Background: Na+, K+ -ATPase (NKA) is a protein complex localized at the cell membrane, the main factor responsible for the establishment of the sodium and potassium gradient across the cell membrane. NKA activity is reduced in several brain pathological disorders, including ischemia, injury, depression and mood disorders, mania, stress, neuronal hyperexcitability, and epilepsy. In a previous study, some of us described a marked reduction (to about 35% of control values) of NKA activity in erythrocytes from ASD children; a more recent study, aimed at understanding what determines the impairment of enzyme activity, suggested that Oxidative Stress-related alterations in plasma membrane organisation could be the cause. 

Objectives: The study aims to confirm this abnormality in a new cohort of children with ASD in comparison to typically developing children. Part of this work is under revision for the journal Communications in Medicine.

Methods: Twenty-six children diagnosed with non-syndromic ASD (all males, mean age = 6.1; age range 3-8 years) and 30 typically developing children (all males; mean age 6.0 years; range 3-8) were recruited by the Child Neuropsychiatric Unit of Bologna University. Inclusion criteria for the ASD group included a diagnosis based on ADOS and DSM-5 and negative results on CGH array testing. Exclusion criteria for both groups were neurological conditions, epilepsy, ongoing or recent (within the last 4 months) infectious diseases, recent (within the last 4 months) surgery or trauma, or antibiotic use and intake of supplements or vitamins in the month preceding sample collection. Na+/K+ -ATPase activity. Na+/K+ -activated Mg2+ -dependent ATPase activity was determined in cell membranes by the Kitao method. ATPase activity, assayed in the presence of 10 mmol/L ouabain, was subtracted from the total Mg2+-dependent ATPase activity to calculate the activity of Na+/K+ -ATPase. Protein concentration was determined as described by Bradford, using serum albumin as a standard. The intra-assay variation was 5.3%, while the inter-assay variation was 8.1%. Data were tested for normality using the D'Agostino-Pearson test, following which appropriate parametric tests (Student's t for independent data) or the nonparametric equivalent (Mann–Whitney) were used. Differences were considered significant at P < 0.05.

Results: As shown in Fig. 1, the mean RBC Na+/K+ -ATPase activity in the ASD children cohort was 3.002 (0.58 SD). The corresponding values in the NT group were 7.053(0.54 SD; t statistics 26.7; p<0.0001). There were no overlapping values between NT and ASD (NT range 5.245-7.577; ASD range 1.197-3.865). In the ASD group, NKA activity was not correlated with clinical features.

Conclusions: This result is an essential confirmation of previous results obtained in a different cohort of NT and ASD children. The marked decrease in NKA with surprisingly excellent features in terms of specificity and sensitivity suggests that abnormalities in antioxidant status and erythrocyte properties could be involved in the patho-mechanisms of ASD and eventually contribute to its clinical manifestations. These findings underscore the relevance of the impairment of NKA activity as a peripheral biomarker of ASD.

Notes: 

(1)Department of Clinical Sciences, Polytechnic University of Marche, Ancona, Italy, (2)Pediatric Surgery Department, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Dipartimento di Scienze Mediche e Chirurgiche (DIMEC), Bologna, Italy, (3)IRCCS Istituto delle Scienze Neurologiche di Bologna, UOC Neuropsichiatria dell’Età Pediatrica, Bologna, Italy, (4)IRCCS Istituto delle Scienze Neurologiche di Bologna, UOC Neuropsichiatria dell’Età Pediatrica, Centro Regionale per i Disturbi della Nutrizione e dell’Alimentazione in Età Evolutiva, Bologna, Emilia-Romagna, Italy, (5)ISOF, Consiglio Nazionale delle Ricerche, Bologna, Italy, (6)Child Psychopathology Unit, Scientific Institute and IRCCS E. Medea, Bosisio, Italy, (7)Fondazione Danelli ONLUS, Lodi, Italy, (8)Autism Research Unit, Villa Santa Maria SCS, Tavernerio, Como, Italy