Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and communication, repetitive behaviors and/or restricted interests(Association, 2013). The overall prevalence of ASD has been consistently increasing in recent years. The early study on autism conducted in the 1960s reported that its prevalence was about 4/10,000(Lotter, 1966), whereas the prevalence of ASD had significantly risen from 13.4 to 1000 children in 2010(CDC, 2014) to 27.9 in 2016 in the USA(Xu et al., 2019). A recent meta-analysis indicated that the prevalence of ASD in China was 26.5/10,000(Liu et al., 2018), although this was significantly lower than the reported prevalence abroad. In 2020, the prevalence of ASD among children aged 6–12 years was about 0.7% in China(Zhou et al., 2020).

Children with ASD might be disabled and required life-long care(Cheuk et al., 2011), which burdens patients, families, and the public-health system. Although the pathogenesis of ASD is still not clear, it has been widely recognized that ASD occurs due to a combination of genetic and environmental factors, with the former being predominant (Bai et al., 2019). However, it has been found that maternal folic acid supplementation strategies, such as intake timing and intake dosage, may aid in reducing in the risk of ASD in offspring(Liu, Zou, Sun, Wu, & Chen, 2021).

In recent years, the metabolic abnormalities in ASD have attracted increasing attention, especially abnormalities related to amino acids. Amino acids are organic compounds containing amino and carboxyl groups, which are the basic protein units that have an essential role in regulating the immune system, cell signaling and metabolism, neurotransmission and so on(Vargason et al., 2018). Although previous studies have found the differences in plasma amino acid levels between autistic children and healthy children(Aldred, Moore, Fitzgerald, & Waring, 2003; D’Eufemia et al., 1995; Hoshino et al., 1984), there are still some disagreements over abnormal plasma amino acid levels between autistic children and healthy children. These inconsistent results in plasma amino acid levels between autistic children and healthy children might be due to the following reasons: firstly, most previous studies were small sample size studies, besides a few relatively large sample sizes in case-control studies (i.e., the sample size in ASD group ≥ 50 cases)(Adams et al., 2011; Cai, Ding, Zhang, Xue, & Wang, 2016; Naushad, Jain, Prasad, Naik, & Akella, 2013; Vargason et al., 2018; ** peers. Res Autism Spectr Disord, 50, 60–72. doi: https://doi.org/10.1016/j.rasd.2018.03.004 ." href="/article/10.1007/s10803-022-05829-z#ref-CR60" id="ref-link-section-d174067502e2131">2018; ** brain. The Journal Of Physiological Sciences: Jps, 66(5), 375–379. doi: https://doi.org/10.1007/s12576-016-0442-7 ." href="/article/10.1007/s10803-022-05829-z#ref-CR26" id="ref-link-section-d174067502e2857">2016; Owens & Kriegstein, 2002). Increased γ-aminobutyric acid and glycine in autistic children can disturb the excitation/inhibition balance in brain, which might lead to autism(Marotta et al., 2020; Zheng, Wang, Li, Rauw, & Baker, 2017).

Essential amino acids in the human body must be supplied by food; however, some autistic children may have eating difficulties and gastrointestinal symptoms, and be very picky about the taste and color of food(Kral, Eriksen, Souders, & Pinto-Martin, 2013). Accordingly, decreased essential amino acids (lysine, tryptophan, phenylalanine, histidine) in autistic children might be partially due to insufficient food intake or poor eating habits. Besides, we also found that the plasma levels of ethanolamine and glutathione (reduced) in the ASD group were decreased, which is consistent with the previous studies(Bala et al., 2016; Geier et al., 2009; James et al., 2004). Ethanolamine is involved in synthesizing phosphatidylethanolamine, and reduced ethanolamine in autistic children might cause chronic oxidative stress via decreased phosphatidylethanolamine synthesis(Wang et al., 2014). Glutathione is a tri-peptide involved in the redox balance of glutathione in the intracellular environment. The intracellular environment is maintained by a high glutathione (reduced)/glutathione (oxidative) ratio(Schafer & Buettner, 2001), which regulates a wide range of cell functions, including the scavenging of oxygen free radicals, cell membrane integrity, signal transduction, and so on(Dickinson et al., 2003). Therefore, reduced glutathione in autistic children may disrupt the redox balance of glutathione, thus further aggravating oxidative stress.

Interestingly, we also found that the plasma levels of sarcosine and δ-aminolevulinic acid were elevated. Sarcosine is an intermediate product of glycine metabolism, and the increased sarcosine level in our study might be due to increased glycine level, although Adams et al (Adams et al., 2011) found no significant difference in the sarcosine plasma level between the autistic children and neurotypical children in their study. However, most measurements of secondary plasmas amino acids and amino acid metabolites, including sarcosine being below the detection limit of 0.05 umoles/100ml and the large range age span of recruited subjects (5-16y) limited the interpretation of the outcomes of their study(Adams et al., 2011). Vargason et al(Vargason et al., 2018) also measured the level of Sarcosine, but omitted it from further analysis due to the subjects’ intervention issue. In addition, besides children, they(Vargason et al., 2018) also recruited adult subjects, and therefore, the age span of included subjects was large (11.8 ± 8.5y), which made it difficult to compare their PPA’s outcomes with current study and other studies that only included children’ subjects, as the level of plasma amino acids substantially varies with age(Lepage et al., 1997).

The plasma level of δ-aminolevulinic acid or pyroglutamic acid was not reported in the previous large sample sizes case-control studies (Table 5). In vivo, δ-aminolevulinic acid as the precursor of heme, is produced by glycine and succinyl-CoA under the δ-amino-γ-levulinic acid (ALA) synthetase(McLeod, Mack, & Brown, 1991), and δ-aminolevulinic acid must activate mitochondria so that it can convert into heme in the cell(Malik & Djaldetti, 1979). Most studies have indicated mitochondrial dysfunction and oxidative stress as the neuropathological basis of autism (Gorman et al., 2015; Rossignol & Frye, 2012). Therefore, the elevated δ-aminolevulinic acid in autistic children might cause mitochondrial dysfunction and oxidative stress. However, a recent animal model study showed that δ-aminolevulinic acid could inhibit oxidative stress and ameliorate autistic-like behaviors for the prenatal valproic acid-exposed rats(Matsuo, Yabuki, & Fukunaga, 2020), which implicate that accumulation of δ-aminolevulinic acid may result from autism-induced mitochondria dysfunction. The further studies needed regarding the relationship between δ-aminolevulinic acid and pathogenesis of autism.

Pyroglutamic acid is cyclized to form lactams from free amino groups of glutamate or glutamine. Pyroglutamic acid can antagonize nerve excitement by inhibiting glutamate(Abraham & Podell, 1981).The reduced plasma level of pyroglutamic acid in autistic children revealed in our study might further aggravate the neuroexcitatory toxicity of autism by reducing the inhibitory effect of pyroglutamate acid on glutamate.

In addition, we found that the levels of homocysteine, hydroxyproline and ornithine in autism were reduced, which were contrary to previous researches’ results (Vargason et al., 2018; Zou et al., The Correlation Between Plasma Amino Acid and Clinical Phenotype

The correlation between plasma amino acid and clinical phenotype was further analyzed using logistic regression analysis in the autistic children group.

In terms of severity of autism, there were positively correlation between severity of autism and the plasma level of tryptophan (Table 4); the higher the plasma tryptophan level, the worse the severity of autism.

The plasma level of tryptophan was reduced in the current study, which was in line with previous studies(Adams et al., 2011; Naushad et al., 2013; ** peers. Res Autism Spectr Disord, 50, 60–72. doi: https://doi.org/10.1016/j.rasd.2018.03.004 ." href="/article/10.1007/s10803-022-05829-z#ref-CR60" id="ref-link-section-d174067502e3103">2018; Zou et al., 2018). The impairment in methylation and oxidative stress affect the occurrence of ASD. Increased serine might affect one-carbon metabolism, thus leading to ASD.

Cai et al.(Cai et al., 2016) found the plasma level of glutamate was positively associated with increasing severity of ASD. In their study, Cai and colleagues showed three possible mechanisms in their study, which were mostly related to the excitotoxicity of glutamate.

In 2020, Zou et al(Zou et al., 2020) also found that the level of homocysteine was positively correlated with the severity of autism, although they evaluated the severity of autistic children with ADOS-CSS (Table 5).

Regarding the regression, although there were no significant differences between plasma amino acids and regression in the current study, the level of glutathione might have a significant trend toward associate with regression (Table 4). Glutathione is a tripeptide, composed of glutamic acid, cysteine, and glycine, involved in the redox balance of glutathione in the intracellular environment. In addition, a previous study showed that the serum glutamate/glutamine ratio was elevated in ASD PIQ ≥ 70 group(**ng et al., 2021).

However, when the correlation was analyzed between plasma amino acids and other respective clinical phenotypes, including cognition and adaptability, there were no significant differences in the current study.

Strength and Limitation

The present study was a relatively large-sample case-control study that recruiting more than 100 cases. We tried to exclude other confounding factors, such as acquired vitamin B12 deficiency, age and sex-matched between groups, to ensure the accuracy and reliability of the study. We also measured the level of amino acids by the advanced detection method of LC-MS/MS. Currently, we measured and analyzed the largest number of PAAs among case-control studies (Table 5). In addition, we further analyzed the correlation between plasma amino acid levels and a set of the clinical phenotypes of autistic children.

However, the present study has the following limitations: (1) this was retrospective research, so prospective large sample cohort studies are needed to further verify the findings of this study in the future; (2) the PAAs level was greatly affected by eating, it might be more accurate to dynamically monitor the changes of amino acids. Also, no information was collected on the patient’s underlying nutritional status; (3) there were no other neurodevelopmental groups, such as the ID group included in the current study, influencing outcomes’ representatives.

Conclusion

There were significant differences in seventeen amino acids between ASD and the control group; Elevated levels of neuroactive amino acids (glutamate) and decreased essential amino acids exhibited mostly distinct characteristics of plasma amino acid in autistic children. Increased level of tryptophan might be associated with the severity of autism.