Our findings show an abnormal utilization of tryptophan as energy source in cells from patients with ASDs, suggesting impaired tryptophan metabolism. Our analysis consisted of three independent sets of experiments in which we measured NADH production in 87 lymphoblastoid cells derived from ASD patients as compared to 78 controls. The difference between the case–control populations was statistically significant in each of these experiments. The statistical analysis was performed in a blind fashion with regard to the presence of ASDs in the patients. The results correlated with the behavioral traits associated with either syndromal or non-syndromal autism, independent of the genetic background of the individual. The low level of NADH generation in the presence of tryptophan was not observed in cell lines from patients with intellectual disability without ASD or schizophrenia, or in conditions showing several similarities with syndromal ASDs except for the behavioral traits.
Metabolism of tryptophan via the serotonin synthesis pathway leads to production of NADH, while metabolism via the kynurenine-quinolinic acid pathway leads to the synthesis of NAD+, the precursor of NADH (Figure 4). The decreased level of NADH generation in the presence of tryptophan may reflect less utilization of tryptophan resulting from downregulation of metabolic reactions along either of these pathways.
Analysis of microarray expression data previously collected on the initial 10 ASD patient cell lines and 10 controls found reduced levels of some genes involved in the serotonin and kynurenine pathways in ASD patients. No two patients exhibited the same expression profile. The enzyme responsible for the conversion of quinolinic acid to nicotinate D-ribonucleotide (quinolinate phosphoribosyltransferase) showed a trend towards increased expression, although this was statistically significant in only seven out of the 10 cell lines analyzed, when each cell line was individually compared to the control group using the Mann–Whitney one sample test (Additional file 5: Table S5). This enzyme links the tryptophan-kynurenine pathway to NAD+ biosynthesis (Figure 4) and is produced by a gene (QPRT) mapping to 16p11.2. Deletions and duplications of this region have been frequently associated with ASDs, suggesting that abnormal dosage of the genes in 16p11.2 may be responsible for autism features .
Pyridoxine and its metabolite, pyridoxal phosphate, play a critical role as co-factors in both the serotonin and kynurenine pathways, so their deficiency may affect tryptophan metabolism. Unfortunately, the PM plates do not contain any compound closely related to pyridoxine. Additionally, our limited microarray data for the enzymes involved in pyridoxine/pyridoxal phosphate metabolism (PHOSPHO2, PDX, PNPO) did not show significant differences when compared to controls in any of the 10 patients.
Our findings support a possible mitochondrial dysfunction as a result of impaired tryptophan metabolism in cells from patients with ASDs  and several biochemical mechanisms could be responsible. NADH is a critical energy carrier for the electron transport chain and tryptophan is the main precursor of the kynurenine-quinolinic acid pathway that leads to NAD+ synthesis. Furthermore, the expression microarray data from a previous study revealed selective under-expression of the mitochondrial isoform of tryptophanyl tRNA synthetase (WARS2) in 6/10 cell lines from ASD patients, and normal expression levels for the cytoplasmic isoform (WARS1). Considering the expression of tRNA genes is sensitive to the availability of the corresponding amino acid, it is possible the tryptophan levels in mitochondria are lower in ASD patients than controls (Figure 4).
In the brain, mitochondrial dysfunction has an effect on neuronal development and morphology, neurite overgrowth, and synaptic plasticity [20–22]. There appears to be a close link between mitochondrial dysfunction and synaptic abnormalities, which are considered one of the main pathogenic events associated with ASDs [5, 23]. Five of the first 10 ASD cases had a mutation in genes involved in glutamatergic synapses (SHANK3 and NLGN4). Also, some of the pathways involved in regulation of synaptic protein expression  are also involved in the expression of protein destined for the mitochondria.
The biochemical pathway that leads to NAD+ synthesis, beginning with tryptophan, is the kynurenine pathway (Figure 4) which has two major final products: quinolinic acid (metabolized to nicotinamide and NAD+), and kynurenic acid . Some of the most frequently reported neuroanatomical findings in ASD brains are increased brain size and relatively increased white matter, particularly in the radiate zone [25, 26]. It is possible that impairment of the quinolinic-kynurenic balance may have consequences on the programmed ‘pruning’ process and lead to an excess of white matter. Microglial cells are considered to be responsible for keeping the proper balance between quinolinic and kynurenic acid, since they are able to secrete both compounds. The expression of those molecules is strongly influenced by the activity of the immune system. Particularly, nitric oxide (NO) plays a critical role in the interaction between inflammation and neuronal circuits and causes mitochondrial dysfunction. It is important to note that elevated NO levels have been reported in ASD patients [27, 28].
Although approximately 99% of the dietary tryptophan intake is metabolized via the kynurenine pathway , tryptophan is also the main precursor for both serotonin and melatonin (Figure 4). Melatonin plays a critical role in the regulation of the circadian rhythm, and anomalies of this rhythm have been associated with some of the signs in the autistic spectrum, like seizures or sleep disorders . Serotonin is a neurotransmitter involved in multiple aspects of brain functions, ranging from the regulation of mood to the control of appetite and social interactions  and its production has been reported as deficient in ASD brains . Tryptophan levels have been demonstrated to directly influence central nervous system (CNS) serotonin levels  and behavior , and altered tryptophan transport has been described in fibroblasts from boys with attention deficit/hyperactivity disorder (ADHD) . It is noteworthy that tryptophan hydroxylase is the rate-limiting enzyme in the biosynthesis of serotonin and the gene coding the isoform 2 of this enzyme (TPH2) was underexpressed in the expression microarray analysis of lymphoblastoid cells from 10 ASD patients as compared to controls (P = 0.0166). In the human brain, both isoforms, TPH1 and TPH2, are ubiquitously expressed with some particular region-specific differences  and TPH2 expression is significantly higher in the raphe nuclei, the core of serotonergic system . Thus, lower expression levels of TPH2 in white cells might reflect abnormal serotonergic activity in the raphe nuclei. Additionally, the MAOA enzyme in the serotonin pathway which generates NADH was found to be underexpressed in cells from ASD patients.
Serotonin also plays a critical role in regulating neuronal morphology and circuitry  and recent work showed that placental cells are able to synthesize serotonin from tryptophan provided by the maternal blood . This exogenous source of serotonin is important in the neurodevelopment of the forebrain in the first month of gestation, because the endogenous source (hindbrain, future serotonergic system) is not sufficient at this stage. Interestingly, disrupted organization of the fronto-temporal lobes is one of the most consistent neuroanatomical findings in ASD patients , and lower expression levels of genes involved in synapses, neurotransmitter transport and neuron projection have been detected in frontal and temporal lobes of ASD brains . At the cellular level, such disrupted organization is reflected by abnormalities in the minicolumns, the basic functional units of the cortex, which have been reported in ASD brains to be narrower, increased in number per cortical area and with reduction of neuropil space, caused by the smaller size of the peripheral interneurons [25, 39].