Measurement of many markers in OAT indicated moderate to severe vitamin B12 deficiency in all individuals with a diagnosis of ASD, as analysed in the current study. Thus, levels of MMA, the “traditional” marker of Adenosyl B12 deficiency, were increased to as much as twenty-four times that of neurotypical individuals (Figure 1). Similarly, many less “traditional” markers associated with Methyl B12 deficiency were also elevated including KA, QA, 5HIAA, VMA, and HVA (Figures 2-3, Table 1).
According to the Equilibrium Law as expounded by Le Chatelier (Le Chatelier’s Principle) any change in conditions that effect an equilibrium will result in a corresponding change to establish a new equilibrium. This principle can be used to “drive” reactions to produce or reduce the products of a reaction or the amount of a particular substrate in a reaction. Hence in a reaction A + B ó C + D, one can increase the amount of product of the reaction (C + D), if you increase [A] or [B] you can increase [C] and/or [D].
This principle has particular relevance to two methylation reactions in the body, the production of Epinephrine (Adrenalin) by the methylation of Norepinephrine by the enzyme Phenylethanolamine-N-Methyl transferase (PNMT)
Norepinephrine + SAM (PNMT) => Epinephrine and the production of Melatonin by the methylation of N-Acetylserotonin by the enzyme Hydroxy-Indole-O-methyl transferase (HIOMT).
N-Acetylserotonin + SAM (HIOMT) => Melatonin
Both enzymes (PNMT and HIOMT) require the methylation cycle product, S-Adenosylmethionine (SAM) for activity.
Studies suggest that in conditions of reduced methylation the body adapts by trying to drive the production of Adrenalin and Melatonin by increasing the amount of the precursors, Dopamine and Nor-epinephrine (Adrenalin precursors) and N-Acetyl Serotonin and Serotonin (Melatonin precursors). This can be measured in OAT by increases in the degradation products HVA and VMA (Fig 2) as well as an increase in the levels of QA and KA (Figure 3) and 5HIAA (Table 1)
In a previous study , it was shown that each of the children assessed in this study was also found to be functionally deficient in vitamin B2. Given the absolute necessity for functional vitamin B2 (as FMN and FAD) for the activity of Methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), two essential enzymes in the regeneration of MethylCo(III)B12 [20, 21, 22, 23], it is intriguing to speculate that it is the original functional vitamin B2 deficiency that subsequently caused the functional vitamin B12 deficiency. In this respect this would mean that the developmental delay, as measured, is not simply a consequence of low vitamin B12 in the mothers, a known predisposing factor for ASD, but rather it could result from functional B2 deficiency, which then causes gradual inactivation of vitamin B12, resulting in functional B12 deficiency, which would be indistinguishable from absolute B12 deficiency in symptoms, but would be dismissed by the clinician due to there being normal or elevated serum vitamin B12 levels. Low functional B2, both through the reduced ability to make active B6 (PLP), also reduces the activity of the enzyme serine- hydroxymethyltransferase (SHMT) and then greatly reduces activity of the methylation cycle.
Hence (see Fig 4).
Reduced levels of active vitamin B2 as FMN and FAD also reduce the ability of the enzyme methionine synthase reductase to maintain the activity of the enzyme Methionine synthase. Thus, under conditions of functional B2 deficiency, the activity of Methionine Synthase reductase rapidly reduces and so too the function of Methionine Synthase. The result is that will be an accumulation of inactive Co(II)B12, which, because of the FMN/FAD deficiency, cannot be re-activated by MTRR and so will contribute to a large circulating pool of inactive serum vitamin B12. Studies looking at individuals with mutations in either methionine synthase or methionine synthase reductase have shown, that developmental delay is common [24, 25, 26, 27, 28). Given that the identity of serum B12 is not routinely measured, it is only by looking at biochemical markers of deficiency, as has been done in this study, that there can be an accurate diagnosis of functional B12 deficiency in these children. Further, the consequence of vitamin B2 deficiency-mediated functional B12 deficiency, is that unless the functional B2 deficiency can be fixed, one can never fix the functional B12 deficiency.
Lack of methylation due to functional methyl B12 deficiency would also reduce the production of the Electron Transport System shuttle vector CoQ10, whose synthesis requires three methylation steps.
This in turn would reduce the energy transfer down the Electron Transport Chain (ETC), and reduce the amount of ATP produced inside the mitochondria of these individuals, and thereby reduce the energy available within the cytoplasm, which would have a profound effect on the highly energy dependent neurons, with a reduction in neuronal conduction speeds, and reduced brain plasticity, network function, and behavioural systems .
Apart from its role in the production of adrenalin, melatonin and CoQ10, over 40% of all SAM-derived methylation within the body is used in the synthesis of creatine . In this process Guanidinoacetate is methylated using S-Adenosylmethionine as the methyl donor, via the enzyme Guanidinoacetate-N-methyltransferase (GAMT, also known as GNMT) to form creatine, with separate enzyme systems in the liver and the brain [31,32]. Lack of production of creatine leads to Cerebral Creatine deficiency, which has been shown to be associated with a group of treatable intellectual development disorders, including many conditions including prominent speech delay, particularly in expressive and cognitive speech , autism, epilepsy, movement disorders, and myopathy [33, 34].
In a previous study we examined the mineral status of many of the ASD individuals within the current cohort and demonstrated that all children with ASD that were tested were deficient in one of more of Iodine, Selenium and Molybdenum. In line with the need for all of Iodine, Selenium and Molybdenum in the activation of vitamin B2, this deficiency was accompanied by an expected deficiency in functional vitamin B2. The current study demonstrates that the functional deficiency in vitamin B2, observed in the test cohort, leads to an expected deficiency in functional vitamin B12
It has been known for over 40 years that vitamin B12 deficiency in the neonate is associated with developmental delay in children [14, 28, 33, 34, 35, 36]. These early studies were somewhat different to the current study in that the children with ASD in the previous studies had an absolute deficiency in serum vitamin B12. In contrast children in the current study have been found to have both a functional deficiency in vitamin B2 and a functional deficiency in vitamin B12 with normal to elevated serum B12. These findings would suggest that potentially the dramatic increase in the rate of ASD over the past 20 years could be due to decreases in the intake of Iodine, Selenium and/or Molybdenum in countries, in which soil deficiencies of these minerals are now more common. Potentially this would suggest that lack of one single nutrient could be the precipitating event that results in the spectrum of developmental delays such as those seen in autism. Of note is the potential that any of the deficiencies would ultimately lead to reduced methylation and cerebral creatine deficiency, with the accompanying symptoms.
The current studies fill a critical gap in the understanding of the mechanism behind the factors involved in developmental delay typical of ASD, and as such greatly aid in the understanding of the cause behind the condition and therefore provide a potential mechanism for the prevention and treatment of ASD.
Firstly, they support earlier studies in which Iodine deficiency was found to be the single most preventable cause of developmental delay, and identify the pathway of vitamin B2 activation whereby a deficiency of any of Iodine or Selenium or Molybdenum deficiency, would result in functional vitamin B2 deficiency.
Secondly, they describe how this functional deficiency in vitamin B2 would ultimately result in the eventual inactivation of vitamin B12 and so result in functional deficiency in vitamin B12, a known predisposing factor for developmental delay as typified in ASD.
Thirdly, through its role in the methylation cycle the studies identify critical methylation products which are essential for neurological development, including the production of melatonin, an essential molecule for the differentiation and maturation of neuronal stem cells into myelin producing oligodendrocytes, and importantly in the production of the energy transfer molecules, CoQ10 and creatine. In this regard a deficiency in creatine alone has been known to result in many of the symptoms typical of ASD.