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Research

 

Research Carried out by our group has shown

  • All children with Modern Day Autism are functionally deficient in vitamin B2

  • All children with Modern Day Autism are functionally deficient in vitamin B12

  • The majority children with Modern Day Autism are functionally deficient in iron

  • The majority of children with Modern Day Autism are deficient in vitamin D at time of testing

  • All children with Modern Day Autism have Paradoxical B12 deficiency

     

    Research Methods

    We have been lucky enough to have available data from around 2000 children with autism. The data has included Hair Metals Test Analsyis, in which potential toxic metals have been estimated, but more importantly essential metals have also been measured. In addition, Organic Acids Test has been  dogma on vitamin B12 deficiency implies a direct link between levels of serum vitamin B12, and sufficiency. Hence as vitamin B12 levels reduce, markers such as MMA and homocysteine increase in an inverse relationship to serum B12 levels (Minerva etal, 2021; Bailey etal, 2011). A

    Paradoxical B12 deficiency

    The current dogma on vitamin B12 deficiency implies a direct link between levels of serum vitamin B12, and sufficiency. Hence as vitamin B12 levels reduce, markers such as MMA and homocysteine increase in an inverse relationship to serum B12 levels (Minerva etal, 2021; Bailey etal, 2011). A consequence of this "Dogma" is that if a person does not have vitamin B12 levels lower than a highly variable amount, dependent upon study(148 pmol/L Minverva etal, 2021; 179 pg/ml Spain Sanz-Cuesta et al, 2012), they cannot be B12 deficient. Hence vitamin B12 deficiency would be mainly due to poor diet, or poor absorption (Elmadfa and Singer, 2009). The traditional correlation of homocystein and MMA with decreasing serum vitamin B12.

    Many people, however, experience symptoms of vitamin B12 deficiency yet their serum levels of vitamin B12 may be normal or much higher than normal. Subsequent examination of biochemical markers such as MMA or homocysteine may show that these markers are moderate to highly elevated. As such the symptoms and biochemical markers are indicative of vitamin B12 deficiency, yet the serum vitamin B12 levels are paradoxically high. Such persons are deemed to have "Paradoxical Vitamin B12 Deficiency". Generally, however, the reason(s) for "Paradoxical B12 deficiency" are not known, even despite its association with a greater increase in "all-cause mortality" (Flores-Guerrero etal. 2020), chronic viral liver disease (Sugihara etal, 2017), Anorexia Nervosa (Corbetta etal, 2014) and death from COVID-19 .

    .

     A typical example of Paradoxical B12 deficiency (as per Dynacare Plus)

    It has been known for over 40 years that measurements for serum vitamin B12 levels can be greatly distorted by the presence of of non-functional cobalamin analogues (Kolhouse etal, 1978; Kane et al, 1978; Igarai et al, 1978), and in many cases measurement of serum B12 levels is not predictive of the levels of functionally active B12 (England and Linnell, 1980; Andrès et al, 2013; Serraj et al, 2011; Ermens et al, 2002; Rochat et a;. 2012l Podzolkov et a;. 2019; Zulfiqar et al, 2019). In addition, low intracellular folate levels can often be associated with the presence of inactive analogues of cobalamin in serum (Sheppard and Ryrie, 1980). Alternatively it can be associated with inflammatory conditions such as Rheumatoid arthritis due to the overproduction of the B12 binding proteins transcobalamin and haptocorin (Christensen et al 1983: Grindulis et al, 1984), Cystic fibrosis (Lindemans etal, 1984), and patients treated with nitrous oxide (Parry etal, 1985). Despite the almost completely non-predictive nature of serum vitamin B12 levels it is still the measurement of choice for vitamin B12 sufficiency. Its utility is arguably restricted to determining conditions such as dietary insufficiency, where serum levels are routinely low. Little wonder that so many physicians miss functional vitamin B12 deficiency in patients!

    In the scattergram above there is no obvious relationship between levels of vitamin B12 in serum, and the standard marker of vitamin B12 deficiency, MMA (MethylMalonic Acid). In absolute vitamin B12 deficiency in serum, MMA starts to increase as vitamin B12 drops below 250 pmol/L.

     

    Causes of Paradoxical B12 deficiency

    The major cause of Paradoxical Vitamin B12 Deficiency appears to lack of functional vitamin B2, which may occur due to overt vitamin B2 deficiency in a person's diet, Hypothyrodism (Habbar etal, 2008), or due to lack of adequate intake of Iodine, Selenium and/or Molybdenum, which in turn leads to insufficient production of the two active forms of vitamin B2, namely FMN and FAD. FMN and FAD both have critical roles in cycling and maintenance of activity of vitamin B12, particularly methyl B12.

    Methyl-Co(III)B12 has a major role in the body in the removal of homocysteine, and in the regeneration of methionine in the methylation cycling using the enzyme methionine synthase reductase (MTR).

    In the reaction, homocysteine + Methyl-Co(III)B12[MTR]  => Methionine + Co(I)B12[MTR].

    The problem with this reaction is that the methyl group is lost from MethylCo(III)B12, which is reduced to Co(I)B12 and so cannot perform further methylation reactions. Theoretically if the reaction only happened once you would need approximately 13.7 gm of MethylCo(III)B12 to remethylate the 1.35 gm of homocysteine formed per day, and around 1.37 kg of the enzyme methionine synthase. Since the daily requirement for vitamin B12 is only around 5 ug, of which around 1.37 ug is MethylCo(III)B12, then clearly this does not happen.

    Regeneration of MethylCo(III)B12 is performed by methionine synthase which transfers the methyl group from 5-methyl-tetrahydrofolate (5MTHF) to Co(I)B12.

    Thus, 5MTHF + Co(I)B12[methionine synthase] => THF + MethylCo(III)B12[methionine synthase].

    If the 5MTHF was only used once, then the body would require around 459 mg of 5MTHF per day, however, the daily requirement for folate is around 1000th of this at 400-500 ug/day, so clearly some other source, apart from diet is required to supply this amount of 5MTHF.

    The solution comes from within the folate cycle. Here the THF, formed above is converted to the folate derivative 5,10-methylene-THF by the enzyme serine hydroxymethyl transferase (SHMT). The enzyme methylene-tetrahydrofolate reducate (MTHFR), then converts the 5,10-methylene group to 5-methyl-THFwhich it transports of the folate cycle into the methylation cycle, in this way a single folate molecule can be recycled over 1000 times into and out of the folate cycle providing the many 5MTHF groups for regeneration of MethylCo(III)B12.

    The reaction 5,10-methylene-THF [MTHFR] => 5-methyl-THF [MTHFR]

    The enzyme, MTHFR, though is critically dependent on FAD and NADPH for enzymatic activity (McNulty etal, 2014) and as levels of FAD drop, the enzyme rapidly loses activity, leading to insufficient 5MTHF for remethylation of Co(I)B12 to MethylCo(III)B12. In this instance the Co(I)B12 is rapidly oxidized to the biologically inactive Co(II)B12. The body does though have a "way around this" and it uses the enzyme methionine synthase reductase plus S-Adenosylmethionine (SAM) to remethylate Co(II)B12.

    Thus, Co(II)B12[MTR] + SAM[MTRR] => MethylCo(III)B12 + SAH + MTRR.

    MTRR, like MTHFR is also a "Flavoprotein" and uses both of the active forms of vitamin B2, FMN and FAD for activity. Once again the activity of the enzyme is critically dependent upon the concentration of FMN and FAD. The activity of the enzyme MTRR is so critical for regeneration of MethylCo(III)B12, that certain mutations in the gene have been found to be conditionally lethal in the womb, or are associated with much higher rates of Down Syndrome, Neural Tube Defects, increased homocysteine (Garcia-Minguillan etal, 2014; DeClerc etal, 1998) and increased cancer risks.

    From the above it can readily be seen that if there is insufficient FMN and/or FAD, there will be a gradual accumulation of the inactive Co(II)B12, which is released from the cell and then starts to accumulate in serum, leading to paradoxically high serum B12.

    Further, the conversion of both hydroxycobalamin and cyanocobalamin to the active Adenosyl and methyl cobalamins, also involve "Flavoproteins" (Obeid etal, 2015) and hence if a person takes or is injected with, high doses of hydroxycobalamin or cyanocobalamin and has functional B2 deficiency, the inactive hydroxycobalamin and cyanocobalamin will accumulate in serum, however the symptoms of vitamin B12 deficiency will not be resolved. Despite the obvious consequences of the functional B2 deficiency in the metabolism of B12, the majority of authors do not seem cognizant of this (Obeid etal, 2015), and seem to believe that B12 deficiency only occurs due to low intake of the vitamin, rather than ineffective processing, and are unaware of the phenomenon of Paradoxical B12 deficiency.

    Paradoxical B12 deficiency is also apparent in certain cancers, such as lung cancer as a result of smoking. In this case one would expect a build up in inactive CN-Cbl due to the cyanide produced during smoking. The higher the serum B12, the higher the associated risk of lung cancer (Fanidi etal, 2019). In addition, inhalation of large quantities of nitrous oxide (Marotta and Keserwani, 2020).

    The other major cause of Paradoxical B12 occurs when people take large oral doses of B12.

    Normally when one either obtains vitamin B12 from digestion in the stomach or from a low dose oral B12 supplement, a protein called haptocorrin (HC), which is secreted into saliva, binds to the B12 and protects it from acid degradation in the stomach. Relatively acid resistant analogues of B12 such as cyanocobalamin have significant hydrolysis at 37oC in the acid environment of the stomach, the more sensitive methylcobalamin, is rapidly hydrolysed in stomach acid. The amount of HC, though is relatively low, and whilst it could protect around 100 ug of B12, there is not enough HC secreted to protect the B12 in some of the high dose oral supplements. Once the B12-HC complex reaches the small intestine the HC is rapidly degraded and the "protected" B12 that has been released is bound by the B12 carrier protein, Intrinsic Factor (IF). IF is relatively non-specific in its binding to B12 and so will bind both intake and hydrolysed B12. If 90%of it is degraded then 90% of the material bound by IF will also be degraded. This mix is then taken up from the intestine and bound by the B12 carrier protein, transcobalamin (TC). TC is even more promiscuous in its binding than IF and so it will transport both the intact and degraded B12 into the cell. Once inside the cell, though the two enzymes MMA-CoA mutase and methionine synthase are very specific for Adenosyl and Methyl cobalamin, respectively, and so any inactive B12 is rapidly expelled from the cells and binds to circulating haptocorrin (HC). As more and more high dose oral B12 is administered the situation gradually gets worse and worse, because now much of the HC secreted in saliva (Collins etal 1999) already has inactive B12 bound to it, and so this HC-B12 complex can no longer protect incoming B12 from acid hydrolysis. Over time, the levels of serum B12 get higher and higher, but more and more of the B12 is inactive.

    Vitamin B2 deficiency and paradoxical B12 deficiency

    From the above it can readily be seen that if levels of the two functional analogues of vitamin B2, namely FMN and FAD are reduced the following will happen.

    1. The activity of MTHFR will decrease proportionally with the decrease in FAD, thus resulting in reduced production of 5MTHF.

    2. The reduced 5MTHF will result in a build-up in Co(I)B12, which over time will oxidize to Co(II)B12.

    3. The reduced amounts of FMN and FAD will in turn reduce the activity of the enzyme MTRR and so the levels of inactive Co(II)B12 will build up inside the cell and will eventually be discarded from the cell resulting in a build up of Co(II)B12 in serum. A condition of Paradoxical B12 Deficiency will result.

    The major cause of Paradoxical Vitamin B12 Deficiency appears to lack of functional vitamin B2, which may occur due to overt vitamin B2 deficiency in a person's diet, or deficiency of Iodine, Selenium and/or Molybdenum. Such deficiencies are very common, and our studies have shown that 50% of people with CFS or ASD are deficient in Iodine, 80% in Selenium and/or 50% in Molybdenum.

    See http://vitaminb12deficiency.info/hypothyroidism.htm for further information.

    In cases of functional vitamin B2 deficiency, there should be a relationship between glutaric acid (a standard marker of vitamin B2 deficiency) and MMA, and other vitamin B12 deficiency markers, HVA, VMA, QA, KA and HMG. There should also be a correlation between MMA and HVA, VMA, QA, KA and HMG. However, like MMA, there should be little relationship between these markers and serum vitamin B12.

    One marker of functional B2 deficiency is glutaric acid. As can be seen as glutaric acid levels increase so too does MMA, indicating reduced activity of vitamin B12. In contrast there was no relationship between glutaric acid and absolute B12 levels.

    HVA is a surrogate marker for methyl B12 deficiency. There was a good correlation between HVA levels and glutaric acid, indicating that there is a correlation between increased B2 deficiency (glutaric acid increasing) and increase methyl B12 deficiency (as per the HVA marker). There was no correlation between levels of HVA and total serum B12.

    VMA is, like HVA a marker of methyl B12 deficiency. As was the case for HVA, increased VMA was correlated with increased glutaric acid (left panel) as well as increased MMA (a classical marker of B12 deficiency).

    QA is also a surrogate marker for methyl B12 deficiency. There was a good correlation between QA levels and glutaric acid, indicating that there is a correlation between increased B2 deficiency (glutaric acid increasing) and increased methyl B12 deficiency (as per the QA marker). There was no correlation between levels of QA and total serum B12.

    KA is also a surrogate marker for methyl B12 deficiency. The good correlation between KA levels and glutaric acid, is not as good as for QA, because you need functional vitamin B2, as FMN to form KA, so in Iodine and/or Selenium deficiency, levels of KA are reduced, and so the correlation to glutaric acid is not as good. In contrast, there was still a good correlation between KA and MMA indicating the relationship between increasing methyl B12 deficiency (KA marker) and increased Adenosyl B12 deficency (MMA marker) (Russell-Jones 2022).

     

    The date presented above demonstrates that serum B12 levels do not correlate at all with levels of functional vitamin B12!!

     

     

    Treatment of Autism

    It is essential that for successful treatment of Paradoxical B12 deficiency that the cause of the functional vitamin B2 deficiency be addressed including supplementation with sufficient vitamin B2, Iodine, Selenium and Molybdenum. Such treatment, though, is generally not performed, but it has immense consequences as far as treatment results, and explains why literature on treatment of vitamin B12 deficiency is rife with examples in which supplementation studies using inactive forms of vitamin B12 (cyanocobalamin or hydroxocobalamin) without the co-administration of the necessary B2/I/Se and Mo have been ineffective in treatment. (see Langan and Goodbred, 2017) There is though a treatment protocol aimed at fixing the functional vitamin B2 deficiency and then the functional B12 deficiency see https://understandingautism.com.au/prevention.htm

     

    Publications from research.

     

    As they are approved, links will be included to PDF copies.

    B12 deficiency in Autism

    B2 deficiency in Autism

    Paradoxical B12 deficiency

    Vitamin B12 deficiency and sleep disorders

    Altered Neurotransmitter Metabolites in vitamin B12 deficiency

     

    Much of the information on the metabolic issues in ASD can also be applied to Dementia and CFS/FM/ME

    Manuscripts in Preparation

    Altered Glucose metabolism in Autism

    Metabolism and Autism

    ;

     

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