Combined¹H-NMR and¹H–¹³C HSQC-NMR to improve urinary screening in autism spectrum disorders

Abstract

Autism spectrum disorders (ASD) are neurodevelopmental diseases with complex genetic and environmental etiological factors. Although genetic causes play a significant part in the etiology of ASD, metabolic disturbances may also play a causal role or modulate the clinical features of ASD. The number of ASD studies involving metabolomics is increasing, and sometime with conflicting findings. We assessed the metabolomics profiling of urine samples to determine a comprehensive biochemical signature of ASD. Furthermore, to date no study has combined metabolic profiles obtained from different analytical techniques to distinguish patient with ASD from healthy individuals. We obtained (1)H-NMR spectra and 2D (1)H-(13)C HSQC NMR spectra from urine samples of patients with ASD or healthy controls. We analyzed these spectra by multivariate statistical data analysis. The OPLS-DA model obtained from (1)H NMR spectra showed a good discrimination between ASD samples and non-ASD samples (R(2)Y(cum) = 0.70 and Q(2) = 0.51). Combining the (1)H NMR spectra and the 2D (1)H-(13)C HSQC NMR spectra increased the overall quality and predictive value of the OPLS-DA model (R(2)Y(cum) = 0.84 and Q(2) = 0.71), leading to a better sensitivity and specificity. Urinary excretion of succinate, glutamate and 3-methyl-histidine differed significantly between ASD and non-ASD samples. Urinary screening of children with neurodevelopmental disorders by combining NMR spectroscopies (1D and 2D) in multivariate analysis is a better sensitive and a straightforward method that could help the diagnosis ASD.

Metabolomics of autism spectrum disorders: early insights regarding mammalian-microbial cometabolites

“…Other possible causes influencing the amino acid metabolism in autism are Down's syndrome, Fragile X syndrome, epilepsy, diet regime or food habits. For example, in ASD patients, two metabolomics studies reported a decreased concentration in urinary glutamate [63,69] and one more study found an increased glutamate blood concentration [70]. These findings comply previous results published in the literature and recently summarized in a brief review [81].…”

“…The increased level of β-hydroxybutyric acid, known as ketone body when it accumulates in blood, was correlated to deficiency in cytochrome oxidase enzymes of the electron transport system [67]. Citric acid was found increased in the urine of ASD patients in four metabolomics studies [65][66][67]69], suggesting the presence of an amino acid deficiency or problems with protein metabolism [67]. Surprisingly, in a study they were observed decreased blood citric acid levels in autistic individuals without giving an account of any evidencebase postulation on this finding [70].…”

“…Therefore there is the need to correlate previous results obtained in longitudinal cohorts [86,87] These approaches are commonly known as chemometric methods. Table 3 contains a list of basic requirements for standardization, indicating whether or not (yes/no) each item has been fulfilled by the eight most relevant metabolomics studies in autism [63][64][65][66][67][68][69][70]. This table makes evident the high heterogeneity of methodologies used to obtain results in autism and this corresponds to a very low level of standardization.…”

“…In autistic subjects, the increased urinary excretion rate of an indigestible carbohydrate, namely D-mannitol [67], supports the so-called 'leaky gut hypothesis' [72], based on the observation that the intestinal mucosa is abnormally permeable in autism. Several studies [63,65,66,69] found changes in the levels of aromatic compounds such as hippurate (a glycine conjugated compound) and phenyacetylglutamine (PAG), suggesting the involvement of gut microflora in autism. Indeed, hippurate and PAG are generated from their precursors, benzoic acid and phenylacetic acid, respectively, by bacterial metabolism in the intestinal lumen.…”

Metabolomics Study of Urine in Autism Spectrum Disorders Using a Multiplatform Analytical Methodology

“…14, 15 The supernatant (500 μL) was then added with H NMR spectra were obtained as previously described using a Bruker DRX-500 spectrometer (Bruker SADIS, Wissembourg, France) operating at 500 MHz, using a "cpmg" pulse program.…”