Serum phenylacetylglutamine and hydroxyphenylacetylglutamine

Serum phenylacetylglutamine and 4-hydroxyphenylacetylglutamine, which provide a major route of excretion of excess nitrogen from the body, were elevated in children with EED. Phenylacetylglutamine is formed from the conjugation of phenylacetate (from the gut microbiome or host) and glutamine (mainly generated from the Krebs (S)-Crizotinib from α-ketoglutarate). Phenylacetate is primarily produced by the gut microbiome by the decarboxylation of phenylalanine in unabsorbed protein residues (Seakins, 1971). Children with EED also had elevated serum phenylacetate. Elevated urinary phenylacetylglutamine has been described in children with inflammatory bowel disease (Martin et al., 2016). Elevated serum phenylacetylglutamine was an independent risk factor for mortality and cardiovascular disease in nearly 500 patients with chronic kidney disease (Poesen et al., 2016). It is unclear whether phenylacetylglutamine has direct toxic effects or is a marker for altered colonic microbial metabolism and/or renal dysfunction (Poesen et al., 2016). Elevated serum 4-hydroxyphenylacetate was found in children with EED in the present study. 4-hydroxyphenylacetate, one of the most abundant phenylpropanoid-derived compounds found in human fecal samples, is derived from fermentation of tyrosine by the gut microbiome (Russell et al., 2013). Our previous studies (Semba et al., 2016b) showed that children with increased gut permeability have lower circulating tryptophan concentrations and elevated serotonin concentrations, which was corroborated in the present study using a different metabolomics platform. A new finding is that increased gut permeability was also associated with low indolelactate, which is a metabolite in tryptophan catabolism through a series of indoles. The present study also corroborates the relationship between increased gut permeability and low citrulline levels, an observation also made in a study of children from Peru and Tanzania (Kosek et al., 2016). We also showed in the previous (Semba et al., 2016b) and present study that children with increased gut permeability had low serum ornithine, a component of the urea cycle. An adequate supply of citrulline and ornithine is required for normal function of the urea cycle. A new finding is that children with increased gut permeability had elevated homocitrulline levels. In the absence of ornithine from the urea cycle, mitochondrial carbamoyl phosphate increases and can generate homocitrulline from lysine via ornithine transcarbamylase. Using the Metabolon platform in the present study, we were unable to corroborate some findings that were significant in the previous study that used the Biocrates metabolomics platform, such as a positive association between serum glutamate and gut permeability. In addition, although the present study showed that carnitine and nine acylcarnitines were positively associated with gut permeability, the previous study using the Biocrates platform did not show a significant association between carnitine and acylcarnitines with gut permeability. The Biocrates p180 kit has a semi-quantitative method that measures 39 specific acylcarnitines, however, in our study only seven acylcarnitines were above the limit of detection, based on our settings of only using metabolites that were detected in >80% of the sample population (Semba et al., 2016b). This underscores the importance of carrying out LC-MS/MS assays that provide absolute quantification specifically for carnitine and acylcarnitines in the future (Giesbertz et al., 2015, Minkler et al., 2015). The low serum mannitol/sorbitol (these two sugars are isomers that cannot be distinguished in the MS assay) concentrations and elevated serum lactose levels provide corroborative evidence for reduced small intestinal surface area and increased gut permeability, respectively, in children with EED. Mannitol and sorbitol are monosaccharides that readily cross the intact gut barrier. Serum mannitol concentrations are reduced proportional to the intestinal surface (Sigalet et al., 2000). Lactose is a disaccharide that, like lactulose, does not cross a normal intact intestinal barrier. Serum lactose was positively correlated with gut permeability. EED is associated with malabsorption of fats. In the present study, low serum concentrations of three fat-soluble substances, α-tocopherol (vitamin E), thymol (a monoterpene phenol found in oils from plants), and isoeugenol sulfate (a substance from plants), are consistent with fat malabsorption in EED.