Transsulfuration and Glutathione

In a presentation made at the Autism One Conference in 2005, Dr. Jill St. James discussed the how the metabolic pathway of transsulfuration appears to be disrupted in autistic children, resulting in autistic children being deficient in critical vitamins and amino acids, including B6, B12 and Glutathione. The following information is derived from her presentation.

Because we are an oxygen-based life form, oxygen both provides life and but at the same time can be harmful to cells (in the form of oxidative stress). To maintain health, our body must not only use oxygen but produce anti-oxidants to prevent the oxygen from damaging tissues within the body.

If not enough anti-oxidants are produced damage can result to the energy-producing mitochondria of cells, to the cell's DNA, to the composition of cell membranes, and to the protein composition. The results of excessive oxidation can include genes being expressed abnormally which can lead to abnormal paths of development, to cells not transmitting information well due to impaired cellular membranes (including neurons in the brain), and to cellular die-off due to toxicity.

Excessive oxidation has also been linked to heart disease, cancer, autoimmune diseases (such as arthritis and colitis) and neurodegenerative conditions such as Multiple Sclerosis, Parkinsons and Alzheimers. Recent research indicates that oxidative stress appears now to also be a key factor in autism.

What is currently being intensively studied by autism researcher is the Methionine/Glutathione Transsulfuration Pathway, which appears to be disrupted for individuals within the autism spectrum. (It is called transsulfuration because the molecules in this pathway are sulfur-bearing.) This pathway is also critical to the body's ability to remove toxins through methylation (binding the toxins to methyl groups and excreting them from the body). A methyl group is one carbon atom bonded to three hydrogen atoms (CH3).

Overview of the Methionine/Glutathione Transsulfuration Pathway

Abbreviations in the above diagram are: THF: tetrahydrofolate; MS: methionine synthase; BHMT: betaine-homocysteine methyltransferase; MAT: methionine adenosyltransferase; SAM: S-adenosylmethionine; SAH S-adenosylhomocysteine; SAHH: SAH hydrolase; ADA: adenosine deaminase; AK: adenosine kinase; CBS: cystathionine beta synthase

Figure 1 - A Normal Transsulfuration Metabolic Pathway

The figure above depicts the series of metabolic reactions that occurs within neurotypical people to convert methionine to glutathione. The items in yellow are catalysts/enzymes that act upon one molecule to create another molecule. For example, Methione is acted upon by MAT to create SAM, which is then used to create SAH, then Homocysteine, Cystathionine, Cysteine, and finally Glutathione. The Glutathione is used to remove toxins from the body and is reconverted into Methionine, during methylation process, and the Methionine/Glutathione Transsulfuration pathway cycle is repeated.

If this transsulfuration pathway is disrupted, oxidative stress can result and lead to various health problems. For example, if Homocysteine isn't converted by Vitamin B6 into Cysteine, high levels of homocysteine can build up, leading to problems such as heart disease.

For autistic children, this metabolic pathway differs from that of neurotypical children in varying ways:

	A decrease in ADA activity has been reported in children with autism.
	Metabolic disruptions in the removal of adenosine or homocysteine lead to an increase in SAH.
	Excessive levels of adenosine inactivate SAHH activity by binding to the active site on the cell and result in SAH accumulating.
	Excessive SAH, in turn, represses the essential methyltransferase reactions, key to the removal of toxins from the body.
	Cysteine is dependent upon having enough methionine. A decrease in methonine is associated with a decrease in cysteine, which is critical to producing glutathione. Autistic children have low levels of methionine and glutathione. Methionine transsulfuration to cysteine and glutathione occurs primarily in the liver, the predominant organ for methionine metabolism.
	Glutathione (GSH, which stands for g-glutamylcysteinylglycine), is one of the body's major antioxidants. Most cells are not able to take up intact glutathione and rely on the uptake of its precursors, cysteine, cysteinylglycine, or g-glutamylcysteine for GSH synthesis inside the cell. Glutathione synthesis occurs primarily in the liver. A decrease in the production of Glutathione in the liver would result in harm to vulnerable tissues that depend upon it to protect them again oxidative stress (including the brain, intestines, and thymus) and would tend to results in neurological, gastrointestinal, and immunilogic problems in autistic children.

Dr. St. James conducted an experiment at Arkansas Children's hospital, whicih included comparing the levels of molecules that are part of the transsulfuration pathway in 20 autistic children with those of 33 neurotypical children. The profile of autistic children was severely abnormal:

Molecule	Neurotypical Children	Autistic Children
Methionine (µmol/L)	30.6 ± 6.5	19.3 ± 9.7
SAM (nmol/L)	90.0 ± 16.2	75.8 ± 16.2
SAH (nmol/L)	20.1 ± 4.3	26.1 ± 5.4
Homocysteine (µmol/L)	6.3 ± 1.2	5.4 ± 0.9
Adenosine (µmol/L)	0.28 ± 0.16	0.39 ± 0.19
Cysteine (µmol/L)	210 ± 18.5	163 ± 14.6
Total Glutathione (µmol/L)	7.9 ± 1.8	4.1 ± 0.5
Oxidized Glutathione (nmol/L)	0.3 ± 0.1	0.55 ± 0.2
GSH/GSSG Ratio	25.5 ± 8.9	8.6 ± 3.5

Most notable are the significantly lower levels of Methionione, Cysteine, and total Glutathione for the autistic children. Because Methionine is a precursor for Cysteine, and Cysteine is a precursor for Glutathione, it is not surprising that all of these molecules were found at lower levels in autistic children. The increase in SAH and adenosine for autistic children compared to the neurotypical children is of concern because SAH inhibits the action of SAM (methyltransferase activity) and cellular methylation reactions which enable the body to remove toxins.

The reduction in Total Glutathione and increase in Oxidized Glutathione resulted in a 3-fold reduction in the ratio of reduced (active) GSH to oxidized (inactive) Glutathione (GSSG). This is problematic it indicates becuase reduced levels of Glutathione mean that there these children have less anti-oxidant protection against oxidative stress.

Cellular consequences of decreased glutathione antioxidant potential are:

	Reduced ability to detoxify environmental toxins and heavy metals, resulting in neurotoxicity
	Oxidation of cysteine thiol (SH) groups in proteins, resulting in altered function of proteins
	Decreased liver GSH synthesis, resulting in reduced cysteine within the brain
	Degeneration of the lining of the intestinal tract, resulting increased gut permeability and autoimmunity
	Increased Th2, alterted thymic T cell subsets, resulting in autoimmune issues
	Reduced S-adenosylmethionine synthesis and increased SAH accumulation, resulting inhibition of methyltransferase and reduced ability to methylate and excrete toxins from the body

Reduced total antioxidant capacity (active Vitamin C and E depend on GSH), which results in worsening all of the other aspects.

Cellular consequences of reduced methylation capacity are:

	Reduced DNA methylation, resulting in damage to cells
	Reduced protein methylation, resulting in altered protein activity/function
	Decreased catecholamine-O-methyltransferase activity, resulting in altered neurotransmitter synthesis
	Reduced membrane phosphatidylcholine synthesis, resulting in impaired membrane fluidity and signaling
	Reduced methylation and detoxification of arsenic, resulting in increased oxidative damage to cells

Dr. St. James' Study with Folinic Acid and Betaine

Dr. St. James attempted to improve the levels of Methionine, Cysteine and Glutathione of the 20 autistic children in her study and by doing so increase their ability to detoxify through the methylation process. Each child was given supplements of 800 mg of folinic acid and 1000 mg betaine (trimethylglycine) over a three week period.

After only three week of supplementation, the changes were quick and dramatic. The blood serum levels of Methionine, Cysteine, and Glutathione resulted in a 2-fold increase in the ratio of reduced (active) to oxidized (inactive) glutathione. Eight of the 20 children continued the intervention with betaine and folinic acid for an additional 3-4 months. After the extended intervention period, SAM levels increased to 112 nmol/L, SAH levels decreased to 17 nmol/L, and adenosine levels decreased to 0.18 µmol/L; all well within normal ranges.

Molecule	Autistic Children Before Supplementation	Autistic Children After Supplementation
Methionine (µmol/L)	19.3 ± 9.7	28.0 ± 7.2
SAM (nmol/L)	75.8 ± 16.2	81.4 ± 10
SAH (nmol/L)	26.1 ± 5.4	25.1 ± 8.4
Homocysteine (µmol/L)	5.4 ± 0.9	7.0 ± 1.0
Adenosine (µmol/L)	0.39 ± 0.19	0.33 ± 0.1
Cysteine (µmol/L)	163 ± 14.6	225 ± 37
Total Glutathione (µmol/L)	4.1 ± 0.5	5.7 ± 1.0
Oxidized Glutathione (nmol/L)	0.55 ± 0.2	0.48 ± 0.2
GSH/GSSG Ratio	8.6 ± 3.5	13.8 ± 4.8

Folinic acid was used instead of Folic acid because it is more easily assimilated into folate metabolism. Folinic acid is converted to 5, 10-methyleneTHF which will support purine and thymidylate synthesis and also methionine synthesis. Betaine provides a folate-independent pathway for regenerating methionine via BHMT (betaine-homocysteine methyltransferase) that occurs primarily in the liver.

These results suggest that supplementation with folinic acid and betaine had a strong positive impact on antioxidant capacity in the autistic children. Although SAM levels were significantly increased, the decrease in SAH and adenosine levels did not reach statistical significance due to individual variability. Targeted nutritional intervention with folinic acid and betaine successfully increased both the ability to methylate capacity and levels of active glutathione in the 20 participating autistic children, especially after 3-4 months of intervention.

According to Dr. St. James, micronutrients supportive of methionine synthesis include:

	Zinc: MS, BHMT, and methyltransferases are zinc-dependent enzymes involved in methionine cycle.
	Folinic Acid: Supports nucleotide synthesis by increasing 5,10-methylene THF; supports methionine recycling by increasing 5-CH3THF.
	Betaine (trimethylglycine): Substrate for BHMT; increases BHMT expression and activity
	Methyl-B12: Replaces need for methyl group transfer from 5methylTHF: potential for trapping folate as 5-methyl-THF and reducing synthesis of metabolically active THF
	Choline: Spares phosphatidylcholine breakdown to betaine for BHMT-mediated methionine synthesis

Dr. St. James also recommends the following micronutrients and precursors to support glutathione synthesis:

	Glutathione methyl ester.
	N-acetylcysteine: Stable form of cysteine that readily crosses cell membrane and increases intracellular gluthatione levels; toxic only at extremely high doses (1.5 gm). (Note from Autism Coach - some children within the autism spectrum do not do well with this supplement.)
	2-oxothiozolidine -4- carboxylate (OTC): Stable derivative of cysteine that is readily converted to cysteine and increases glutathione synthesis.
	Vitamin B6: CBS and cystathionine lyase are both B6 dependent enzymes that are involved in the transsulfuration of homocysteine to glutathione.
	Selenium: Glutathione peroxidase is a selenium-dependent enzyme; selenium deficiency is common in malabsorption and gastrointestinal disease.
	Glutamine: Glutamine enhances gut glutathione production. (Note from Autism Coach - glutamine can be problematic for some children within the autism spectrum.)
	Antioxidants: Vitamin E, vitamin C, lipoic acid.

Dr. Megson also hypothesizes that the reason why more males than females are diagnosed as autistic has to do with the female hormone, estrogen. Estrogen has been shown to increase blood levels of glutathione in experimental animals. Mitochondrial glutathione levels and antioxidant capacity have been reported to be higher in females than in males. Taken together, the evidence suggests that both cellular methylation capacity and antioxidant activity are higher in females than males. The increased rate of methionine transsulfuration and glutathione antioxidant activity in females may protect females from developing autism.