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Methylcobalamin and formation of Iron-sulphur proteins

Iron-sulphur proteins

Iron-sulphur proteins are a group of non-heme, iron containing proteins, in which elemental iron is complexed with sulphur. They are characterized by the presence of iron-sulfur clusters containing sulfide-linked di-, tri-, and tetra-iron centers in variable oxidation states. They are some of the most primitive structures in the body and evolved when the earth was devoid of oxygen and energy transfer was mainly through iron and sulphur complexes. Notable examples include the Krebs cycle proteins, aconitase and succinate dehydrogenase, plus ferrodoxin (adrenodoxin - involved in activation of vitamin D) and  complex I, II and III of the electron transport chain. In addition iron-sulphur complexes are present in enzymes responsible for structure, including lysyl-hydroxylase

Iron-sulphur complexes

Iron-sulphur protein, Adrenodoxin. Note the iron-sulphur complexes

Iron-sulphur protein, aconitase. Note the iron-sulphur complexes

 

Reduced iron deficiency, particularly when coupled with vitamin B12 deficiency is associated with lower activity of the Krebs cycle enzyme, Aconitase, which in turn results in lower energy entering Krebs cycle, higher urinary citrate, and lower Mini Mental score. Lower aconitase activity has been found in the cerebellum and Brodmann areas of the brains in those with autism.

MMSE score against the activity of the enzyme aconitase (Figure. Data from Mangialasche etal,2015)

Reduced Aconitase activity in the cerebellum and Brodman area of the brain in control and those with autism (Fig from Rose etal, 2012). Note the reduced ratio of GSH:GSSG between those with autism and the NT controls

Energetics of Iron Deficiency

Iron has a major role in the conversion of energy generated from the metabolism of fats, sugar and protein, and as iron levels drop (as measured by serum ferritin), the energy conversion via the iron-sulpur enzyme aconitase drops rapidly and there is an increased excretion of citrate in urine - representing metabolic energy that cannot be processed. Iron deficiency in the diet is an environmental disaster, as it represents a highly inefficient use of consumed calories, with some children "wasting" upwards of 80% of consumed calories. The corollary to this is that the highly energy dependent neurons in the brain are effectively starved of energy as iron levels drop. This then results in impaired mitochondrial respiration, lower intracellular ATP production, and leads to chronic neuronal energy insufficiency (Raichle and Gusnard, 2002), with a subsequent reduction in mitochondrial speed with reduced hippocampal neuronal development (Bastian etal, 2019). Decreasing levels of ferritin in serum have been correlated with higher levels of "wasted" citrate in urine - Personal obvervations)

Relationship between serum ferritin (vertical axis) and urinary citrate (horizontal axis).

Energetics of Methyl B12 Deficiency

One of the standard markers of Methyl B12 deficiency is homocysteine. Urinary Organic Acids testing (OAT) has shown a correlation between functional vitamin B12 deficiency and elevations in the neurotransmitter metabolite, HVA. Because OAT does not measure homocysteine, HVA has been used as a surrogate marker for HVA (see https://b12oils.com/b12.htm)  Formation of CoQ10 requires 3 methylation reactions. In functional methyl B12 deficiency, levels of the CoQ10 precursor HMG are elevated, and energy transfer along the electron transport chain is reduced. As can be seen from the scattergram, the levels of the B12 deficiency marker, HVA correlated with those of the CoQ10 deficiency marker, HMG

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Relationship between HVA (vertical axis) and HMG (horizontal axis)

Methyl B12 also has an indirect association with the formation of iron-sulphur proteins. Hence the sulphur from methionine is the dietary source of the sulphur in cysteine, glutathione, and in iron-sulphur proteins, such as aconitase and succinate dehydrogenase. Movement of the sulphur in methionine into the sulphation pathway requires stimulation of the enzyme, cystathionine beta synthase (CBS), by S-Adenosylmethionine. In conditions of low methylation due to Methyl B12 deficiency, the activity of CBS is reduced, and so too is the movement of sulphur into the sulphation pathway, and there is a reduction in the capacity to make iron-sulphur proteins, due to a sulphur deficiency.

Comparison between HVA (vertical axis) and citrate (horizontal axis) showed a correlation between increasing HVA and increasing citrate. Suggesting that methyl B12 deficiency results in lower production of, or lower activity of aconitase. It should be noted, however, that there will be a co-dependency upon levels of iron and sulphur from methionine intake, thus reducing the R2 value.

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Relationship between HVA (vertical axis) and citrate (horizontal axis)

Similarly, methyl B12 deficiency also resulted in lower activity of the iron-sulphur protein succinate dehydrogenase. Increased Methyl B12 deficiency, as represented by increasing HVA (verical axis), resulted in increased succinic acid (horizontal axis).

Relationship between HVA (vertical axis) and succinate (horizontal axis)

Reduced activation of vitamin D in Methyl B12 Deficiency

Conversion of 25-OH vitamin D, to the active form, 1,25-diOHvitamin D, requires a complex interaction between the enzymes alpha-hydroxylase, and the iron sulphur protein, adrenodoxin, and the FAD-dependent enzyme adrenodoxin reductase. Deficiency in active vitamin D can be estimated by an increase in phosphoric acid. Decreased methyl B12, as estimated by HVA levels (horizontal axis), resulted in reduced activation of 25-OH-D to 1,25 diOH D, with reduced production of calcium-phosphate resulting in increased levels of phosphoric acid in urine (vertical axis).

Reduced activation of vitamin D as determined by an increase in urinary phosphoric acid (vertical axis) when compared with HVA (Horizontal axis)

Reduced production of Glutathione in Methyl B12 Deficiency

Production of glutathione requires the conjugation of glutamate, cysteine and glycine. In methyl B12 deficiency, the lower activity of the enzyme cystathionine beta synthase (CBS) results in reduced transfer of the sulphur in homocysteine into the sulphation cycle, with a resultant reduction in the levels of cysteine. In the absence of cysteine, the molecule, glutathioine (GluCysGly) cannot form and pyroglutamate is formed. Methyl B12 deficiency as determined by increased HVA (vertical axis) was correlated with a marked n increase in levels of urinary pyroglutamate (horizontal axis).

Reduced production of GSH in methyl B12 deficiency. Methyl B12 deficiency, as determined from increased urinary HVA (vertical axis) corrleated with an increase in pyroglutamic acid (horizontal axis)

Methyl B12 and Lysyl-hydroxylase

The iron-sulphur protein, lysine hydroxylase, functions to modify lysyl residues in proteins such as collagen, allowing proper hydrogen-bonding of neighbouring strands of collagen, thereby strengthening the structure. Reduced lysyl-hydroxylase activity is associated with laxity of joints such as occurs in Ehlers Danlos syndrome, and in many children with autism.

Summary

Reduced levels of methyl B12 have been associated with lower energy transfer in the electron transport chain, due to lower production of the electron shuttle vector, CoQ10.

Reduced levels of methyl B12 have been associated a reduced production of iron-sulphur proteins, resulting in lower activity of the Krebs cycle enzymes aconitase and succinate dehydrogenase.

Reduced levels of methyl B12 have been associated with a lower production of the intracellular anti-oxidant and disulphide shuttle vector, glutathione, and an increased production of pyroglutamic acid.

Reduced levels of methyl B12 have been associated with lower activity of lysine hydroxylate and an increased laxity of joints.

 

References

 

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