Sulfur in CFS: signals in the noise?

Some findings presented at a recent CFS conference got me interested in sulfur again. What is the current picture? How does it fit with everything else? Here’s a mini-review of my reading.

Sulfur is the 7th most abundant element in the body ¹. Most has been presumed to come from dietary proteins, specifically the two sulfur-containing amino acids: methionine and cysteine. However, a substantial amount also comes from other organosulfur compounds in plants (e.g. allium and cruciferous veg) and inorganic sulphates (i.e. water and food) ².

Methionine is the most essential sulfur-containing nutrient and can be converted to other sulfur metabolites. Initially, methionine is adenosylated (by MAT) to s-adenosyl-methionine (SAM, aka AdoMet), a methyl-donor, which is then demethylated to s-adenosyl-homocysteine (SAH) and hydrolysed (by SAHH) to homocysteine. Homocysteine can be remethylated back to methionine (MS and BHMT pathways) or catabolised by via the transsulfuration pathway (CBS and CGL) to other sulfur metabolites (e.g. cysteine/GSH, taurine and H₂S). Enzymes in these pathways use energetic and B vitamin-derived cofactors/substrates—e.g. MTHFR (B₂, B₃, B₉), MS (B₉, B₁₂), MSR (B₂, B₃), MAT (ATP), SAHH (B₃), CBS (B₆) and CGL (B₆).

Homocysteine is the infamous marker of sulfur metabolism, since elevated levels (hyperhomocysteinemia, e.g. >15uM ³) are associated with many diseases. As above, homocysteine metabolism is dependent on 1-carbon metabolism (i.e. folate and methionine/SAM cycles) and transsulfuration pathways, which are regulated by cell activity and metabolic homeostasis (e.g. SAM and redox) ^4–8. In healthy people, blood homocysteine fluctuates throughout the day in relation to circadian rhythms (~7–10uM) ⁹ and can be transiently increased by protein/methionine intake ^10–12. Elevated homocysteine may result from many factors including genetics, age, diet and disease processes.

…in ME/CFS

There are not many studies which have looked at sulfur metabolism in ME/CFS. Here I have pulled together the most relevant findings I am aware of in the published literature and presented at conferences.

Peripheral findings

Since the late 90s, there have been several amino/organic acid studies on blood and urine. Some early studies found changes to sulfur metabolites (i.e. methionine, cysteine, taurine or sulfate) in vaguely defined ^13,14 and standard criteria ME/CFS ¹⁵. More recently, the largest study so far (n=153) found lower levels of many serum amino acids (incl. methionine, but not cysteine) in females only ¹⁶; for comments see ¹⁷. Other recent metabolomic studies found some changes implicating sulfur-related pathways, including methylation (MetS, B₁₂ and betaine) ^18,19 and taurine ²⁰.

Surprisingly, homocysteine has rarely been measured. Over 20 years ago, in 1997, a small study on 12 women with CFS/FM found a couple (17%) had elevated blood homocysteine ²¹. Most recently, at a 2017 OMF conference, Dr Moreau reported finding very elevated plasma homocysteine levels (~30uM) in 20% of >100 CFS patients, which correlated illness severity. There were no changes to B vitamins (B₆, B₉ and B₁₂), but low vitamin C in males (YouTube). He will further investigate the cause of the high homocysteine. Note, plasma homocysteine largely results from liver and kidney metabolism, so may implicate these organs.

Several early studies measured RBC glutathione (GSH) and found either no, low or varied changes in CFS subgroups (reviewed in ²²). Moreover, in a very small muscle biopsy study, total GSH was elevated, alongside markers of oxidative stress, suggesting a compensatory mechanism ²³.

At the 2009 IACFS/ME conference, interesting results of an uncontrolled pilot study on 21 women with CFS/FM were presented ²². This study found lower plasma GSH/GSSG, RBC SAM and plasma/cell folates. Biomarkers and symptoms improved with 6 months of multi-vitamin supplementation. This result supported a link between methylation and GSH status, similar to that previously found in autism ²².

Central findings

In the early 1997 study above on women with CFS/FM, while blood homocysteine was only elevated in a minority, they all had increased cerebrospinal fluid (CSF) homocysteine (avg. 3x) and most ‘suspiciously low’ CSF B₁₂, both of which correlated symptoms ²¹. Note, there were no changes to CSF methionine, cystathionine and MMA. These results suggested impaired B₁₂-dependant remethylation of homocysteine in the CNS ²¹.

In 2009, an initial neuroimaging study failed to find significant changes to brain GSH in ME/CFS ²⁴. However, subsequent studies found brain GSH was low and related to ventricular lactate and symptoms in CFS (as well as controls and MDD) ^25,26. Note, GSH had a much stronger association with CFS symptoms than lactate ²⁶. Further, a validation study reported that N-acetyl-cysteine (NAC) could boost brain GSH and markedly improve symptoms (conference abstract ²⁷). Note, NAC not only supplies cysteine for GSH synthesis, but may also promote homocysteine catabolism to cystathionine and H₂S ²⁸.

Causes?

Why might sulfur metabolism be affected in some with CFS? Here are some possibilities:

• Genetics? Common gene variations can influence sulfur metabolism (e.g. MTHFR and MSR snps) ²⁹. Some interaction between MTHFR snps and the response to B₁₂/folate supplementation was reported in CFS ³⁰.
• Cofactor deficiencies? As mentioned above, enzymes of sulfur metabolism require B vitamin-derived cofactors. In CFS, some studies have found lower B₁₂ ^19,21, folate ³¹, B₆ ³², FAD ¹⁹, NAD^{+ 33} and NAD(P)H ³⁴. ATP is also required for the synthesis of SAM and GSH (which are required to form methyl-B₁₂ ^35,36), and the conversion of vitamins B₂, B₃ and B₆ to their active cofactor forms. Note, MAT may depend on de novo ATP synthesis from serine ³⁷. Some studies find evidence of bioenergetic dysfunction and low ATP in ME/CFS ^20,33,38. Note however, neuroimaging studies have found no changes to brain ATP (at rest) ²⁶, while NAC boosts brain GSH ²⁷.
• Gut microbiome? The gut microbiome appears to regulate systemic bioavailability and metabolism of H₂S ³⁹. Gut bacteria may also produce and supply significant folate to the body ⁴⁰, but compete for B₁₂ ⁴¹. In particular, SIBO has been reported in CFS ⁴², which has been associated with low B₁₂ status in other conditions ⁴¹.
• Oxidative stress? Oxidative stress upregulates transsulfuration flux ⁴. Further, since both folate and B₁₂ are sensitive to oxidation ^35,43, oxidative stress has been suggested to impair MS-dependant remethylation activity in various conditions ^44–47, including CFS ^21,22.
• Inflammation? Conditions of chronic inflammation and tissue breakdown are associated with increased homocysteine levels and sulfur excretion in humans ^1,6. Stimulation of immune cells can increase sulfur metabolism ⁴⁸ and homocysteine release ⁴⁹. In animal models, sepsis increases methionine transsulfuration to cysteine ⁵⁰ and inflammation can deplete vitamin B₆ ⁵¹. In ME/CFS, immune activity may be related to illness duration ⁵², severity ⁵³ and bacterial translocation ^54,55.
• Autoimmunity? Autoantibodies may block proteins mediating nutrient uptake, such as the folate receptor ⁵⁶ and megalin ^30,45. The latter was mentioned in relation to brain B₁₂ in CFS ³⁰.

Effects?

What are the effects of altered sulfur metabolism in CFS? Various theories have considered GSH ^26,57–59, methylation ^21,59 and H₂S in CFS ⁶⁰. Here I will briefly review some possibilities.

GSH

Brain GSH correlated symptoms in CFS, suggesting clinical significance ²⁶. Moreover, a small validation study on 16 people with CFS reported that 4 weeks of NAC treatment normalised brain GSH and markedly improved symptoms; whereas brain GSH did not change in a healthy control group on the same treatment (conference abstract ²⁷). This might support causality between GSH and symptoms in CFS—but by what mechanism? GSH is the substrate of major intracellular antioxidant systems regulating redox signalling and buffering oxidative stress ⁶¹. Consequently, low GSH could have many detrimental effects on cell functions and viability ⁵⁹, and is associated with neurological diseases ^45,57. Interestingly, brain GSH inversely correlated lactate in CFS (and combined study group), suggesting a relationship with bioenergetics ²⁶. The authors suggested oxidative stress may impair brain blood flow and energy metabolism in CFS ²⁶.

Low GSH could affect many redox-sensitive metabolic and signalling pathways. Of particular interest here, low GSH may promote a functional B₁₂ deficiency, since glutathionylcobalamin serves as a precursor to methylcobalamin, the cofactor for MS ³⁵. Further, neuronal mitochondrial oxidative stress can deplete methyl-folate ⁴³. Therefore low GSH and oxidative stress might impair B₁₂/folate-dependant remethylation of homocysteine, aggravating the dysfunctions discussed below.

Homocysteine

Both plasma homocysteine (Moreau) and CSF homocysteine/B₁₂ ²¹ also correlated symptoms in CFS. Further, some preliminary therapeutic reports suggest B₁₂ and folate supplementation can improve biomarkers and symptoms in ME/CFS ^22,30,62. For instance, a small uncontrolled pilot study on 21 women with CFS/FM reported that 6 months treatment with B vitamins normalised several biomarkers (i.e. SAM, GSH and folates), while lowering and improving symptoms (i.e. energy, sleep, mental clarity, pain and wellbeing) (conference report ²²).

Plasma homocysteine levels may be as high as ~30uM in severe CFS. These levels and far higher (>100uM) are seen in some other disorders (e.g. advanced kidney disease ⁶³, severe nutrient deficiency and genetic diseases ^64,65). The clinical significance probably depends on aetiology—what is blood homocysteine a marker of and which tissues are affected ^66–69? Levels of around 30uM won’t necessarily be associated with CFS-like symptoms (e.g. nitrous oxide abuse ⁷⁰). But perhaps this anecdote is noteworthy. The recent BBC series 'doctor in the house' included someone struggling with long-standing health issues, including a history of stroke and ongoing fatigue, exhaustion and sleep issues. Nothing had shown up in tests, until his homocysteine came back at 35uM. This seemed related to a gene variation, while multi-vitamin supplementation rapidly improved his condition and dropped his homocysteine to 7uM (YouTube).

Homocysteine has many effects in cell and animal models (e.g. hypomethylation, homocysteinylation, NMDA receptor and oxidative stress), which may occur at different levels. Interestingly, the acute effects of homocysteine have been studied in humans via methionine or homocysteine-loading with assessment of vascular function ^12,71–74 and metabolic, lipid or inflammatory markers ^75,76. For instance, in young healthy adults, oral methionine or homocysteine acutely increases homocysteine (e.g. from 10 to 25, or 50uM respectively) and impairs blood flow (i.e. brachial artery flow-mediated dilation) ^12,71,72. This occurs even at low increments of homocysteine (i.e. 2–3uM) ¹² and is most tightly related to the reduced form of homocysteine ⁷². Of additional relevance to severe CFS, this vascular dysfunction is prevented by vitamin C ⁷¹, and may be related to homocysteine-induced oxidative stress and depletion of nitric oxide ^77,78. Note, not all methionine-loading studies find these changes to peripheral or cerebral blood flow ⁷³, while in others there may be age-dependence ⁷⁴, suggesting underlying physiology/health (antioxidant status?) is important. Consistent with vascular changes, the methionine-loading test also frequently induces clinical symptoms, such as dizziness, resulting in impaired perception and vigilance ⁷⁹.

Methylation

In the methionine/SAM cycle, accumulation of homocysteine can promote feedback inhibition or reversal of the SAHH reaction and increase SAH ^80,81, a potent inhibitor of most SAM-dependent methylation reactions, thus lowering the methylation potential (SAM/SAH) ^82,83. SAM is involved in over 50 methyl-transfer reactions to DNA, proteins, phospholipids and other small metabolites ⁸². Quantitatively, the major methyl consumers are biosynthesis of phosphatidylcholine (membranes, lipoproteins, bile, mucus, etc.) and creatine (energy recycling, etc.) ^84–86. Sensitivity to SAH is methyltransferase and tissue-specific ⁸³. Some pathways in particular have been implicated. For instance, in human studies, plasma homocysteine directly correlates SAH, which negatively correlates phosphatidylcholine (level and PUFA/DHA content) in blood/RBC ^87,88 and DNA methylation in lymphocytes (Fig) ⁸³. Note, SAM also plays a non-methyl role in polyamine biosynthesis (i.e. spermidine and spermine); although in cell studies SAM depletion had a greater effect on methylation ^89,90.

Dysregulation of the methionine cycle has further repercussions for other connected metabolites/pathways. For instance, elevated homocysteine and inhibition/reversal of SAHH activity may deplete adenosine by trapping it in SAH ⁹¹. Conditions of low SAM or high homocysteine may promote compensatory BHMT activity at the expense of betaine ^64,92. Low B₁₂/MS activity and SAM may favour higher MTHFR activity and trapping of folate as methyl-THF. Low SAM may slow transsulfuration flux (i.e. CBS ⁵) and GSH metabolism (i.e. Nrf2/GCL ^93,94 and GST activity ⁹⁵), which could link methylation and redox status in some conditions ^5,96. Note, depletion of transsulfuration products (e.g. GSH/GSSG ⁴ and taurine ⁹⁷) will increase CBS activity, but this may be limited by SAM, which stabilises CBS ⁵.

Long-term…?

In some studies, CFS has been associated with risk factors for cardiovascular disease (CVD) ^98–100, the primary cause of death globally. CVD-related changes in CFS include increased arterial stiffness ¹⁰⁰ and endothelial dysfunction (i.e. brachial artery flow-mediated dilation and forearm skin vasodilation) ⁹⁸, which in turn, may be related to several factors including age, inflammation and oxidative stress ^98–100.

Sulfur metabolism is of major relevance to ageing and related diseases. Methionine restriction and transsulfuration pathways promote stress resistance, healthspan and longevity in animal models ^101,102. Conversely, in humans elevated homocysteine (even more so SAH) is an independent risk factor for CVD ⁶⁹, as well as other conditions including osteoporosis ¹⁰³ and cognitive decline ^104,105. Similarly, loss of thiol redox (incl. Cys/CySS and GSH/GSSG) is associated with CVD ⁶¹ and other age-related diseases ¹⁰⁶. Note, low RBC GSH was associated with CVD risk factors in CFS ¹⁰⁷.

So could sulfur be important in some with ME/CFS?

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