Dr. Naila Rabbani was interviewed by Seth Bittker over email in March 2018 on her recent research on blood and urine-based biomarkers for autism spectrum disorders. Dr. Rabbani is an expert in Translational Medicine at University of Warwick. Her work focuses on disease mechanisms, particularly the study of damage to the proteome by glycation, oxidation and nitration. © 2018
SB: Dr. Rabbani, congratulations on your paper “Advanced glycation endproducts, dityrosine and arginine transporter dysfunction in autism – a source of biomarkers for clinical diagnosis.” What do you see as its most important findings?
NR: Thank you. I believe the most important finding is the emergence of the basis of a blood-based diagnostic test for autism that could be made widely available. This requires validation in a further large group of children with and without autism.
SB: There has been a lot of discussion of a need for discriminatory biomarkers for ASD. A lot of biomarker research has focused on neurological biomarkers such as electroencephalography or eye-tracking. Other research, such as yours, focuses on levels of compounds in blood or urine. Can you comment on the relative utility of these two types of biomarkers?
NR: A blood-based test or urine-based test may be performed in the clinical chemistry departments of well-equipped hospitals and test results may be interpreted without the need for a specialist in child development.
SB: It is interesting to compare some of the findings in your research to similar studies. You find lower arginine in urine in ASD with statistical significance. West, et al. found decreased homocitrulline in the plasma in ASD with great statistical significance (West et al., 2014). Is there a connection between West’s findings on homocitrulline and your findings on arginine?
NR: Actually, we found increased arginine in plasma and urine of children with autism, compared to those without. Renal clearance of arginine was lower in children with autism than those without. This relates to dysfunctional handling of arginine by the kidney. There is no link of homocitrulline to arginine. We used the most robust analytical method for mass spectrometry metabolite analysis – a technique called stable isotopic dilution analysis. The studies by West et al. should preferably be repeated using this method for surety of the outcome they found.
Our approach of studying spontaneously modified or “damaged” proteins and amino acids is more powerful than previous studies of unmodified proteins and amino acids. For proteins, this is because the combination of changes in protein modifications is likely reflecting changes in processes that drive ASD or are markers of them. For damaged amino acids, these are released from modified proteins by proteolysis. Unlike unmodified amino acids, when released they are not re-incorporated into proteins and accumulate – providing the basis of the diagnosis of ASD.
SB: Some research suggests that ASD is associated with methylation deficits and sulfation deficits (James et al., 2004; Adams et al., 2011). Is there a connection between the markers you have found in ASD and methylation or sulfation deficits?
NR: No connection that I am aware of.
SB: A lot of research has shown ASD is associated with increased autoimmune activity (Mostafa et al., 2014; Careaga et al., 2013 for example). Do you see some of the biochemical markers you have found as potentially a result of autoimmune activity? Do you see some of the biochemical markets you have found as increasing the risk of development of autoimmunity?
NR: Dityrosine is formed mainly by the enzyme DUOX which has an important role in host immunity. Increased DUOX may be associated with autoimmunity in some cases.
SB: There is a lot of research on generic abnormalities and genetic polymorphisms that increase risk of ASD. Some of this research is on genes that affect neuronal development. Your findings do not seem to be directly connected to neuronal development. Is there a connection? If not, does this suggest development of ASD in some cases is dependent upon two hits (susceptibility to neuronal dysfunction in combination with metabolic dysfunction)?
NR: We were encouraged in that our findings support previous evidence from genetic studies – particularly those linked to amino acid transport proteins, arginine transport proteins particularly. Genetic polymorphism of arginine transport proteins affects all cells in the body, of course, and a non-invasive way to assess change in amino acid transport is to measure renal clearance which, with normal glomerular filtration rate, is influenced by amino acid re-uptake from renal tubules mediated by amino acid transporters. The decreased renal clearance of arginine and Nω-carboxymethylarginine (CMA) in children with ASD is consistent with a change in function of arginine transporter proteins as may occur in amino acid transporter genetic polymorphism.
Also, although we studied modifications of plasma protein in part of our study, the major protein in plasma – albumin – exchanges with albumin in cerebrospinal fluid. So some of the modifications we find of protein in plasma may have occurred whilst that protein was in cerebrospinal fluid in the ventricles of the brain. The increased CMA free adduct found in plasma filtrate may have originated in part from proteolysis of increased CMA-modified protein in the brain. Further studies are required to follow up the mechanisms that underlie these interesting changes found in ASD.
SB: Does your research on advanced glycation endproducts and ASD suggest anything about diets that should be researched further in cases of ASD?
NR: Advanced glycation endproducts (AGEs) in the diet are absorbed into the body as glycated amino acids. These are not incorporated into our proteins; they are rather excreted by the kidney. This likely poses limited challenge to body function with the changes found of renal function in ASD. The changes of AGE content of plasma protein reflects endogenous AGE formation – formation of AGEs in the body. For AGEs in the diagnostic algorithm, CML and CMA were increased in ASD and a further AGE, 3DG-H, was decreased in ASD. So, there is not a general increase in AGEs but rather an increase in AGEs formed by a specific precursor – glyoxal, and decrease of AGE formed by a different precursor – 3-deoxyglucosone (3-DG). We interpret this as likely increased glyoxal exposure from lipid peroxidation in ASD and increased activity of enzymes that metabolize 3-DG in ASD – which may be a protective response to increased 3-DG. The latter is a metabolic state called dicarbonyl stress. This complexity of response likely makes the characteristics of the changes specific to ASD and may account for the high accuracy of the diagnostic test based thereon. Again, further studies are required to follow up the mechanisms that underlie these interesting changes found in ASD.
SB: Are there other environmental factors that might contribute to the biochemical gestalt your research suggests is characteristic of ASD?
NR: We have not studied this and therefore have no comment.
SB: Was it difficult to get funding for this research?
NR: The study was part funded by grants obtained by our collaborators at the University of Bologna and part-funded by our own research group resources at the University of Warwick. Innovative research of this type is always difficult to fund via peer review system of obtaining grant funding. We hope now the advances made have been published and publicised by the media, funding bodies and reviewers supporting studies of ASD will be aware that there are initial-stage data and findings of potential diagnostic importance to build on and funding for further investigations will be forthcoming in due course. Our innovative research will continue, I would like to repeat and validate our blood and urine test and translate it to benefit parents, grandparents and autistic community, who have been so desperately waiting for blood test like this for potentially a rapid diagnosis of their loved ones.
SB: Do you have any suggestions on how those who fund ASD research should determine which studies that they fund?
NR: I encourage all research funding bodies and funding application reviewers to maintain an openness to new developments, findings and ideas that may make a step-advance in diagnosis and understanding of ASD. If something new appears that may not fall within the current strategy of funding bodies, then I encourage sufficient flexibility of strategic considerations to adapt to change and support follow-up of the new findings in service and benefit of the ASD community.
SB: Is there anything else that you would like people to understand about this research?
NR: I would like to thank the ASD community for the many correspondences of support and encouragement given to me and my colleagues since our recent publication. We will be working to take this initial step further for improved diagnosis to support clinical care and services for ASD. I will be setting up a www page to provide further information on our current study and further advances and for those who may like to support our further research.
This is the first-time low-level damage to proteins and amino acids in blood and urine have been studied in autism. The research found a link between ASD and damage to proteins in blood plasma by oxidation and modifications by glyoxal and sugar. The changes found suggest a link to low level inflammation, lipid peroxidation and a protective response to potentially damaging sugars (dicarbonyl stress). The most reliable of the tests used is protein in blood plasma.
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