UA-45667900-1
Showing posts with label Autism Biomarkers. Show all posts
Showing posts with label Autism Biomarkers. Show all posts

Wednesday, 21 October 2015

Biomarkers in Autism






This post has been sitting unfinished for a while, so I decided to publish it before I forget all about it

The two papers discussed today really confirm much of what we have already established in this blog, but they are very useful as a recap and for those with limited time.

The first paper is extremely comprehensive and, if you go through it very slowly, really tells you much of what you need to know about the biology of autism.  It is some wonder that so few clinicians are aware of these findings.



Abstract

Autism spectrum disorders (ASDs) are complex, heterogeneous disorders caused by an interaction between genetic vulnerability and environmental factors. In an effort to better target the underlying roots of ASD for diagnosis and treatment, efforts to identify reliable biomarkers in genetics, neuroimaging, gene expression, and measures of the body’s metabolism are growing. For this article, we review the published studies of potential biomarkers in autism and conclude that while there is increasing promise of finding biomarkers that can help us target treatment, there are none with enough evidence to support routine clinical use unless medical illness is suspected. Promising biomarkers include those for mitochondrial function, oxidative stress, and immune function. Genetic clusters are also suggesting the potential for useful biomarkers.



Here are the key parts; I do suggest you read the full text of the paper.


Metabolic Biomarkers


There are no autism-defining, metabolic biomarkers, but examining the biomarkers of pathways associated with ASD can point to potentially treatable metabolic abnormalities and provide a baseline that can be tracked over time. Each child may have different metabolic pathologies related to SNPs, nutrient deficiencies, and toxic exposures. Examples of metabolic disorders that can lead to an autistic-like presentation include phenylketonuria (PKU) (37), disorders of purine metabolism (38), biotinidase deficiency (39), cerebral folate deficiency (40), creatine deficiency (41), and excess propionic acid (which is produced by Clostridium) (42, 43).

A recent review assessed the research on physiological abnormalities associated with ASD (44). The authors identified four main mechanisms that have been increasingly studied during the past decade: immunologic/inflammation, oxidative stress, environmental toxicants, and mitochondrial abnormalities. In addition, there is accumulating research on the lipid, GI systems, microglial activation, and the microbiome, and how these can also contribute to generating biomarkers associated with ASD.

The brain is highly vulnerable to oxidative stress (51), particularly in children (52) during the early part of development (47). As environmental events and metabolic imbalances affect oxidative stress and methylation, they also can affect the expression of genes.

Several studies have detected altered levels of a large collection of substances in body-based fluids from ASD subjects compared to controls (e.g., serum, whole-blood, and CSF) (53). These findings encompass either of two main disease-provoking mechanisms: a CNS disorder that is being detected peripherally [e.g., serotonin and its metabolites, sulfate (54), low platelet levels of gamma-aminobutyric acid (GABA) (55), low oxytocin (which affects social affiliation) (56), and low vitamin D levels (57, 58)] or a systemic abnormality that has repercussions in the brain (59).

Oxidative stress markers

Oxidative stress can be detected by studying antioxidant status, antioxidant enzymes, lipid peroxidation, and protein/DNA oxidation, all of which have been found to be elevated in children with autism (Table (Table2).2). Different subgroups of children with ASD have different redox abnormalities, which may arise from various sources



Measurements of antioxidant status include measurement of glutathione, the primary antioxidant in the protection against oxidative stress, neuroinflammation, and mitochondrial damage (68, 69). Glutathione is instrumental in regulating detoxification pathways and modulates the production of precursors to advanced glycation end products (AGEs) (70). Measuring reduced glutathione, oxidized glutathione, or the ratio of reduced glutathione to oxidized glutathione helps determine the patient’s oxidation status. In many patients with ASD, the ratio of reduced glutathione to oxidized glutathione is decreased, indicating a poor oxidation status

The enzyme glutathione peroxidase has been used as a marker and is typically reduced. There are mixed results concerning the enzyme levels of superoxide dismutase (SOD) (72). Other markers for glutathione inadequacy include alpha hydroxybutyrate, pyroglutamate, and sulfate, which can be assessed in an organic acid test. Lipid peroxidation refers to the oxidative degradation of cell membranes. There is a significant correlation between the severity autism and urinary lipid peroxidation products (67), which are increased in patients with ASD

Plasma F2t-Isoprostanes (F2-IsoPs) are the most sensitive indicator of redox dysfunction and are considered by some to be the gold standard measure of oxidative stress (73). They are increased in patients with ASD and are even higher when accompanied by gastrointestinal dysfunction (73).

Decreased levels of major antioxidant serum proteins transferrin (iron-binding protein) and ceruloplasmin (copper binding protein) have been observed in patients with ASD. The levels of reduction in these proteins correlate with loss of previously acquired language (47) although there are mixed reviews of the significance of this (66).


Plasma 3-chlortyrosine (3CT), a measure of reactive nitrogen species and myeloperoxidase activity, is an established biomarker of chronic inflammatory response. Plasma 3CT levels reportedly increased with age for those with ASD and mitochondrial dysfunction but not for those with ASD without mitochondrial dysfunction (65).

3-Nitrotyrosine (3NT) is a plasma measure of chronic immune activation and is a biomarker of oxidative protein damage and neuron death. This measure correlates with several measures of cognitive function, development, and behavior for subjects with ASD and mitochondrial dysfunction but not for subjects with ASD without a mitochondrial dysfunction (65).


Mitochondrial dysfunction markers

Mitochondrial dysfunction is marked by impaired energy production. Some children with ASD are reported to have a spectrum of mitochondrial dysfunction of differing severity (44) (Table (Table3).3). Mitochondrial dysfunction, most likely an early event in neurodegeneration (76), is one of the more common dysfunctions found in autism (77) and is more common than in typical controls (78). There is no reliable biomarker to identify all cases of mitochondrial dysfunction (79). It is possible that up to 80% of the mitochondrial dysfunction in patients with both ASD and a mitochondrial disorder are acquired rather than inherited (44).

Mitochondrial dysfunction can be a downstream consequence of many proposed factors including dysreactive immunity and altered calcium (Ca2+) signaling (80), increased nitric oxide and peroxynitrite (68), propionyl CoA (81), malnutrition (82), vitamin B6 or iron deficiencies (83), toxic metals (83), elevated nitric acid (84, 85), oxidative stress (86), exposure to environmental toxicants, such as heavy metals (8789), chemicals (90), polychlorinated biphenyls (PCBs) (91), pesticides (92, 93), persistent organic pollutants (POPs) (94), and radiofrequency radiation (95). Other sources of mitochondrial distress include medications such as valproic acid (VPA), which inhibits oxidative phosphorylation (96) and neuroleptics (97, 98).

Markers of mitochondrial dysfunction include lactate, pyruvate and lactate-to-pyruvate ratio, carnitine (free and total), quantitative plasma amino acids, ubiquinone, ammonia, CD, AST, ALT, CO2 glucose, and creatine kinase (CK) (44). Many studies of ASD report elevations in lactate and pyruvate, others report a decrease in carnitine, while others report abnormal alanine in ASD patients (44) or elevations in aspartate aminotransferase and serum CK (99). Increases in lactate are not specific and may only occur during illness, after exercise or struggling during a blood draw (100).


Rossignol and Frye (44) recommend a mitochondrial function screening algorithm. This includes fasting morning labs of lactate, pyruvate, carnitine (free and total), acyl carnitine panel, quantitative plasma amino acids, ubiquinone, ammonia, CK, AST/ALT, CO2, and glucose (44). The interpretation of such a panel and the indications for specific treatments has not yet been established.

Methylation

The methylation pathway provides methyl groups for many functions, including the methylation of genes, which can result in the epigenetic changes of turning genes on and off (Table (Table4).4). This transfer occurs when S-adenosylmethionine (SAM) donates a methyl group and is transformed to S-adenosylhomocysteine (SAH). SAH can be transferred to homocysteine, which can either be re-methylated to methionine or be transferred by the sulfuration pathway to cysteine to create glutathione. With increased oxidative stress, SAH might be diverted away from the methylation pathway to the sulfuration pathway in order to make more glutathione. This will result in less methionine and less methylation ability.

A marker of methylation dysfunction is decreased SAM/SAH ratio in patients with ASD. Fasting plasma methionine decreases since through SAM it is the main methyl donor. Fasting plasma cysteine, a sulfur containing amino acid is the rate-limiting step in the production of glutathione and is significantly decreased. Plasma sulfate is decreased, which may impair detoxification pathways. Homocysteine is generally increased, but the studies are mixed (66). Vitamin B12 and folate are required for the methylation pathway. The MTHFR genetic SNP is reported to heavily influence the methylation pathway (66).

Immune dysregulation


Cytokine evaluation


Chronic inflammation and microglia cell activation is present in autopsied brains of people with ASD (101, 102) (Table (Table5).5). Factors that increase the risk of activating brain microglia include traumatic brain injury (TBI) (103) reactive oxygen species (104) and a dysfunctional blood brain barrier (105). The blood brain barrier can be compromised by oxidative stress (106), acutely stressful situations (107), elevated homocysteine (108), diabetes (109), and hyperglycemia (110). Cytokines can pass through a permeable blood brain barrier and start this process (111). Hence, cytokines can serve as a marker of the immune dysregulation, which can further complicate ASD.


Autoimmunity and maternal antibodies


Autoimmune autistic disorder is proposed as a major subset of autism (118), and autoimmunity may play a role in the pathogenesis of language and social developmental abnormalities in a subset of children with these disorders (119). There are many autoantibodies found in the nervous system of children with ASD who have a high level of brain antibodies (120, 121). These can be measured as biomarkers in this subset of ASD patients. The anti ganglioside M1 antibodies (122), antineuronal antibodies (123), and serum anti-nuclear antibodies (123, 124) correlate with the severity of autism. Other autoantibodies postulated to play a pathological role in autism include: anti neuron-axon filament protein (anti-NAFP) and glial fibrillary acidic protein (anti-GFAP) (125), antibodies to brain endothelial cells and nuclei (119), antibodies against myelin basic protein (126, 127), and anti myelin associated glycoprotein, an index for autoimmunity in the brain (128). BDNF antibodies were found higher in ASD (129), and low BDNF levels may be involved in the pathophysiology of ASD (130).

Antibodies in patients with autism are found to cells in the caudate nucleus (131), cerebellum (132, 133), hypothalamus and thalamus (121), the cingulate gyrus (134), and to cerebral folate receptors (135). Children with cerebellar autoantibodies had lower adaptive and cognitive function as well as increased aberrant behaviors compared to children without these antibodies (132).

Mother’s immune status


Research studies indicate an association between viral or bacterial infections in expectant mothers and their ASD offspring (136, 137). Maternal antibodies cross the underdeveloped blood brain barrier of the fetus (138) leading to impaired fetal neurodevelopment and long-term neurodegeneration, neurobehavioral, and cognitive difficulties (139).

Dysbiosis


When the gut becomes inflamed, it breaks down and becomes permeable, sometimes referred to as dysbiosis. Dysbiosis is reported to be an upstream contributing factor to autoimmune conditions and inflammation. Markers under consideration include circulating antibodies against tight junction proteins, LPS, actomyosin (145) calprotectin (146), and lactoferrin (147). Dysbiosis was found in 25.6% of patients with ASD (148). It is proposed to have a direct effect on the brain as it is a hypothesized source of inflammation (149151) and autoimmunity (152, 153), possibly through molecular mimicry (154). Diet is one source of dysbiosis (155).


Amino acids and neuropeptides


Platelet hyperserotonemia is considered one of the most consistent neuromodulator findings in patients with ASD (Table (Table6).6). As for other neuropeptides, a recent review reported approximately 15 components that are altered in ASD compared to controls (53). Among them, interesting research has been done on glutamate, GABA, BDNF, and dopamine and noradrenaline systems. A recent study reported a positive correlation between severity of clinical symptoms and plasma GABA levels in patients with ASD, supporting the idea of a disrupted GABAergic system (156).

 

Fatty acid analysis


Abnormal fatty acid metabolism may play a role in the pathogenesis of ASD and may suggest some metabolic or dietary abnormalities in the regressive form of autism (42, 157). There is evidence of a relationship between changes in brain lipid profiles and the occurrence of ASD-like behaviors using a rodent model of autism (42). Hyperactivity in patients was inversely related to the fluidity of the erythrocyte membrane and membrane polyunsaturated fatty acid (PUFA) levels (158). Imbalances of membrane fatty acid composition and PUFA loss can affect ion channels and opiate, adrenergic, insulin receptors (159) and the modulation of (Na + K)-ATPase activity (160). Analysis of red blood cell membrane fatty acids is a very sensitive indicator of tissue status and may reflect the brain fatty acid composition (161).
Seventeen percent of children with ASD manifest biomarkers of abnormal mitochondrial fatty acid metabolism, the majority of which are not accounted for by genetic mechanisms (162). Patients with ASD had reduced percentages of highly unsaturated fatty acids (163) and an increase in ω6/ω3 ratio (158).

Biomedical Interventions


There are no published studies of interventions for ASD that use neuroimaging or genetic biomarkers in a prospective manner to guide treatment. Biomedical interventions based on body fluid/product biomarkers have been used in a small but growing numbers of well designed, published studies. Several recent reviews summarize these.

+ + +



If you have managed to digest all of that information, here is another very interesting paper.

The researchers are, as so often, from Johns Hopkins.  This time they propose an idea to simplify the understanding of the bewildering number of autism sub-types.

I have frequently commented in this blog that in many identified underlying dysfunctions, being hyper (too much) or hypo (two little) causes the same effect, i.e. autism.

They split autism into:-

·        hyper-active pro-growth signaling pathways (e.g. big heads)
·        hypo-active pro-growth signaling pathways  (e.g. small heads)


So the first question is whether the patient is type A or type B.









It is definitely a step forward in simplifying what is going on, so that one day a clinician, without being a Nobel Laureate, could treat autism without just using trial and error.  If the clinician had also read, and understood, the first paper, he/she really would be able to help the patient.






The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions.









Monday, 30 September 2013

Biomarkers in Autism: Mercury – Science, Bad Science & GSH (again)

You do not need to have any particular view about vaccines and autism; but there are some very strange connections between mercury and autism.

I came back to look at this subject, having noticed that one of the more rational/objective researchers included a chelating agent in his patent for autism treatment.   Chelating agents remove heavy metals like mercury or lead from the body, but they also remove important elements like calcium.  Very high or low levels of electrolytes like Ca or K can kill you.

In 2006 clinical trials on chelation therapy in autism were halted by the US National Institute of Health on “safety reasons”.  But in 2012, a much bigger 5 year long, $30 million study called Trial to Assess ChelationTherapy (TACT) in coronary heart disease reported back that this “fringe” therapy did indeed work, though for reasons unknown.


The autism trial was to use a chemical called DMSA,  while the coronary heart disease trial used a chemical called EDTA.  The 5 year trial appeared to show EDTA was safe.


Measuring Mercury

There are various ways of measuring for mercury; you can measure for it directly in urine, blood, hair and even teeth.  You can also measure for biomarkers of mercury and the popular one is called Porphyrin Testing.

The problem is that if you have been subject of some serious heavy metal contamination the metal may no longer be in your blood or urine in elevated levels.  This is why forensic science laboratories look at hair and teeth.

At this point the bad science and the science start to get mixed up.  There is a chemical called precoproporphyrin, an atypical porphyrin previously identified only in adult humans and animals with prolonged exposure to Mercury or compounds containing mercury.  It is often present in substantial concentrations in urine of younger children with autism.

This has created a nice business with laboratories charging $120 to measure porphyrin in the urine of autistic children.  A handful of researchers keep writing studies about mercury in autism, using porphyrin to “measure” them.

One of the labs used is surprisingly in France.  It seems many US citizens are mailing samples to Laboratoire Philippe Auguste in Paris.

But, at the same time, another group of scientists take the opposite approach and say that urinary porphyrins are biomarkers of autistic spectrum disorder, because a subset of people with ASD have disordered porphyrin excretion as a metabolic characteristic.  They have gone so far as to patent their idea as a test for autism.  By this logic paying $120 to test a kid known to have ASD would be pretty pointless.


The researcher suggests that the elevated Urinary porphyrins have nothing to do with mercury at all.


… Several possibilities might account for these differences. Not to be bound by theory, Hg exposure appears unlikely to play a role in this effect, because no significant differences were observed between NT and AUT subjects for indices of past exposure to Hg from dental or medical sources, as reported by parents/caregivers. Additionally, urinary Hg concentrations, measures of recent Hg exposure, were very low among all subjects in this study (Table 2), and no significant differences between diagnostic groups were observed …


… the present findings indicate that porphyrin metabolism, particularly in preadolescent children, may be too disordered or differently regulated to permit detection of the Hg-mediated changes in urinary porphyrin excretion that are apparent in adult subjects …


… another factor that may account for the differences in urinary porphyrin levels between AUT and NT children is mitochondrial dysfunction, a disorder commonly associated with autism …


Where is the Mercury coming from?

The sources put forward as to where the mercury is coming from include:-

·        Mother’s dental fillings containing mercury

·        Any amalgam fillings the child has

·        Mercury in the environment

·        Mercury in vaccines

If your body is unable remove mercury as fast as it is absorbing it, then the total amount of mercury in your body will increase.  So it is your cumulative past exposure, minus what you have removed, that is the key figure.

The body’s main antioxidant, glutathione (GSH), is its key resource to deal with disposing of heavy metals.  It has been established for years that GSH levels are reduced in almost all cases of autism.  Incidentally, GSH levels are also reduced in old age and so those subjects in the TACT clinical trial for chelation in heart disease that benefited, did do (according to Peter) because the chelator is an antioxidant.  It lowered their oxidative stress and raised their GSH level.


Mercury in Hair Samples

An interesting study measured the level of mercury in babies’ first haircuts.  This is about when the baby is 17 months old.

The study showed much lower levels of mercury in the ASD babies than in the control babies.  This is probably the opposite of what you might have expected.  There is also a nice chart correlating the level of mercury in the control babies with the number of amalgam fillings in the mother.


The authors proposed that the kids with ASD must have higher levels of mercury in their bodies, because they are unable to eliminate mercury like typical children.

“If reduced overall mercury elimination is related to hair elimination, then autistic infants will retain significantly higher levels of mercury in tissue, including the brain, than normal infants.”



  
A later study has some equally surprising findings.  The study in Poland, looked at kids aged 3-4 and also 7-9.  They found, as in the baby study, that the youngest kids had lower levels of mercury in their hair than the typical kids.  But the older kids had higher mercury levels in their hair than the kids in the control group. 


The conclusion was that:-
The results suggest that autistic children differ from healthy children in metabolism of mercury, which seems to change with age.

Mercury in baby teeth

 So now we come to teeth.  If the ASD kids have low mercury, it will be claimed that this means they must have high internal levels since they have not eliminated it in their teeth.  If they have high mercury then they will say that this proves there is a high level of mercury in kids with ASD.  Read on and find out.

Well the study tells us that baby teeth are a good measure of cumulative exposure to toxic metals during fetal development and early infancy.  They found that 6 year old children with autism had twice as much mercury in their teeth as neurotypical children.



This study determined the level of mercury, lead, and zinc in baby teeth of children with autism spectrum disorder (n = 15, age 6.1 +/- 2.2 yr) and typically developing children (n = 11, age = 7 +/- 1.7 yr). Children with autism had significantly (2.1-fold) higher levels of mercury but similar levels of lead and similar levels of zinc. Children with autism also had significantly higher usage of oral antibiotics during their first 12 mo of life, and possibly higher usage of oral antibiotics during their first 36 mo of life. Baby teeth are a good measure of cumulative exposure to toxic metals during fetal development and early infancy, so this study suggests that children with autism had a higher body burden of mercury during fetal/infant development. Antibiotic use is known to almost completely inhibit excretion of mercury in rats due to alteration of gut flora. Thus, higher use of oral antibiotics in the children with autism may have reduced their ability to excrete mercury, and hence may partially explain the higher level in baby teeth. Higher usage of oral antibiotics in infancy may also partially explain the high incidence of chronic gastrointestinal problems in individuals with autism.


How much Mercury is bad for you?

Mercury is definitely not good for you, but just how much is actually bad for you?

Eating a lot of fish will raise maternal levels of mercury, so in the US women are advised to eat less fish during pregnancy.

In the Seychelles (islands in the Indian Ocean) the diet included 10 times as much fish and since they eat big fish, mercury consumption is 20 times higher.  The level of vaccination was near 100% and the vaccines contained thimerosal.



Using linear and nonlinear regression analyses, the researchers found no consistent correlation between prenatal exposure to methyl mercury and scores on ASD screening instruments.

Parent feedback

If you look on the web, it is pretty clear that many parents think their chelation therapy had a positive impact.  There is even a very unscientific survey showing this somewhere; I cannot find it today.


Since the chelation is like a big anti-oxidant infusion, I would expect to see a big positive improvement, regardless of whether mercury has anything at all to do with it.

Big Sceptics

There are some big sceptics about chelation.  Here is one site called chelation watch
and here is an interesting article by a Doctor who followed ”his dark side” into the world of alternative therapy and emerged a big sceptic.

James R. Laidler, MD    -  My Involvement with Autism Quackery

My personal journey through the looking glass has ended. I stepped into “alternative” medicine up to my neck and waded out again, poorer but wiser. I now realize that the thing the “alternative” practitioners are really selling is hope—usually false hope—and hope is a very seductive thing to those who have lost it.

Other research

There is plenty of other research on the subject of my post.  Normally you can tell by who funded the study or who worked on it, what the likely conclusion is to be.



This paper again shows that urinary porphyrins are a biomarker for autism, rather than mercury.


This paper repeats the story about urinary porphyrins indicating high mercury in autism  



Conclusion

If the US National Institute of Health removed its ban on the clinical trial of chelation in autism, then there would be some high quality facts to judge.  Sadly, this all seems to be linked to “big brother” trying to halt the debate about autism and vaccinations, all for the very sound reason of public health.

I think it is quite possible that the culprit is oxidative stress and low GSH and that the bizarre results of mercury levels in hair, teeth and urine are in fact no more than a consequence of low levels of GSH.  The oxidative stress is clearly damaging, perhaps the slightly elevated levels of heavy metals are themselves harmless.

Perhaps the best thing would be to measure the level of GSH (GSH redox) in babies, children and then again after middle age.  High levels of oxidative stress, whether linked to autism or other conditions could then be treated.

There is a cheap and effective antioxidant called NAC (N-acetyl cysteine), it is known to raise GSH.  If you want to call it a chelating agent, you would also be correct.

Since mercury is known to be a very harmful substance, we should of course try to minimize it in humans.