Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder

Abstract

Autism spectrum disorder (ASD) has been associated with mitochondrial disease (MD). Interestingly, most individuals with ASD and MD do not have a specific genetic mutation to explain the MD, raising the possibility of that MD may be acquired, at least in a subgroup of children with ASD. Acquired MD has been demonstrated in a rodent ASD model in which propionic acid (PPA), an enteric bacterial fermentation product of ASD-associated gut bacteria, is infused intracerebroventricularly. This animal model shows validity as it demonstrates many behavioral, metabolic, neuropathologic and neurophysiologic abnormalities associated with ASD. This animal model also demonstrates a unique pattern of elevations in short-chain and long-chain acyl-carnitines suggesting abnormalities in fatty-acid metabolism. To determine if the same pattern of biomarkers of abnormal fatty-acid metabolism are present in children with ASD, the laboratory results from a large cohort of children with ASD (n=213) who underwent screening for metabolic disorders, including mitochondrial and fatty-acid oxidation disorders, in a medically based autism clinic were reviewed. Acyl-carnitine panels were determined to be abnormal if three or more individual acyl-carnitine species were abnormal in the panel and these abnormalities were verified by repeated testing. Overall, 17% of individuals with ASD demonstrated consistently abnormal acyl-carnitine panels. Next, it was determined if specific acyl-carnitine species were consistently elevated across the individuals with consistently abnormal acyl-carnitine panels. Significant elevations in short-chain and long-chain, but not medium-chain, acyl-carnitines were found in the ASD individuals with consistently abnormal acyl-carnitine panels-a pattern consistent with the PPA rodent ASD model. Examination of electron transport chain function in muscle and fibroblast culture, histological and electron microscopy examination of muscle and other biomarkers of mitochondrial metabolism revealed a pattern consistent with the notion that PPA could be interfering with mitochondrial metabolism at the level of the tricarboxylic-acid cycle (TCAC). The function of the fatty-acid oxidation pathway in fibroblast cultures and biomarkers for abnormalities in non-mitochondrial fatty-acid metabolism were not consistently abnormal across the subgroup of ASD children, consistent with the notion that the abnormalities in fatty-acid metabolism found in this subgroup of children with ASD were secondary to TCAC abnormalities. Glutathione metabolism was abnormal in the subset of ASD individuals with consistent acyl-carnitine panel abnormalities in a pattern similar to glutathione abnormalities found in the PPA rodent model of ASD. These data suggest that there are similar pathological processes between a subset of ASD children and an animal model of ASD with acquired mitochondrial dysfunction. Future studies need to identify additional parallels between the PPA rodent model of ASD and this subset of ASD individuals with this unique pattern of acyl-carnitine abnormalities. A better understanding of this animal model and subset of children with ASD should lead to better insight in mechanisms behind environmentally induced ASD pathophysiology and should provide guidance for developing preventive and symptomatic treatments.

Figure 1

Acyl-carnitine elevations in the brain of rats treated intracerebroventricularly with propionic acid. Notice that the majority of fatty-acid elevations were in short-chain (2–5 carbon length) and long-chain (13–18 carbon length) fatty-acids as compared with the medium-chain (6–12 carbon length) fatty-acids. This is adapted from Thomas et al. where it was presented as a table.

Figure 2

Algorithm for metabolic workup of autistic spectrum disease patients evaluated in the medically based autism clinic. Patients are screened with biomarkers of abnormal mitochondrial function in the fasting state. Abnormalities are verified with repeat fasting biomarker testing. For patients with biomarkers for a fatty-acid oxidation defect, other disorders of fatty-acid metabolism are ruled-out before further workup for a mitochondrial disorder. Patients with consistent biomarkers for mitochondrial dysfunction are first investigated for genetic causes of their mitochondrial disorder before considering a muscle and/or skin biopsy. mtDNA, mitochondrial deoxyribonucleic acid; RBC, red-blood cell.

Figure 3

Average acyl-carnitine values (with s.e. bars) from 20 patients with consistent abnormal elevations in multiple acyl-carnitines. Acyl-carnitine values are represented as percent upper limit of normal for each acyl-carnitine species. Notice that C4OH, C14 and C16:1 are significantly elevated as compared with the maximum upper limit of normal.

Figure 4

Gluthathione abnormalities in four children with consistent elevations in multiple acyl-carnitine species. Notice that the patients have lower total (tGSH, μM) and free (fGSH, μM) reduced gluthathione, as well as lower tGSH/fGSSG (free-oxidized gluthathione, μM) and fGSH/fGSSG ratios and higher fGSSG as compared with typically developing controls, suggesting both a reduction in the production of gluthathione and increase in gluthathione utilization by reactive species.

Figure 5

Electron transport chain (ETC) function of muscle (a, b) and fibroblast culture (c, d), as well as function of the fatty-acid oxidation pathway in fibroblast cultures (e, f). Graph values represent percent of normal ETC function, uncorrected (a, c) or corrected for citrate synthase (b, d). Muscle ETC results suggest a partial defect in complexes I/III and I/III rotenone sensitive (RS) while fibroblast culture ETC function suggests a partial defect in complex II/III activity. In fibroblast culture ETC studies complexes I/III RS and IV demonstrate considerable variability due to overactivity (>200% of the mean) in complex I/III RS in three patients and complex IV in one case. Fatty-acid oxidation values represent mean of specific acyl-carnitine species (higher is worse) uncorrected (e) and corrected for citrate synthase (f). Elevation in the short-chain fatty-acid D3-C4 was due to three patients demonstrating high D4-C4 values. The one patient with a significantly elevated D4-C4 value was found not to have a mutation in ;the short-chain acyl-CoA dehydrogenase gene suggesting that the abnormalities in fatty-acids in fibroblast culture were due to other mitochondrial metabolism abnormalities.

Figure 6

The tricarboxylic-acid cycle during (a) typical metabolism and (b) with high levels of propionic acid. Propionic acid is metabolized to propionyl-CoA, which inhibits the proximal portion of the tricarboxylic-acid cycle and enhances the distal portion of the tricarboxylic-acid cycle (see discussion for details). FADH_2, flavin adenine dinucleotide; NADH, nicotinamide adenine dinucleotide.