B12 and Folate (BSH 2014)


Cobalamin Deficiency (B12)




All Ages

  • Infection – H. pylori, Giardia, Tapeworm

  • Malabsorption – Pernicious Anaemia

  • Co-morbidities – Gastric resection / banding, Coeliac, Tropical Sprue, Crohn’s


  • Congenital – Transcobalamin deficiency, Imerslund Grasbeck Syndrome


  • Due to higher requirements


  • Malabsorption – Achlorhydria 2o to PPI or atrophic gastritis


Clinical Features


Neurological presentation in absence of haematological abnormality is common.

Patients with clear clinical signs of deficiency may have serum cobalamin levels seemingly within the normal range, further testing required.

Hx – Anaemia, Dietary history, AI disease (vitiligo, hypothyroid, pernicious anaemia), Glossitis, Paresthesia, peripheral neuropathy, GI malabsorption symptoms (pale stool, abdo pain, nocturnal motions, mouth/anal ulceration), GI surgical history, Neurocognitive symptoms, Alcohol, Drugs (metformin, PPI, COCP), Pregnancy.


Primary Tests


FBC – macrocytic anaemia (neuro impairment occurs with a normal MCV in 25% of cases)

Film - >5% neutrophils with ³5 lobes, oval macrocytes, megaloblasts, megametamyelocytes.


Serum Cobalamin

  • Quantifies inactive transcobalamin I and III-bound (holohaptocorrin) and active transcobalamin-II-bound (holotranscobalamin)

  • Widely available, cheap & automated

  • Lacks sensitivity and specificity

  • False normal results occur in presence of high titre anti-intrinsic factor antibodies

  • No clear ‘normal’ result, but <148 pmol/l used.


Secondary Tests


Plasma Methylmelonic Acid (MMA)

  • Increased in cobalamin deficiency (in particular, very high levels of >0.75umol/l)

  • Also increases in renal disease, bacterial overgrowth and haemoconcentration


Plasma total homocysteine (tHcy)

  • Increased in cobalamin deficiency, often rises early in deficiency

  • Also increases in renal disease, folate & B6 deficiency, hypothyroidism


Holotranscobalamin (HoloTc) - <32pmol/l suggest deficiency

  • Immunoassay for the ‘active’ fraction. More sensitive and specific

Investigating the Cause


Anti-Intrinsic Factor Antibodies (Anti-IFAB)

  • Automated chemiluminescent assay

  • 95% positive predictive value for pernicious anaemia, ie low false positive rate.

  • But, anti-IFAB only present in 40-60% of pernicious anaemia cases

  • False positive may occur following recent cobalamin injection


Anti-Gastric parietal cell Antibodies (Anti-GPC)

  • Positive in 80% of pernicious anaemia, and 10% of normal population


Schilling test no longer in use.





Normal dose

  • 1mg IM 3x per week for 2 weeks, then every 3 months

Neuro dose

  • 1mg IM alt days until Sx stop improving, minimum 3 weeks, then every 2 months


  • Reticulocyte count should rise within 7-10 days, reassess diagnosis if not

Subclinical Deficiency

  • Low dose 50microg PO daily

Side Effects

  • Transient hypokalaemia, itch, rash, fever, nausea, dizziness, anaphylaxis


Specific Scenarios


Metformin – Mechanism not known. Ix and treat as per all other patients


HRT – Asymptomatic women on OCP or HRT with a level 110-148 pmol/l do not need further Ix


Pregnancy – Levels fall in pregnanct, short course of treatment if symptomatic


Vegetarian – Consider supplements, esp when pregnant or breast feeding


Post-GI surgery – Monitor long term for deficiency and consider low dose replacement


Infancy – Check MMA or tHcy if normal serum levels but deficiency suspected. Consider genetics.


BSH recommended investigation pathways


If strong suspicion of B12 deficiency with objective parameters, e.g. anaemia

  • B12 <148 pmol/l --> Check Anti-IF Ab and start B12 replacement

  • B12 >148 pmol/l --> Check Anti-IF AB, MMA, Check MMA, tHcy or HoloTC and start B12 replacement whilst awaiting results.


If B12 has been check for non-specific symptoms in absence of objective parameters

  • B12 <110 pmol/l --> Management as above

  • B12 >110 pmol/l --> Repeat level in 1-2 months --> persistent low level, check anti-IF Ab and consider 4 weeks low dose oral B12 with repeat level in 3-4 months --> peristent low level, check MMA, tHcy or HoloTC and consider treating as antibody-negative pernicious anaemia.


Folate Deficiency


Folate = term encompassing all biologically active forms of the vitamin

Folic Acid = the synthetic preparation


Both are absorbed in the small intestine & half of body folate found in the liver.


Folate is required for DNA synthesis and therefore earliest signs of deficiency present in the most rapidly dividing cells – bone marrow and GI tract




False negatives – test performed soon after oral folate intake

False positives – anorexia, acute alcohol intake, pregnancy, anticonvulsant therapy


Serum folate

  • Chemiluminescent test for folate binding protein

  • No consensus on normal range

  • <7nmol/l (3ug/l) often used

  • 7-10nmol/l (3-4.5ug/l) is indeterminate zone


Red cell folate

  • Assessment of tissue folate status over lifetime of the red cell

  • Great number of pre-analytical and analytical variables


Plasma total Homocysteine (tHcy)

  • Increases with folate deficiency

  • Also rises with B12/B6 def, renal disease, hypothyroidism.


Clinical Approach


Levels fall with several weeks of folate deprivation but not clear how long have to remain deficient for to cause clinical effects.


Isolated folate deficiency is very rare. Look for other nutrient deficiencies.



  • Poor diet, alcoholism, GI disease, Increased red cell destruction, Exfoliative skin disorders, Pregnancy, MTHFR gene mutation

  • Goat’s milk much lower folate content than cow’s milk

  • >80g of ethanol a day increases risk of defiency (e.g. 8 glasses of wine per day)

  • In pregnancy, folate requirements increase and preferentially delivered to fetus




5mg daily for 4 months

15mg daily in malabsorptive states

200-500 ug daily prophylaxis in pregnancy

5mg daily as prophylaxis in haemolysis or chronic dialysis