B12 or not B12? That is the question. The answer, well that s tricky. Cat Ronayne Senior Teaching Fellow Dunedin School of Medicine
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1 B12 or not B12? That is the question. The answer, well that s tricky. Cat Ronayne Senior Teaching Fellow Dunedin School of Medicine
2 The perils of bread The principal food, but far from being the most proper Bread mixed with water & kept in a degree of heat to that of the human stomach, soon turns sour From: Domestic medicine, Dr William Buchan, 1803.
3 Tea is evil! Ladies it will ruin you for men! The mischief occasioned by tea arises chiefly from its being substituted for solid food. This is so much the case at present, that, had I time to spare, I think it could not be better employed than in writing against this destructive drug. From: Domestic medicine, Dr William Buchan, 1803.
4 B12 deficiency is a haematology thing, right? 1850s - Dr Thomas Addison Described a lethal form of macrocytic anaemia, with glossitis & neurological symptoms No treatment, leaving patients exhausted, with no hope of a cure Image by H. Watkins via Wikimedia Commons, CC BY 4.0
5 ?Could something in food help 1923 Drs George Richard Minot & George Whipple Bled dogs to make them anaemic Treated them with different foods - Liver was the most effective 1926 Drs Minot & William Parry Murphy Cured 45 patients by feeding them lots of raw liver - Clinical improvement within 2 weeks Dog, DBCLS 統合 TV, CC BY 4.0; Liver, M Häggström, CC-0 via Wikimedia Commons
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7 From descriptive art to interdisciplinary science 1937 Dr William B Castle Intrinsic factor in the gastric mucosa is needed for absorption of extrinsic factor 1948 Extrinsic factor is vitamin B12! Crystalline form isolated from liver & published within weeks of each other by researchers at Merck & Glaxo 1956 Dr Dorothy Hodgkin described the structure of B12 Nobel prize for Chemistry in Dr Robert Woodward successfully synthesized B12 Patients given injections of pure B12 recovered completely WB Castle, ASH, DMC Hodgkin & R Woodward,
8 Fig Haematology: Clinical principles & applications. Rodak, Fritsma & Keohane. 4e, Elsevier 2012
9 Classic haematological findings Hallmark features Oval macrocytes Hypersegmented neutrophils May also see Cabot rings Howell-Jolly bodies Pappenheimer bodies Pancytopenia Circulating megaloblasts C Ronayne
10 Oval shaped cells Megaloblastic anaemia Iron deficiency anaemia Membrane defects Hereditary elliptocytosis, SEA ovalocytosis Bone marrow disease, infiltration MDS, myelofibrosis, leukaemia Drug therapy Hydroxyurea, RT inhibitors MBA, C Ronayne IDA, C Ronayne MF, C Ronayne MDS, C Ronayne SEO, NZIMLS Standardised Reporting
11 Hypersegmented neutrophils Megaloblastic anaemia Infection (right shift) MDS/MPD, GCSF therapy Drugs, uremia, heat stroke & burns MBA, C Ronayne MDS-MPD, C Ronayne Chemotherapy drug effect, C Ronayne Sepsis, C Ronayne Heat stroke & amphetamines, DA Nolte & MA Proytcheva, ASH Imagebank
12 Cabot rings Remnants of microtubules from the mitotic spindle or nuclear membrane Also seen in lead poisoning & other RBC disorders A feature of normal camelids C Ronayne Cabot rings in normal llama. Salah M Azwai, Veterinarski Arhiv 77(1), 69-79,
13 Howell-Jolly bodies - Nuclear remnants - Also seen in hyposplenism Basophilic stippling - Ribosomal aggregates - Also seen in lots of other disorders Pappenheimer bodies - Iron-containing inclusions - Also seen in lead poisoning, hyposplenism, MDS & other disorders Pancytopenia - Low RBC, WBC & platelets - Also seen in lots of other disorders Circulating megaloblasts - Abnormally large RBC precursors - Also seen in folate deficiency & BM infiltration C Ronayne ASH Imagebank C Ronayne
14 Clinical features vary widely Many patients have a normal MCV & Hb Hallmark features may be masked by other conditions More common than previously estimated & increasing Variable clinical manifestation due to rate, severity & pathophysiology of deficiency Alesia Hunt et al. BMJ 2014;349:bmj.g5226
15 B12 absorption is complex! B12 released from food by peptic acid & binds to salivary haptocorrin (HC) In small intestine, B12 released from HC & binds to IF In the terminal ileum, endocytosis of B12-IF complex via the Cubam receptor Released to plasma & binds to transcobalamin (holotc) Any interference in this process increases the risk of vitamin B12 deficiency! Green, R. et al. (2017) Vitamin B 12 deficiency Nat. Rev. Dis. Primers doi: /nrdp
16 Once B12 is in cells B12 is an essential co-factor in for 2 enzymes: Methylmalonyl-CoA mutase Methylmalonyl CoA -> Succinyl CoA - For energy pathways e.g. TCA Methionine synthase Homocysteine -> Methionine - Generates universal methyl donors MethylTHF -> Tetrahydrofolate (THF) - For thymidine production Green, R. et al. (2017) Vitamin B 12 deficiency Nat. Rev. Dis. Primers doi: /nrdp
17
18 So how can we test for B12 deficiency? How should we test for B12 deficiency?
19 Methylmalonic acid & homocysteine Substrates of B12-dependent reactions - Increased in >90% of B12 deficient patients - Test are analytically challenging & expensive MMA is considered the gold standard - More sensitive & specific for B12 deficiency - Affected by renal impairment, bacterial overgrowth & inherited enzyme defects - Test on serum or plasma Hcy is less sensitive & specific - Increases in hypothyroidism, folate & pyroxidine deficiency - Test on fasting plasma Green, R. et al. (2017) Vitamin B 12 deficiency Nat. Rev. Dis. Primers doi: /nrdp MMA
20 Serum B12 testing Serum B12 = HC-B12 + TC-B % Haptocorrin-bound B12 Biounavailable 10-30% Transcobalamin-bound B12 Bioavailable Poor sensitivity & specificity for deficiency May miss up to 45% of B12 deficient subjects if used as a screening test - Often normal in deficiency - May be low when normal tissue B12 & high dose ascorbic acid, inherited HC deficiency & folate deficiency
21 Roche Elecsys Vitamin B12 II Competitive Immunoassay Pretreatment (9 minutes): Sample + Pretreatments R1 + 2 liberate bound B12 Reaction 1 (9 minutes): Addition of R1 (IF*) Reaction 2 (9 minutes): Addition of microparticles + R2 (B12+biotin) 3e601 ECL Technology, Cobas 6000 analyser series, Roche Diagnostics
22 Serum holotranscobalamin The transcobalamin bound-b12 fraction of serum B12 - aka holotc or active B12 A better reflection of functional B12 status than total B12 - An early marker of B12 depletion - Stable in pregnancy - Falsely low in some TCN2 polymorphisms - Debate about cut-off values Green, R. et al. (2017) Vitamin B 12 deficiency Nat. Rev. Dis. Primers doi: /nrdp
23 Roche Elecsys Active B12 Sandwich Immunoassay Step 1 (9 minutes): Sample + R1 + R2 Step 2 (9 minutes): Addition of microparticles 3e601 ECL Technology, Cobas 6000 analyser series, Roche Diagnostics
24 So, what s the answer?
25 Probably Active B12 (holotc) - As a screening test, then MMA if required 90yo male, Hyposplenism, low iron & borderline B12 & folate. Image by Chris Greenwood.
26 Probably Active B12 (holotc) - As a screening test, then MMA if required - Probably not haematology, as odd cells are associated with lots of different diseases Thank-you! 90yo male, Hyposplenism, low iron & borderline B12 & folate. Image by Chris Greenwood.
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