of abnormal absorption: 500 mcg/day of CNCbl — serum levels should be monitored periodically
• For those who can absorb vitamin B₁₂, oral administration of 500–1000 μg may maintain serum levels; however, serum B₁₂, homocysteine, and methylmalonic acid levels should be obtained at 6-month intervals for 2 years to ensure adequate absorption
• Passive absorption of ingested vitamin B₁₂ 1000 μg in the absence of intrinsic factor provides about 3 μg/day, supporting oral replacement in some pernicious anaemia patients
• Some persons cannot convert cyanocobalamin to hydroxocobalamin because of a genetic defect — these individuals do well with hydroxocobalamin replacement therapy

In Dr. Gerwin's experience, fatigue and sleep disturbance improve after 2–4 weeks of cobalamin replacement therapy, and reduction in the irritability of myofascial trigger points takes 4–6 weeks.

Folic Acid

Folic acid replacement and maintenance dose recommendations are determined both by the daily requirement needed to minimise the occurrence of neural tube defects in newborns, and by the concern that high doses of folic acid will aggravate the neurological deficits of vitamin B₁₂ and obscure the early haematological signs warning of possible combined degeneration of the spinal cord by correcting the megaloblastic anaemia alone.

CRITICAL: every physician should know NOT to administer folic acid without checking the vitamin B₁₂ level. Daily intake of 400 μg of folic acid can aggravate the effects of vitamin B₁₂ deficiency and will also reduce elevated homocysteine levels associated with folic acid deficiency. However, reduction of elevated homocysteine levels to the point that there is no increased mortality from cardiac and cerebral thrombosis requires a higher daily dose of about 700 μg.

• Hence, a daily dose of 1 mg has been considered adequate
• Higher doses of folic acid may in fact be required, and may be determined by the level of homocysteine, but should be given only if vitamin B₁₂ levels are normal as well
• Patients should be cautioned that folic acid absorption is impaired by the simultaneous ingestion of antacids
• It is wise to routinely prescribe adequate amounts of vitamin B₁₂ and folic acid together, not just one — they are both water-soluble vitamins, inexpensive, available without prescription, and can be taken orally as a 500 mg tablet of B₁₂ and a 1 mg tablet of folic acid daily. This dosage is safe and effective

Practical Considerations for B₁₂ Testing

Vitamin B₁₂ inadequacy or deficiency should be considered in persons with clinical evidence of peripheral neuropathy, in vegans or persons on a predominantly vegetarian diet who do not supplement their diet with vitamin B₁₂, diabetics and others who may not absorb cobalamin, and in persons over the age of 50, since gastric mucosal atrophy is progressive as age increases and impairs vitamin B₁₂ absorption. Persons with a macrocytic anaemia are also suspect.

Whenever serum levels of vitamin B₁₂ are less than 300 pg/ml, supplementation with cyanocobalamin should be given. When serum B₁₂ levels are between 300–400 pg/ml, serum and urine homocysteine and methylmalonic acid levels are obtained, and if any one of them are elevated, supplementation should be given.

The Biology of Starvation: B₁₂ and Folate

Vitamin B₁₂ and folic acid occupy a unique position in the biology of starvation because they govern DNA synthesis itself — the fundamental capacity of dividing cells to replicate. When dietary supplies of either vitamin are restricted, the consequences appear first in the most rapidly dividing cells:

1. Bone marrow — megaloblastic changes appear within weeks of folate depletion; erythropoiesis becomes inefficient, producing large, fragile red cells that carry less oxygen per unit of cell volume; the resulting anaemia deprives working muscle of oxygen and directly aggravates the energy crisis at TrPs
2. Gastrointestinal mucosa — enterocytes fail to replicate efficiently, causing malabsorption; this creates a vicious cycle in which deficiency impairs the absorption machinery needed to correct the deficiency
3. Neural tissue — in cobalamin deficiency specifically, the progressive failure of myelin synthesis destroys the structural integrity of long axons; the spinal cord lesions (subacute combined degeneration) represent the most catastrophic end-stage of a process that begins as subtle neuropathy affecting peripheral nerves — exactly the nerves most relevant to TrP irritability

The evolutionary context of this vulnerability is that neither vitamin can be synthesised by the human body. Vitamin B₁₂ is synthesised only by bacteria; folate by plants. In ancestral environments, both were readily available — animal products for B₁₂, fresh vegetation for folate. Modern food processing, cooking, and restrictive diets strip both away with remarkable efficiency.

In the context of chronic pain management, this means that a patient with even months-long restriction of animal products (B₁₂) or with a diet dominated by cooked, refined, or processed foods (folate) is accumulating a metabolic debt that will eventually manifest as increased TrP irritability, impaired treatment response, and diffuse muscle pain — often without the classic haematological markers that clinicians rely upon.

Related Pages

• Perpetuating Factors — Overview
• Folic Acid and Trigger Points
• Vitamin B₆ (Pyridoxine) and Trigger Points
• Iron and Trigger Points
• Cobalamin — Wikipedia
• Vitamin B₁₂ deficiency — Wikipedia
• Pernicious anaemia — Wikipedia
• Subacute combined degeneration — Wikipedia
• Folate deficiency — Wikipedia
• Methylmalonic acid — Wikipedia
• Homocysteine — Wikipedia
• Methyl-folate trap — Wikipedia

References

• Travell JG, Simons DG. Myofascial Pain and Dysfunction: The Trigger Point Manual, Volume 1. 2nd ed. Baltimore: Williams & Wilkins; 1999. Chapter 4, Section C.