Redox signalling occurs when a biological system alters in response to a change in the level of a particular reactive oxygen species (ROS) or the shift in redox state of a responsive group such as a dithiol–disulphide couple (D'Autreaux and Toledano, 2007; Finkel, 2011; Fourquet et al., 2008; Janssen-Heininger et al., 2008; Rhee, 2006). Although ROS are best known as damaging agents in pathology, a more nuanced view has developed. It is now clear that some ROS, such as hydrogen peroxide (H2O2), can act as messengers both in the extracellular environment and within cells (D'Autreaux and Toledano, 2007; Fourquet et al., 2008; Janssen-Heininger et al., 2008; Rhee, 2006). Mitochondria seem to be an important redox signalling node, partly because of the flux of the ROS superoxide (O2·−) generated by the respiratory chain and other core metabolic machineries within mitochondria (Balaban et al., 2005; Finkel, 2011; Murphy, 2009a). In addition, the mitochondrial matrix is central to metabolism, as oxidative phosphorylation, the citric acid cycle, fatty acid oxidation, the urea cycle and the biosynthesis of iron sulphur centres and haem take place there. Furthermore, mitochondria have key roles in apoptosis, calcium homeostasis and oxygen sensing (Duchen, 2004; Murphy, 2009a; Murphy, 2009b). Consequently, mitochondria are at the core of many biological processes, and redox signals to and from this organelle help to integrate mitochondrial function with that of the cell and organism. In this Cell Science at a Glance article we outline how mitochondrial redox signals are produced and modulated, the mechanisms by which redox signals can alter mitochondrial function and the experimental procedures available to assess this.
Production and modulation of redox signals to and from mitochondria
The initial ROS formed within mitochondria is O2·−, which is generated by the respiratory chain and other enzymatic components within the mitochondrion (Finkel, 2011; Murphy, 2009a). Mitochondrial O2·− generation provides an indication of functional status because its production is altered by many cellular factors. These include the membrane potential, the reduction state of electron carriers and post-translational modification or damage to the respiratory chain (Murphy, 2009a). However, O2·− itself is not the main ROS signal within mitochondria because it is mostly converted to H2O2 by manganese superoxide dismutase (MnSOD), which reacts very rapidly with O2·− and is present at a high concentration within the matrix (Balaban et al., 2005; Chance et al., 1979; Finkel, 2005; Murphy, 2009a). As H2O2 can pass easily through mitochondrial membranes, it can act as a redox signal from mitochondria to the rest of the cell and vice versa (Balaban et al., 2005; D'Autreaux and Toledano, 2007; Droge, 2002; Fourquet et al., 2008; Janssen-Heininger et al., 2008; Murphy, 2009a).
Respiratory complex III can also release O2·− into the intermembrane space (St-Pierre et al., 2002; Muller et al., 2004; Han et al., 2001). The intermembrane space enzyme p66Shc (the 66 kDa isoform of the growth factor adapter Shc) can also generate O2·−, which can regulate apoptotic cell death (Giorgio et al., 2005). The O2·− can diffuse from the intermembrane space to the cytosol or be converted to H2O2 by an intermembrane space Cu,Zn-SOD (Okado-Matsumoto and Fridovich, 2001). The Mia40p and Erv1p system of the intermembrane space, which inserts disulphide bonds into intermembrane space proteins during import, also generates H2O2 (Koehler et al., 2006), but the potential of this for redox signalling is unclear.
Matrix H2O2 concentration is further regulated by degradation through peroxiredoxin 3 and 5 (Prx3 and Prx5, respectively) and glutathione peroxidase 1 (Gpx1), with Prx3 being the most significant because of its relative abundance and reactivity (Cox et al., 2010). Prx proteins degrade H2O2 using the mitochondrial thioredoxin 2 (Trx2) system as a reducing source, whereas Gpx1 uses the mitochondrial glutathione (GSH) pool (Cox et al., 2010). During its reaction cycle, dimeric Prx3 forms an inter-subunit disulphide that is reduced back to the dithiol form by Trx2 (Rhee, 2006; Rhee et al., 2001). Exposure to H2O2 can lead to a significant fraction of Prx3 being in the disulphide form at any given time, thereby affecting H2O2 release from mitochondria (Cox et al., 2009; Cox et al., 2008). The activity of Prx3 might also be affected by post-translational modification or by the extent of its oligomerisation (Rhee et al., 2001; Rhee et al., 2005b; Cox et al., 2010). The extent of this H2O2 signal can be modulated both by its production, which is highly responsive to mitochondrial status (Murphy, 2009a), and by the rate of its degradation by matrix peroxidases – predominantly Prx3 – and diffusion into and out of the organelle.
The H2O2 that is produced by one mitochondrion can diffuse to another, coordinating or relaying signals between the organelles (Murphy, 2009a). Additionally, H2O2 can diffuse to mitochondria from the cell surface through the activation of NADPH oxidase (NOX) enzymes by growth factors (Janssen-Heininger et al., 2008; Rhee et al., 2005a; Rhee et al., 2005b).
The main ROS involved in redox signalling to and from mitochondria seems to be H2O2; however, other forms of ROS can also contribute. Nitric oxide (NO) is generated by NO synthases, and can diffuse into mitochondria and modulate mitochondrial function by competing with O2 at respiratory complex IV – thereby slowing respiration – and by the S-nitrosation of mitochondrial thiol groups (Moncada and Erusalimsky, 2002). Iron sulphur centres in proteins such as aconitase can react rapidly with O2·− (D'Autreaux and Toledano, 2007), thereby modifying activity independently of H2O. In addition, O2·− can diffuse from the intermembrane space through the outer membrane voltage-dependent anion channel to the cytosol, where it can act as a redox signal (Zhou et al., 2010). However, as O2·− is shorter lived and less diffusible than H2O2, its signalling roles are thought to be more limited. A number of other redox signals might also be produced within mitochondria, including peroxynitrite (ONOO−) and the products of mitochondrial lipid peroxidation, such as prostaglandin-like molecules and 4-hydroxynonenal (HNE) (Levonen et al., 2004). These compounds can modify mitochondrial protein thiols and, thereby, affect their activity; however, the metabolic significance of these interactions is unclear.
Post-translational protein modification by H2O2 and NO
To act as effective biological messengers, molecules such as H2O2 and NO have to bring about a reversible change in the activity of a protein. Generally, this involves modification of a thiol group on a cysteine residue that mediates redox signalling (Eaton, 2006; Gilbert, 1990; Gilbert, 1995; Schafer and Buettner, 2001). For example, when H2O2 acts as a redox signal it oxidises the thiol group on the target protein to a disulphide group, thereby changing the function of the protein; once the level of H2O2 has returned to basal levels the alteration is reversed and the activity of the protein reverts to its initial level (Beltran et al., 2000; D'Autreaux and Toledano, 2007; Hess et al., 2001; Jacob et al., 2003; Janssen-Heininger et al., 2008; Ziegler, 1985). If the modification is to an active-site thiol, for example oxidation of the crucial thiol in tyrosine phosphatases (Boivin et al., 2010), then the impact on the protein is a clear loss of function. However, thiol oxidation can alter proteins and, thereby, mediate the redox signal in other ways, such as by changing binding affinity to another protein, altering its action as a transcription factor, or by modifying the activity of a transporter or channel (Balaban et al., 2005; D'Autreaux and Toledano, 2007; Droge, 2002; Fourquet et al., 2008; Murphy, 2009a; Rhee, 2006; Rhee et al., 2000).
Generally, in response to H2O2, protein thiol groups will initially form a sulphenic acid (−SOH) (Brennan et al., 2004; Charles et al., 2007; Cotgreave and Gerdes, 1998; Fratelli et al., 2004; Leonard et al., 2009; Seres et al., 1996; Ziegler, 1985; Dalle-Donne et al., 2008; Dalle-Donne et al., 2009), which can occur by direct reaction of H2O2 with the thiolate (−S−). This reaction is dependent on the local environment of the thiol and also its pKa, which can lead to certain thiols being particularly sensitive to oxidation. Once formed, the sulphenic acid can itself be a relevant post-translational modification, or it can form other post-translational modifications by reacting with a GSH to form a glutathionylated protein, with an adjacent thiol to form a disulphide (Brennan et al., 2004; Charles et al., 2007; Dalle-Donne et al., 2009; Delaunay et al., 2002; Hurd et al., 2008), or with amides within the protein to form a sulphenyl amide (Sivaramakrishnan et al., 2010). An alternative route to thiol oxidation during redox signalling is the single-electron oxidation of a thiol to a thiyl radical (−S·), which can then react to form disulphide bonds with GSH or with another protein thiol (Wardman and Von Sonntag, 1995; Winterbourn, 1993).
NO metabolism can also modify a protein thiol group into an S-nitrosothiol group (SNO) in a process known as S-nitrosation or S-nitrosylation (Beltran et al., 2000; Hess et al., 2001; Hogg, 2002; Stamler, 1994; Stamler and Hausladen, 1998). The mechanism of SNO formation in vivo is obscure (Hogg, 2002) but, once generated, the SNO can be passed between thiols by transnitrosation, with the formation and stability of the SNO determined by protein sequence motifs that surround the modified cysteine residue (Benhar et al., 2009; Doulias et al., 2010; Hou et al., 1996; Marino and Gladyshev, 2010; Nikitovic and Holmgren, 1996). In addition, an initial SNO on a protein can be modified into other thiol-based groups, such as disulphide, sulphenic acid or into a glutathionylated protein (Nikitovic and Holmgren, 1996; Stamler et al., 1992).
All of these post-translational modifications can potentially act as ‘redox switches’ (Cabiscol and Levine, 1996; Mallis et al., 2000; Schafer and Buettner, 2001; Zheng et al., 1998), altering the function of a protein and, thereby, enabling it to respond sensitively to the reduction potential of a particular redox couple or to the production of a particular ROS. Although structural alterations brought about by these modifications can potentially have a major effect on protein function, in only a few cases have detailed structural analyses shown clearly how this occurs. To be effective signals, these thiol alterations must be readily reversible. This is achieved by the glutathione-reductase–GSH–glutaredoxin (Grx2) system or by the thioredoxin reductase (TrxR2)–Trx2 system that is present in the mitochondrial matrix (Dalle-Donne et al., 2009; Hurd et al., 2005a; Hurd et al., 2005b; Schafer and Buettner, 2001).
Protein thiols can be modified by a direct reaction with H2O2, independently of bulk changes to the redox state of thiol pools. Alternatively, protein thiol modifications can occur through reactions with another thiol–disulphide redox couple. An example of this is the change in the extent of glutathionylation of particular protein thiols in response to changes in the ratio of glutathione to glutathione disulphide (GSH:GSSG), mediated by Grx2 (Costa et al., 2003; Schafer and Buettner, 2001; Beer et al., 2004). However, as this process requires the GSH pool to be significantly oxidised, this situation probably does not occur under most physiological conditions. Alterations to the redox state of Trx2 might also lead to further modifications to protein thiols, provided that a sufficiently oxidised reduction potential can be achieved by the Trx2 pool. More generally, other dithiol proteins – such as peroxidases with appropriate reduction potentials relative to both oxidants and target proteins – can affect the activity of target proteins by introducing internal disulphides (Delaunay et al., 2002).
There are other potential modes of redox signalling in addition to the reversible modification of protein thiols. Proteins can be modified irreversibly by the alkylation of thiols. This is exemplified in the cytosolic pathway of nuclear factor erythroid 2-related factor 2 (NRF2) and Kelch-like ECH-associated protein 1 (KEAP1) (NRF2–KEAP1 pathway), in which one of the KEAP1 thiol groups can react irreversibly with electrophiles to release the NRF2 transcription factor. NRF2 then translocates to the nucleus where it induces transcription of genes under the control of promoters that contain the antioxidant response element (ARE) (Hayes et al., 2010; Kobayashi and Yamamoto, 2006). Alternatively, other interactions are possible, such as the competition of NO with O2 in binding to respiratory complex IV and, thus, altering mitochondrial respiration and the redox state of the respiratory chain (Brown, 1995; Moncada and Erusalimsky, 2002). These and other modes of redox signalling might complement or extend the central role of reversible thiol oxidation.
Biologically important mitochondrial redox signals
The concept of redox signalling in biology initially emerged from studies on ROS production from NOXs and on the interactions of NO with biological systems (reviewed by, Finkel, 2011; Rhee, 2006; Janssen-Heininger et al., 2008). Since then, mitochondria have emerged as an important node of redox signalling in numerous biologically important areas. Among the most intriguing is the role of mitochondrial ROS in O2 sensing, especially during hypoxia (Guzy and Schumacker, 2006; Guzy et al., 2008; Patten et al., 2010; Brunelle et al., 2005). In this process, it seems that the production of O2·− by the respiratory chain increases under conditions of low O2 levels (Chandel et al., 1998; Chandel et al., 2000; Guzy et al., 2005). The site of the O2·− production is thought to be respiratory complex III, but the mechanism is unclear (Chandel et al., 2000; Guzy et al., 2005). The elevated mitochondrial O2·− is converted to H2O2 in the mitochondrial matrix, followed by diffusion into the cytosol where it stabilises hypoxia-inducible factor-1α (HIF-1α), thus leading to the transcription of genes that enable the cell to respond to hypoxia (Sanjuán-Pla et al., 2005). Redox signalling by mitochondrial ROS is now implicated in a disparate range of biologically important areas, including as a determinant of chronological lifespan (the time cells in a stationary phase culture remain viable) in yeast (Bonawitz et al., 2007; Pan et al., 2011; Bell et al., 2007), a factor controlling lifespan in Caenorhabditis elegans (Lee et al., 2010; Yang and Hekimi, 2010; Schulz et al., 2007; Hekimi et al., 2011), in the regulation of the immune system (West et al., 2011; Zhou et al., 2011; Wang et al., 2010), in angiotensin II signalling (Dai et al., 2011), in insulin secretion (Leloup et al., 2009) and mitochondrial homeostasis (St-Pierre et al., 2006).
How to investigate redox signalling pathways
Although there is considerable evidence indicating the importance of mitochondrial redox signalling, changes in ROS concentration or a thiol modification also occur during pathologies. Consequently, it is imperative not to assume that such events are necessarily evidence of a redox signal, and to show that changes in the levels of a particular ROS and the subsequent modification of target proteins correlate with and are sufficient to explain the biological modification. However, assessing changes in ROS and protein redox modifications in biological systems is technically demanding and requires an understanding of the underlying chemistry (Murphy et al., 2011). Despite this, considerable evidence demonstrates the presence of protein thiols within mitochondria that can be modified by H2O2 and S-nitrosating agents (Chouchani et al., 2010; Hurd et al., 2005a; Hurd et al., 2005b; Hurd et al., 2007; Prime et al., 2009; Sun et al., 2007). There are now a variety of methods that can be used to assess the levels of particular ROS within mitochondria, and these include mitochondria-targeted small-molecule fluorescence probes (Dickinson et al., 2010a; Dickinson et al., 2010b; Robinson et al., 2006), the use of mitochondria-targeted proteins derived from green fluorescent protein – whose fluorescence is redox sensitive (Meyer and Dick, 2010), and mitochondria-targeted mass spectrometry probes that enable mitochondrial ROS levels to be estimated in vivo (Cochemé et al., 2011). The proteins modified and the nature of the thiol modification can also be determined by using a number of redox proteomic techniques (Chouchani et al., 2010; Dahm et al., 2006; Danielson et al., 2011; Taylor et al., 2003; Held et al., 2010; Hurd et al., 2007).
Once the involved cysteine residues have been determined it is vital to quantify the extent of the modification to ensure that it correlates with a change in protein activity that is sufficient to account for the phenotypic change (Murphy et al., 2011). Mass spectrometric techniques to assess this are now available (Danielson et al., 2011; Held et al., 2010). Proteomic approaches have also been extended to in-vivo models and a range of mitochondrial proteins have been identified that have reversible modifications (Burwell et al., 2006; Doulias et al., 2010; Charles et al., 2007; Fratelli et al., 2003; Murray et al., 2011; Schroder and Eaton, 2008; Sun and Murphy, 2010; Nadtochiy et al., 2007). Without such measurements it might be that the changes in the level of the putative signalling ROS and in the protein redox modification merely correlate with the change in activity, rather than cause it.
There is increased recognition that protein modifications that are induced by certain ROS, such as H2O2 and NO, are not solely damaging events in biological systems, but might also be important components of feedback and signalling pathways. Mitochondria are at the heart of metabolism and cell death and are, therefore, important for many physiological pathways. It is also clear that ROS and redox modifications of proteins enable mitochondria to respond to and modulate function(s) of cells and whole organisms. However, despite the development of a more nuanced view of the role of ROS and redox modification in biology, caution is still warranted. This is owing to the technical difficulties in measuring and quantifying ROS and protein redox modifications in biological systems. Consequently, it is important to make sure that any changes measured are responsible for the biological changes and are not merely correlates with no signalling function – such as a response to damage or a repair process.
Often redox signalling is compared, explicitly or tacitly, with signalling by reversible protein phosphorylation. However, it is important to bear in mind that with phosphorylation there is a large thermodynamic driving force for the modification of serine, threonine or tyrosine residues that is channelled and kinetically controlled by tightly regulated kinases. The introduction of a bulky, charged phosphate group has a significant effect on the target protein, resulting in a change in its function or location. The reversal of the modification is also tightly regulated by specific phosphatases. Few redox signalling pathways are as well-defined as established phosphorylation signalling pathways, with most only matching a few aspects. Often, the processes that lead to the redox modifications are less specific as there is no kinase equivalent that can selectively modify proteins, with thiol sensitivity usually owing to the influence of local sequence and structural motifs on the pKa and reactivity of the thiol. Consequently, many thiols are susceptible to redox modification, but only a few are important in genuine signalling pathways. This can lead to all redox changes being interpreted as signalling events through a kind of ‘phosphorylation envy’ that has to be guarded against, so the true significance of redox signalling and modifications in mitochondrial biology can emerge.
This article is part of a Minifocus on Mitochondria. For further reading, please see related articles: ‘PINK1 and Parkin-mediated mitophagy at a glance’ by Seok M. Jin and Richard J. Youle (J. Cell Sci.125, 795-799) and ‘Mitochondria and cell signalling’ by Stephen Tait and Douglas Green (J. Cell Sci.125, 807-815).
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
A high-resolution version of the poster is available for downloading in the online version of this article at jcs.biologists.com. Individual poster panels are available as JPEG files at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.098475/-/DC1
I wrote about ASEA in August, 2012. To quote the company’s website, “ASEA is trillions of stable, perfectly balanced Redox Signaling Molecules suspended in a pristine saline solution—the same molecules that exist in the cells of the human body.” Molecules that supposedly have all kinds of antioxidant benefits for health and for athletic performance through “redox signalling.” They claim it is “a mixture of 16 chemically recombined products of salt and water with completely new chemical properties.” But they never specify exactly which molecules those are, what they mean by balanced, or how they can determine that they remain stable. The product label only lists salt and water. If those 16 recombined molecules are really in the product, the FDA can and should act against them for false labeling.
An ASEA distributor (part of the company’s multi-level marketing cadre) recently wrote an e-mail, not to me, and not to the editors of SBM, but to an assistant editor, to demand that my article be taken down, or that at least the comments for that article be re-opened. Since the e-mail was not sent to me, and I don’t have the writer’s permission, I won’t name him or quote him directly but will paraphrase what he said. He said my article had prevented thousands of people from benefitting from the health effects of ASEA. Thousands? I don’t think I’m that influential; I only wish I were! Anyway, it has not been established that ASEA offers any health benefits. He complains that I don’t have any evidence that ASEA doesn’t work, and of course I don’t. The burden of proof is not on me to prove it doesn’t work, but on those making the claims to prove it does.
He says there is real scientific evidence showing that it does work. My article said there was nothing about ASEA listed in PubMed, and he countered that there are 102 mentions. I was skeptical, so I checked for myself. What I found left me rolling on the floor in paroxysms of laughter.
There are indeed 102 citations on PubMed when you search for “ASEA”:
- 84 of them were listed because they were written by an author whose last name was Asea!
- 2 were about alfalfa extracts
- 1 about acoustics
- 1 about nuclear power plants
- 1 about percutaneous coronary intervention for heart attacks
- 1 about the response of Bacillus subtilis to metal ion stress
- 1 about radiotherapy for glioma
- 1 about coronary angiograms
- 1 about training surgeons
- 1 about ceramics to repair skull bone defects in a rabbit
- 1 about another kind of “ASEA”: Alkaline soluble Trypanosoma cruzi epimastigote antigen
- 1 about balancing corporate power
- 1 about industrial robots
- 1 about the driver’s cab in the Rc5 engine
- 1 about electromyography
- 1 about industrial robots
- 1 about an enzyme in the white bloods cells of cattle
- 1 about back disorders
- 0 (that’s ZERO) about the product ASEA or even with any remote connection to redox signaling
And the dates of these articles go back to the 1950’s. According to the company’s website, ASEA wasn’t invented until 6 years ago.
I am still flabbergasted. What was he thinking? Did he even bother looking to see what the articles were about, or did he simply stop reading after he located the number of citations? Did he think we would take his word for it without looking? Does he think the number of citations for a search word means anything about the efficacy of his product? Apparently he does, since he goes on to cite the 11,121 mentions of “redox signaling” on PubMed. So what? There are 20,292 citations for homeopathy. There are 1,665,853 citations for “bacteria;” perhaps he imagines that citing those numbers would be sufficient to prove that any new antibiotic was effective.
One controlled human study
The company website brags about a 2012 study done in the Appalachian State Human Performance Laboratory in North Carolina. The e-mail writer emphasizes that the laboratory and the researchers were reputable, and that the company agreed to publish the results of the tests regardless of whether the findings were favorable. I don’t question any of that. I do question the results and significance.
It was a double blind crossover study of 20 cyclists. Athletes drank either 4 oz of ASEA or a placebo daily for a week. They measured VO2Max, body composition, and 43 metabolites before and after strenuous exercise. Compared to those on placebo, the group taking ASEA had higher levels of myristic acid, palmitic acid, oleic acid, stearic acid, palmitelaidic acid, capric acid, glycerol, lower levels of 7 amino acids, higher levels of fumarate, citrate, and malate, lower levels of ascorbic acid, fructose and threonic acid, and increases in aminomalonic acid, serum creatinine and urea. These are all reported as “least square mean area” (why?) and no measure of significance is provided (why not?).
They found that ASEA did not cause any changes in creatinine, BUN, bilirubin, alkaline phosphatase, AST and ALT; these are reported in mg/dl and “treatment x time p-values” are provided, ranging from 0.7717 to 0.9971. You will notice that creatinine is listed as both higher and as unchanged: it was higher by least square mean area but not by mg/dl.
The study was not published except in the form of slides and videos on the company website. I don’t know whether it was ever even submitted to a peer-reviewed journal for publication; but if it was, I think any reviewer worth his salt would have sent it back for significant revision. They said they knew they could not publish without first analyzing ASEA to see what was in it, and they said their analysis found “signaling molecules,” one of which was a superoxide. They don’t tell us which superoxide or what the other molecules were. I doubt if that would be enough to satisfy any journal editor. At any rate, before we can say anything about such a study it must be published so other experts can review it and offer comments and criticisms and can attempt to replicate the study if they are interested; that’s how science works. The information that they provide online is insufficient to judge the quality of the study. They don’t describe their methods in the detail expected for a published study, and we don’t know whether subjects could distinguish ASEA from placebo by taste, or what their previous experience and beliefs about ASEA were.
What does it mean?
What does all that data mean? I have no idea. The researchers themselves professed to be completely surprised by their results, which they did not expect from what they knew about the effects of the redox signaling molecules that they presumed were in the product. But of course that didn’t stop them from speculating about ASEA’s mechanism of action and its possible clinical benefits. When a study gives such unexpected results, it is prudent to question whether they might be spurious. There are any number of things that can go wrong in a study to generate false findings.
We need to hear from experts in the field who can comment on what these results might mean and whether the study methodology and statistical analyses were appropriate. This was an unpublished, small, preliminary “pilot” study; this kind of study is all too often followed by larger, better studies that reverse the findings. Studies like this are not sufficient to guide clinical practice; all we can do at this point is to suspend judgment and wait for further evidence. Customers who rely on this evidence might want to ask themselves whether they would want to take a prescription drug if the evidence from human trials consisted of a single trial with 20 patients.
I would want to ask those experts about some additional things. They found elevated levels of free fatty acids (FFAs). Is this a good thing? FFAs are elevated in obesity; they cause insulin resistance and may play a role in coronary atherosclerosis. Elevated levels suppress muscle glucose transport, leading to reduced muscle glycogen synthesis and glycolysis. Lowering FFAs should help treat obesity and type 2 diabetes. If this effect of ASEA is real, wouldn’t it be possibly harmful? How accurate are the measurements? I found one comment that “It is very easy to generate FFAs artefactually by faulty storage or extraction.”
My critic says they have “a boatload” of patents. I guess so, if you define a boatload as 4 (maybe it’s a small boat); but the existence of a patent says nothing about whether the product is effective. The company tells us that the foundational technology for ASEA is completely protected by US patents 5,507,932; 5,674,537; 6,007,686; and 6,117,285. It’s easy enough to look them up by number in the patent database. I did, and I found nothing about ASEA or about generating redox signaling molecules.
- 5,507,932 This patent is for an apparatus for electrolyzing fluids, to produce disinfecting agents such as chlorine and ozone.
- 5,674,537 Electrolyzed saline solution containing concentrated amounts of ozone and chlorine species for in vitro treatment of microbial infections.
- 6,007,686 An apparatus for electrolyzing fluids to disinfect blood, dental drills, and other materials.
- 6,117,285 A system for carrying out sterilization of equipment.
These patents say nothing about ASEA or about a unique proprietary method of producing redox signaling molecules. They simply show that the company has patents on a method of electrolysis that offers some advantages for the production of chlorine and ozone. Whoa! Those substances are toxic. There is no evidence to support their claims that the molecules in ASEA are the same ones as in the human body, that they have been somehow stabilized in solution (this would be very hard to do, since they are very reactive molecules), that the “clusters” described on their website exist, that the solution is “balanced” in any sense except in the trivial one that electrolysis necessarily produces equal numbers of positive and negative ions, that it produces “16 chemically recombined products of salt and water with completely new chemical properties,” or that it has any health benefits for humans.
They claim that ASEA is “Safer than water” (?!) but they have no evidence to support that claim. They list the safety studies here. They are studies in cell culture of hamster ovary cells, bacterial cells, rabbits, several beagle dogs, and some mice. They have not studied safety in humans. If, as I suspect, ASEA amounts to nothing but salt water, adverse effects would not be expected unless large quantities were ingested. Again, I would ask customers if they would be willing to take a prescription drug that had not been shown toxic to animals but that had never been tested for safety in humans.
This study attempted to show that there must be therapeutic molecules in ASEA because it “did something” to 43 metabolites. It reminded me of a study for Vitamin O. The product supposedly contained oxygen, but independent laboratory analysis found no oxygen in it. The company argued that there was so much oxygen in it that it didn’t register on the machines! So they did an experiment to show that there must be oxygen in vitamin O because vitamin O raised the abnormally low blood oxygen pressure (PaO2) of anemic patients (even though PaO2 is not abnormally low in anemia). The study was perhaps the most flawed one I have ever read. They got results that were not only “surprising” but impossible, given what we know about physiology.
The e-mail writer goes on to use the fallacious argument from popularity (lots of happy customers) with no understanding of how people might come to believe an ineffective product is helping them. Apparently he doesn’t realize that water scams and quack remedies all have plenty of testimonials from grateful customers. He accuses SBM of being in the business of publishing lies, and he suggests we are doing it for the money (!?), which is pretty ironic given that he is the one selling ASEA to make money and SBM writers are not paid.
Did I lie? I don’t think so. I may have been guilty of a mis-statement in saying there were “no” placebo-controlled studies; but there certainly were no published placebo-controlled studies, which, for the purpose of scientific evidence, amounts to the same thing. I still have no reason to think ASEA is anything more than expensive water.
Now that I have written a new article, my critic and his fellow ASEA distributors and their customers are welcome to join in the comments. They may want to further demonstrate the level of ignorance that led him to cite the irrelevant 102 PubMed articles. I didn’t particularly want to write about ASEA again, but that was just too delicious not to share. I’m still laughing about it! It’s almost enough to make me wonder if ASEA causes brain damage…