Pernicious Anaemia Society
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New research articles

New simpler/cheaper test for MMA:

J Nutr. 2014 Aug 20. pii: jn.114.194829. [Epub ahead of print]

Methylmalonic Acid Quantified in Dried Blood Spots Provides a Precise, Valid, and Stable Measure of Functional Vitamin B-12 Status in Healthy Women.


Methylmalonic acid (MMA) is a sensitive and specific functional biomarker of vitamin B-12 status, commonly assessed in plasma or serum. Dried blood spots (DBSs) allow simpler and more cost-efficient blood sampling than plasma. To facilitate convenient testing for vitamin B-12 deficiency in large-scale surveys and in population groups from remote areas, we developed a method for MMA quantification in DBSs and tested its applicability as well as the long-term stability of MMA in DBSs at various temperatures. MMA was extracted from an 8-mm DBS punch with water:methanol (95:5, v:v) and methyl-d3-malonic acid as the internal standard. After sample cleanup by ultrafiltration and hexane extraction, MMA was quantified by using reversed-phase LC-tandem mass spectrometry. Extraction conditions were optimized to maximize the detection signal and achieve DBS extract concentrations above the lowest limit of quantification (signal-to-noise ratio ≥ 10) of 10 nmol/L. Recovery was between 93% and 96%. Intra- and interassay variation (CV%) for DBS MMA was 0.49% and 2.3%, respectively. Calibrators showed linearity (R2 = 0.998) between 10 and 10,000 nmol/L. In 94 healthy women, MMA concentrations in DBS extract (min-max: 10.2-80.5 nmol/L) and plasma (min-max: 68-950 nmol/L) were correlated (ρ = 0.90) (P < 0.001). MMA concentrations in DBSs were stable at room temperature for 1 wk, in the refrigerator for 8 wk, and at -80°C for (at least) 1 y. This simple and robust method allows quantification of MMA in DBSs of healthy individuals. The linear relation between plasma and DBS MMA suggests that DBS MMA could predict plasma MMA, the current reference indicator for functional vitamin B-12 deficiency. With the advantages of minimally invasive specimen collection and no need for laborious blood processing steps, this method has the potential to be a reliable, convenient, and field-applicable alternative for assessment of vitamin B-12 status.

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This is not new research, but new to me to see in writing that PA/ B12 def patients do very different things/ big variations with the same amount of injected B12, see:

Patient Variation in Pernicious Anaemia, as Shown in a Clinical Trial of Cyanocobalamin, Hydroxocobalamin and Cyanocobalamin–Zinc Tannate


Cyanocobalamin, hydroxocobalamin and cyanocobalamin-zinc tannate were given in turn, as a single dose equivalent to 500 μg. of vitamin B12, to twelve patients who had untreated Addisonian pemicious anaemia. The order in which the drugs were given to each patient was determined by a series of random numbers. Following the first injection, frequent measurements were made of serum vitamin B12 activity until this was reduced to 100 pg./ml. Then the second drug was given with the same follow-up, and ultimately the third drug.

In every case, serum concentrations of vitamin B12 greater than 140 and 100 pg./ml. persisted after hydroxocobalamin and cyanocobalamin-zinc tannate for twice as long as after cyanocobalamin. However, differences between patients were very great, such that the entire trial with the three drugs was completed in 11 months in one case but lasted more than 4 years in another. Variation between patients makes it impossible to anticipate the duration of effect of a single injection of one of these drugs in any patient.


Eur J Clin Nutr. 2015 Jan 7. doi: 10.1038/ejcn.2014.272. [Epub ahead of print]

Functional cobalamin (vitamin B12) deficiency: role of advanced age and disorders associated with increased oxidative stress.

Solomon LR1.


Background/Objective:Functional cobalamin (Cbl; vitamin B12) deficiency (that is, high levels of the Cbl-dependent metabolites, methylmalonic acid (MMA) and homocysteine (HCys), despite normal serum Cbl values) is common in the elderly and is associated with neurocognitive abnormalities, but its cause is unknown. As only reduced Cbls are metabolically active, the possibility that functional Cbl deficiency is associated with disorders having biomarkers indicative of increased oxidative stress (oxidant risks) was considered.Subjects/Methods:A retrospective record review of community-dwelling adults evaluated over a 12-year period for Cbl deficiency in a primary care setting who had serum Cbl values ⩾400 pg/ml (n=170).Results:When no oxidant risks were present, older subjects (⩾70 years) had higher metabolite values than younger individuals (<70 years). MMA values were even higher in the elderly when one oxidant risk was present and in younger subjects when two or more oxidant risks were present. Even at Cbl levels ⩾800 pg/ml, MMA values were increased in 73% of elderly subjects with at least one oxidant risk. HCys values were also higher in both age groups when at least two oxidant risks were present. Cyanocobalamin therapy decreased MMA and HCys values in 86 and 76% of subjects, respectively, with nonresponders more likely to have two or more oxidant risks.Conclusion:Functional Cbl deficiency is associated with disorders marked by increased oxidative stress particularly in the elderly; it occurs even when Cbl levels are high and is not consistently corrected with high-dose cyanocobalamin therapy. Thus, current approaches to recognizing and managing this disorder may be inadequate.European Journal of Clinical Nutrition advance online publication, 7 January 2015; doi:10.1038/ejcn.2014.272.


Orphanet J Rare Dis. 2014 Nov 15;9(1):161. [Epub ahead of print]

Three new cases of late-onset cblC defect and review of the literature illustrating when to consider inborn errors of metabolism beyond infancy.


BackgroundThe cblC defect is a rare inborn error of intracellular cobalamin metabolism. Biochemical hallmarks are elevated homocysteine and low methionine in plasma accompanied by methylmalonic aciduria. Due to the heterogeneous clinical picture, patients with the late-onset form of the disease (onset >12 months) come to the attention of diverse medical specialists, e.g. paediatricians, neurologists, nephrologists, psychiatrists or haematologists. The report reviews the published clinical data and adds three new cases to raise awareness for this severe but often treatable disease.MethodsThe Pubmed and the Cochrane databases were searched for clinical reports on cblC patients and three unreported cases are presented to illustrate the clinical spectrum.ResultsReports on 58 cases (30 females, 22 males, 6¿=¿no information) and the three new cases underlined the clinical heterogeneity of the disease. Time between first symptoms and diagnosis ranged from three months to more than 20 years. Haemolytic uraemic syndrome and pulmonary hypertension were main presenting symptoms in preschool children. In older children / adolescents, psychiatric symptoms, cognitive impairment, ataxia and myelopathy were frequently observed while thromboembolic events and glomerulopathies were almost exclusively seen in adults. Brain atrophy, white matter lesions and myelopathy were frequently encountered. The majority of patients showed marked biochemical and clinical response to treatment with parenteral hydroxocobalamin combined with oral betaine, folate, carnitine and rarely methionine. The course was less favourable in late treated or untreated patients.ConclusionsThe late-onset cblC defect is a rare disease and unfortunately, diagnosis is often delayed. Raising awareness for this disorder can significantly improve patients¿ outcome and perspective by timely initiation of targeted treatment. Newborn screening (NBS) for the cblC defect might be of benefit especially for late-onset patients since treatment seems efficient when initiated before irreversible organ damage. In general, inborn errors of metabolisms should be considered in unexplained medical cases at any age, especially in patients with multisystemic disease. More specifically, total homocysteine in plasma and methylmalonic acid in urine / plasma should be measured in unexplained neurologic, psychiatric, renal, haematologic and thromboembolic disease.

PMID: 25398587 [PubMed - as supplied by publisher] PMCID: PMC4255922 Free PMC Article

39 cases, all very different, it makes interesting, but frightening reading; Case 2 with normal serum B12 and normal active B12, but high homocysteine, low methionine and high MMA, and evidence of neurological issues described in the full text version see a bit pasted below, Marre.

Full free text:

"but due to repeatedly normal vitamin B12 serum levels, the hypothesis was no longer pursued. At this time, the patient additionally complained about an unintended weight loss of 8 kg due to loss of appetite; impaired short-term memory, concentration problems and confusion. In parallel, the patient developed deep vein thrombosis, promoted by reduced physical activity following an ankle joint injury. Further investigations revealed peripheral pulmonary embolism, attributed to a heterozygous mutation in the prothrombin gene. Several months later due to progressive neurological and psychiatric symptoms, the patient was transferred for a second opinion to a tertiary center. Besides the sensory ataxic gait disorder due to neuropathy and myelopathy, the patient presented with depression and cognitive impairment including slow mentation, memory impairment, attention deficits, and executive impairment. Metabolic investigations considerably increased tHcy (228–264 μmol/L) low Met (7.7 μmol/L) and highly elevated urinary MMA excretion in qualitative analysis in the presence of normal vitamin B12 and holotranscobolamin levels. The cblC defect was proven by enzymatic and complementation studies in cultured fibroblasts as described [31]. Molecular genetic analysis of the MMACHC gene revealed a splice site mutation in intron 1 (c.82-1G > A) and a missense mutation in exon 4 (c.482G > A).

Treatment with OH-Cbl 3×2 mg/week IM, betaine 2×6 g PO/day, Folic acid 1×5 mg PO/day was initiated and resulted in resolved cognitive and psychiatric symptoms and improved myelopathy. Biochemical response was marked; tHcy values and MMA excretion immediately decreased while Met remained rather low. Therefore, OH-Cbl treatment was adapted to 3×5 mg IM/ week and methionine 2×125 mg/d was supplemented. Following this scheme, the patient widely recovered but residues of myelopathy remained. His main complaints are impairment of sensory function and hypoaesthesia in the gluteal region and predominantly the lower legs while motor functions recovered almost fully. Evoked potentials show a significant improvement but no resolution of the axonal damage. No thrombosis occurred during four years of follow-up; renal, liver and cardiac function as well as visual acuity and ophthalmoscopy findings were normal. THcy concentrations remain stable between 50 and 60 μmol/L, Met in the upper normal range between 40 and 50 μmol/L and MMA excretion has decreased to 65 mmol/mol creatinine (reference range <10). "


This is a new recap of everything known about PA/ B12 def, which I find easy to read and for me explains bit more about kidney involvement etc, see:

Advances in mechanisms, diagnosis, and treatment of pernicious anemia.

Rojas Hernandez CM1, Oo TH1.


Pernicious anemia (PA) is an entity initially described in 1849 as a condition that consisted of pallor, weakness, and progressive health decline. Since then several advances led to the conclusion that PA is an autoimmune disease characterized by the deficient absorption of dietary cobalamin. It is currently recognized as the most common cause of cobalamin deficiency worldwide. We hereby review the current understanding of the disease and its neurological, hematological, and biochemical manifestations with emphasis on the diagnostic approach, treatment, and monitoring strategies. We propose an algorithm for the diagnostic approach considering the current performance and limitations of the available diagnostic tools for evaluation of cobalamin status and the presence of autoimmune chronic atrophic gastritis (CAG). Patients with PA require lifelong treatment with cobalamin replacement therapy. The current widely available treatment can be provided through enteral or parenteral cobalamin supplements, with comparable efficacy and tolerability.

Full Free text:

Cristhiam M Rojas Hernandez

Advances in Mechanisms, Diagnosis, and Treatment of Pernicious Anemia

Interesting to me bit about difference liver and kidney B12 :


It is estimated that the normal daily cobalamin requirement for an adult corresponds to 1-2 nmoL (Stabler and Allen, 2004). The absorption and transporting mechanisms are highly efficient and dependent on three key proteins: haptocorrin, intrinsic factor (IF) (Yang et al., 2010), and transcobalamin (Yang et al., 2010). Initially, the dietary cobalamin is bound to the salivary protein haptocorrin. The haptocorrin-cobalamin complex reaches the stomach where the effect of the pH and the proteolytic activity of pepsin results in the liberation of approximately 70% of the cobalamin (Fedosov, 2012). Cobalamin is then bound to the IF (Yang et al., 2010) and transported to the ileum. Human IF is a glycoprotein secreted by the gastric parietal cells (PC) and it has a high binding affinity for cobalamin (Alpers and Russell-Jones, 2013). In humans, the absorption of cobalamin occurs via endocytosis mediated by the cubilin receptors located throughout the entire ileum surface (Gueant et al., 1992; Moestrup, 2006).

The enterally absorbed cobalamin is bound to haptocorrin as holohaptocorrin and to transcobalamin (TC) as holotranscobalamin (HTC) (Moestrup, 2006). HTC is initially absorbed by the liver, degraded inside the hepatocytes, stored in the liver, and a fraction is secreted back to blood and is bound to TC (Bor et al., 2004). The HTC delivers its cobalamin to tissues so rapidly that <30% of plasma cobalamin exists as HTC at any time (Carmel, 2011). The role of the plasma haptocorrin has not been yet clarified, and the serum cobalamin bound to haptocorrin seems to be only absorbed by the liver. Therefore, the liver seems to serve as a checkpoint of the absorbed cobalamin before distribution among other tissues and enzymes (Allen, 1975).

The kidney filters both TC and HTC. The filtered TC is reabsorbed in the renal tubules. The reabsorbed HTC is degraded and the cobalamin is returned to the serum bound to freshly synthesized TC (Moestrup, 2006). The TC receptor is expressed by many different types of cells and the role of cobalamin in human health has been reviewed extensively elsewhere (O’Leary and Samman, 2010). The effects of cobalamin in the Krebs cycle and its role in DNA synthesis have impact on hematopoiesis, nervous system development and function, cardiovascular health, bone health, and aging (Koury and Ponka, 2004; O’Leary and Samman, 2010)."

And another new recap but this one is not specifically only about classical PA, also B12 def, see:

Vitamin B12 deficiency - A 21st century perspective .

Shipton MJ1, Thachil J2.


Vitamin B12 deficiency is a common condition which can present with non-specific clinical features, and in severe cases with neurological or haematological abnormalities. Although classically caused by pernicious anaemia, this condition now accounts for a minority of cases and vitamin B12 deficiency occurs most often due to food-bound cobalamin malabsorption. Since missing the diagnosis can result in potentially severe complications, including degeneration of the spinal cord and pancytopaenia, vitamin B12 deficiency must be diagnosed early and managed appropriately. Intramuscular injections have been the mainstay of treatment, but oral replacement therapy can be effective in many cases. There is accumulating evidence that high vitamin B12 levels (values varied from 350-1,200 pmol/l) are associated with haematological and hepatic disorders, in particular with malignancy. This review focuses on the developments in the clinical features and management of vitamin B12 deficiency over the last decade.

© 2015 Royal College of Physicians.

Full Free text:

Interesting bit (to me):

"The conundrum of subclinical cobalamin deficiency

In addition to a symptomatic lack of vitamin B12, there exists a state in which, despite the biochemical anomalies, no clinical manifestations are present, termed ‘subclinical cobalamin deficiency’ (SCCD).20 SCCD is more common than symptomatic vitamin B12 deficiency. Rather than being associated with an IF-mediated inability to absorb vitamin B12, in 30–40% of cases SCCD occurs as a consequence of FBCM. At present, it is thought that SCCD is a transient phenomenon that does not progress to overt vitamin B12 deficiency. Even so, a search should be instituted to exclude treatable causes. Patients with a serum cobalamin within the range of 110–148 pmol/l should be re-checked in 1–2 months, and those with subsequently normal cobalamin levels do not require further investigation.2 However, patients with persistently suboptimal cobalamin levels should be tested for anti-IF antibody titres and given low-dose oral cobalamin as cover. These patients are recommended to return to the doctor if neurological symptoms develop. If anti-IF levels are positive then the patient should be managed as for pernicious anaemia; if negative, then cobalamin levels should be reassessed after 3–4 months and, if persistently low, further biochemical investigations are warranted to confirm biochemical deficiency"


Learning points

• Food-bound cobalamin malabsorption is the commonest cause of low serum cobalamin levels, while pernicious anaemia accounts for the majority of cases of severe vitamin B12 deficiency.

• Neurological symptoms can develop in patients without haematological manifestations like anaemia and macrocytosis.

• Sequelae of untreated vitamin B12 deficiency includes not only permanent neurological symptoms, but also osteoporosis and cardiovascular disease.

• In the UK, current management relies exclusively on intramuscular injection, but evidence suggests that oral therapy is as efficacious as parental treatment.

• High serum cobalamin levels are associated with liver disease, malignancy and haematological dyscrasias, and may be positively correlated with risk of mortality.

© 2015 Royal College of Physicians".

Both have things in them that seems to contradict the other is usual, nothing is 100% with PA/B12.


Interesting re treatment with hydroxo/ cyano and methylcobalamin. It implies as is more often seen that some need hydroxo/cyanocobalamin to also get adenosylcobalamin (AdCbl).


Eur J Clin Nutr. 2015 Jan;69(1):1-2. doi: 10.1038/ejcn.2014.165. Epub 2014 Aug 13.

Treatment of vitamin B12 deficiency-methylcobalamine? Cyancobalamine? Hydroxocobalamin?-clearing the confusion.


Vitamin B12 (cyancobalamin, Cbl) has two active co-enzyme forms, methylcobalamin (MeCbl) and adenosylcobalamin (AdCbl). There has been a paradigm shift in the treatment of vitamin B12 deficiency such that MeCbl is being extensively used and promoted. This is despite the fact that both MeCbl and AdCbl are essential and have distinct metabolic fates and functions. MeCbl is primarily involved along with folate in hematopiesis and development of the brain during childhood. Whereas deficiency of AdCbl disturbs the carbohydrate, fat and amino-acid metabolism, and hence interferes with the formation of myelin. Thereby, it is important to treat vitamin B12 deficiency with a combination of MeCbl and AdCbl or hydroxocobalamin or Cbl. Regarding the route, it has been proved that the oral route is comparable to the intramuscular route for rectifying vitamin B12 deficiency.


Of interest I expect to some: Only 18.9% of patients with vitamin B12 deficiency are discovered to have PA by the WHO definition. It does not mean they are not B12 def, and they still need treatment, just like any one who is diagnosed with PA, the treatment is the same.

J Oral Pathol Med. 2015 May 18. doi: 10.1111/jop.12330. [Epub ahead of print]

Do all the patients with vitamin B12 deficiency have pernicious anemia?



Vitamin B12 deficiency may result in pernicious anemia (PA). This study evaluated whether all the patients with vitamin B12 deficiency had PA.


The blood hemoglobin (Hb), iron, vitamin B12, folic acid, and homocysteine concentrations and mean corpuscular volume (MCV) in 90 vitamin B12-deficient patients were measured and compared with the corresponding data in 180 age- and sex-matched healthy control subjects. PA was defined by World Health Organization (WHO) as having an Hb concentration <13 g/dl for men and <12 g/dl for women, an MCV ≧ 100 fl, a serum vitamin B12 level <200 pg/ml, and serum gastric parietal cell antibody (GPCA) positivity.


We found that 35 (38.9%) and 20 (22.2%) patients with vitamin B12 deficiency had deficiencies of Hb (men <13 g/dl, women <12 g/dl) and iron (<60 μg/dl), respectively. Moreover, 65 (72.2%) and 37 (41.1%) patients with vitamin B12 deficiency had abnormally high blood homocysteine level (>12.7 μM) and high MCV (≧100 fl), respectively. In addition, 43 (47.8%) vitamin B12-deficient patients with had GPCA positivity. Patients with vitamin B12 deficiency had a significantly higher frequency of Hb or iron deficiency, of abnormally elevated blood homocysteine level or high MCV, and of GPCA positivity than healthy control subjects (all P-values < 0.001). However, only 17 (18.9%) of 90 vitamin B12-deficient patients were diagnosed as having PA by the WHO definition.


Only 18.9% of patients with vitamin B12 deficiency are discovered to have PA by the WHO definition.


Good article marre

So 72.2% of people with B12 deficiency have raised homocysteine so will come to some harm sooner or later if that continues, but only 18.9% are likely to get treated- that fits with most experiences here then!!! Over 50% people will go untreated.


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