The total protein concentration and aminoacis profile in urine showed to be good cantidates for biomarkers or managment of individuals with TEA, these data were a stimulating startpoint for populational studies that bring new information for clinical use.
Autism spectrum disorder (ASD) is a developmental disorder characterized by conditions that involve areas of social interaction, communication and behavior as well as sensory sensitivity. At the beginning of the eighties researchs indicated the ASD of association with changes in protein metabolism and amino acids. To identify protein and amino acid profile in the urine of children with ASD and compare with a control group of urine samples. Prospective cross-sectional study, with a case-control design. The cases were children (n = 22) with ASD, male in the age group of 3 to 10 years who were compared with children (n = 22) with typical development, matched by sex and age. The quantification of total proteins was performed using the Bradford method and determination of the amount and amino acid composition by ultra-efficient liquid chromatography (CLUE). The results showed changes in the concentration of protein and amino acids arginine, glycine, leucine, threonine, aspartic acid, alanine, histidine, tyrosine in the urine of children with ASD. The total protein concentration and amino acid profile in urine are good candidates for biomarkers for individuals with ASD, but for this to be concrete, there is a need to develop studies with a larger number of participants.
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Methods A total of 44 children were recruited. Of these, 22 boys diagnosed with ASD and 22 boys with typical development. Urine collection / preparation The first urine of the day was collected and stored in a sterile universal collector (temperature of - 80 ° C). The samples were slowly thawed at a temperature of 4 ° C, homogenized, filtered through a 45 μm PVDF filter (Merck Millipore S / A ®, Cotia, SP, Brazil) . Protein quantification of Bradford method The measurement of the total protein concentration was performed using the Bradford method (1976). The BioRad protein assay (Bio-Rad Laboratories ®, Richmond, CA, USA) was used in the analysis bovine serum albumin (BSA) (Sigma-Aldrich, São Paulo, SP, Brazil) was adopted as a reference standard. The concentration of the samples was estimated by comparison with a standard curve of BSA (1.0 - 0.8 - 0.6 - 0.4 - 0.2 - 0.1 - 0.05 mg / mL), plotted and analyzed by linear regression. Separation and determination of the amino acid composition by ultra-efficient liquid chromatography (CLUE) For the analysis for determining concentration of amino acids were used Ethanol and acetonitrile purchased from JT Baker (Xalostoc, Mexico) trietilamine, phenylisothiocyanate, and trifluoroacetic acid TFA produced by Sigma-Aldrich (St. Louis, USA), will glacial acetic acid 100 % purchased from Merck (Darmstadt Germany), and solution EDTA (pH 8.0) and sodium acetate: marketed by VETEC (Duque Caxias, Rio de Janeiro, Brazil). In vials, 40 μl of sample and 30 μl of the derivatizing solution were added (70% v / v ethanol, 10% v / v Milli-Q water, 10% v / v triethylamine and 10% v / v phenylisothiocyanate), then homogenized and left to stand at room temperature for 15 minutes, then 170 μl of Milli-Q water was added. A volume of 50 μl was injected and analyzed in an ultra-efficient liquid chromatographysystem (CLUE) (LC20 AT, (Shimadzu®, Kyoto, Kinai, Japan). Column C 18 250mm 4.6.7μm was used for the analysis and a guard column of the same material. the mobile phase a consisted of 470 ml buffer solution + 30 ml of ACN mobile phase B and 400mL ACN + 100 ul EDTA solution (pH 8.0). the s olution buffer was prepared c with 19 g of sodium acetate + 1000 ml Milli-Q water + 0.5 ml triethylamine + 200μL EDTA solution (pH 8.0). The elution gradient had a flow rate of 0.3 ml. And the mobile phase gradiente protocol as follows: 0 to 24 minutes 46% of phase a 24 to 30 minutes at phase a has increased its concentration to 100%, the system maintained balanced with phase A at 100% for 8 minutes. The total analysis time was 47 minutes. The reading was performed with a PDAUV - Vis detector at a wavelength of 254nm. Amino acid quantification was determined based on the retention time and peak area obtained from the known concentrations of the amino acids aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine and lysine.