Evaluation of Metabolic Changes in Acute Intermittent Porphyria Patients by Targeted Metabolomics

Abstract

Acute intermittent porphyria (AIP) is an inherited rare hepatic disorder due to mutations within the hydroxymethylbilane gene. AIP patients with active disease overproduce aminolevulinic acid (ALA) and porphobilinogen (PBG) in the liver which are exported inducing severe neurological attacks. Different hepatic metabolic abnormalities have been described to be associated with this condition. The goal of this research was to explore the metabolome of symptomatic AIP patients by state-of-the art liquid chromatography-tandem mass spectrometry (LC-MS/MS). A case versus control study including 18 symptomatic AIP patients and 33 healthy controls was performed. Plasmatic levels of 51 metabolites and 16 ratios belonging to four metabolic pathways were determined. The results showed that the AIP patients presented significant changes in the two main areas of the metabolome under study: (a) the tryptophan/kynurenine pathway with an increase of tryptophan in plasma together with increase of the kynurenine/tryptophan ratio; and (b) changes in the tricarboxylic acid cycle (TCA) including increase of succinic acid and decrease of the fumaric acid/succinic acid ratio. We performed a complementary in vitro study adding ALA to hepatocytes media that showed some of the effects on the TCA cycle were parallel to those observed in vivo. Our study confirms in plasma previous results obtained in urine showing that AIP patients present a moderate increase of the kynurenine/tryptophan ratio possibly associated with inflammation. In addition, it also reports changes in the mitochondrial TCA cycle that, despite requiring further research, could be associated with an energy misbalance due to sustained overproduction of heme-precursors in the liver.

Keywords: LC-MS/MS; acute intermittent porphyria; kynurenine; metabolomics; tricarboxylic acid cycle; tryptophan.

Figure 1

OrthoPLS-DA analysis. Comparison between HC and AIP cohorts. (a) Scores plot of orthoPLS-DA and (b) signature feature. Plots of the orthoPLS-DA reveal clear separation between groups and the altered metabolic pathways (Trp and TCA cycle). Abbreviations: Kynurenic acid/kynurenine (KA/Kyn); fumaric acid/succinic acid (FA/SA); isocitric acid/citric acid (IA/CA); 5-hydroxyindoleacetic acid/serotonin (5HIAA/5HT); serotonin (5HT); serotonin/tryptophan (5HT/Trp); isocitric acid (IA); malic acid (MA); fumaric acid (FA).

Figure 2

Boxplots showing differences between HC and AIP patients. Representation of (a) kynurenine, (b) kynurenine/tryptophan, (c) succinic acid, and (d) fumaric acid/succinic acid. Asterisks code: * p-value between 0.01–0.05; ** p-value between 0.001–0.01; *** p-value < 0.001. Abbreviation: HC: healthy controls, AIP: acute intermittent porphyria.

Figure 3

Effect of ALA addition into human hepatocytes metabolism. Production of (a) succinic acid by hepatocytes after addition of vehicle, 0.5 of ALA and 5 of ALA, and (b) malic acid by hepatocytes after addition of vehicle, 0.5 of ALA and 5 of ALA. Abbreviations: ALA: δ-aminolevulinic acid, n.s.: no significant.

Figure 4

Tryptophan metabolism. Abbreviations: MAO: monoamine oxidase, IDO: indoleamine 2,3-dioxygenase, TDO: tryptophan 2,3-dioxygenase, KMO: kynurenine 3-monooxygenase, KAT: kynurenine aminotransferase. Color code: blue: decreased in AIP patients, white: unaltered in AIP patients, red: increased in AIP patients.

Figure 5

TCA cycle. Abbreviations: LDH: lactate dehydrogenase, IDH: isocitrate dehydrogenase, α-KDH: α-ketoglutarate dehydrogenase, SCS: succinyl Co-A synthetase, SDH: succinate dehydrogenase, MDH: malate dehydrogenase, CS: citrate synthase. Color code: blue: decreased in AIP patients, white: unaltered in AIP patients, red: increased in AIP patients.