GC/MS in recent years has defined the normal and clinically disordered steroidome: will it soon be surpassed by LC/Tandem MS in this role?

Gas chromatography/mass spectrometry (GC/MS) has been used for steroid analysis since the 1960s. The advent of protective derivatization, capillary columns, and inexpensive electron ionization bench-top single quadrupole soon made it the method of choice for studying disorders of steroid synthesis and metabolism. However, the lengthy sample workup prevented GC/MS from becoming routine for steroid hormone measurement, which was dominated by radioimmunoassay. It was the emergence of liquid chromatography/tandem MS (LC/MS/MS) that sparked a renewed interest in GC/MS for the multicomponent analysis of steroids. GC/MS is excellent at providing an integrated picture of a person's steroid metabolome, or steroidome, as we term it. We review the recent work on newly described disorders and discuss the technical advances such as GC coupling to triple quadrupole and ion trap analyzers, two-dimensional GC/MS, and alternative ionization and detection systems such as atmospheric pressure chemical ionization (APCI) and time of flight. We believe that no novel GC/MS-based technique has the power of GC(electron ionization)/MS/MS as a "discovery tool," although APCI might provide ultimate sensitivity, which might be required in tissue steroidomics. Finally, we discuss the role of LC/MS/MS in steroidomics. This remains a challenge but offers shorter analysis times and advantages in the detection and discovery of steroids with a known structure. We describe recent advances in LC/MS steroidomics of hydrolyzed and intact steroid conjugates and suggest the technique is catching up with GC/MS in this area. However, in the end, both techniques will likely remain complementary and both should be available in advanced analytical laboratories

Evaluation of metabolic changes in acute intermittent porphyria patients by targeted metabolomics

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

Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus

Background: Penicillium digitatum is a fungal necrotroph causing a common citrus postharvest disease known as green mold. In order to gain insight into the genetic bases of its virulence mechanisms and its high degree of host-specificity, the genomes of two P. digitatum strains that differ in their antifungal resistance traits have been sequenced and compared with those of 28 other Pezizomycotina. Results: The two sequenced genomes are highly similar, but important differences between them include the presence of a unique gene cluster in the resistant strain, and mutations previously shown to confer fungicide resistance. The two strains, which were isolated in Spain, and another isolated in China have identical mitochondrial genome sequences suggesting a recent worldwide expansion of the species. Comparison with the closely-related but non-phytopathogenic P. chrysogenum reveals a much smaller gene content in P. digitatum, consistent with a more specialized lifestyle. We show that large regions of the P. chrysogenum genome, including entire supercontigs, are absent from P. digitatum, and that this is the result of large gene family expansions rather than acquisition through horizontal gene transfer. Our analysis of the P. digitatum genome is indicative of heterothallic sexual reproduction and reveals the molecular basis for the inability of this species to assimilate nitrate or produce the metabolites patulin and penicillin. Finally, we identify the predicted secretome, which provides a first approximation to the protein repertoire used during invasive growth. Conclusions: The complete genome of P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.TG research is funded in part by a grant from the Spanish Ministry of Economy and Innovation (BFU2009-09168). JFM research is funded in part by the Spanish Ministry of Science (BIO2009-12919). LGC research is funded in part by the Spanish Ministry of Economy and Innovation (AGL-2008-04828-C03-02 and AGL2011-30519-C03-01) and the Generalitat Valenciana (PROMETEO/2010/010 and ACOMP/2011/250). ARB acknowledges the support of the JAE-Doc program from CSIC and the European FEDER fund

Adrenal hormonal imbalance in acute intermittent porphyria patients: results of a case control study

Background: Acute Intermittent Porphyria (AIP) is a rare disease that results from a deficiency of hydroxymethylbilane synthase, the third enzyme of the heme biosynthetic pathway. AIP carriers are at risk of presenting acute life-threatening neurovisceral attacks. The disease induces overproduction of heme precursors in the liver and long-lasting deregulation of metabolic networks. The clinical history of AIP suggests a strong endocrine influence, being neurovisceral attacks more common in women than in men and very rare before puberty. To asses the hypothesis that steroidogenesis may be modified in AIP patients with biochemically active disease, we undertook a comprehensive analysis of the urinary steroid metabolome. Methods: A case–control study was performed by collecting spot morning urine from 24 AIP patients and 24 healthy controls. Steroids in urine were quantified by liquid chromatography-tandem mass spectrometry. Parent steroids (17-hydroxyprogesterone; deoxycorticosterone; corticoesterone; 11-dehydrocorticosterone; cortisol and cortisone) and a large number of metabolites (N = 55) were investigated. Correlations between the different steroids analyzed and biomarkers of porphyria biochemical status (urinary heme precursors) were also evaluated. The Mann–Whitney U test and Spearman’s correlation with a two tailed test were used for statistical analyses. Results: Forty-one steroids were found to be decreased in the urine of AIP patients (P  0.51, P < 0.01). Conclusions: Comprehensive study of the urinary steroid metabolome showed that AIP patients present an imbalance in adrenal steroidogenesis, affecting the biosynthesis of cortisol and resulting in decreased out-put of cortisol and metabolites. This may result from alterations of central origin and/or may originate in specific decreased enzymatic activity in the adrenal gland. An imbalance in steroidogenesis may be related to the maintenance of an active disease state among AIP patients.This work was supported by grants from Instituto de Salud Carlos III FEDER, (CP/10/00576) and the Spanish “Fondo de Investigación Sanitaria” (PI11/00767) to Jordi To-Figuera

Adrenal hormonal imbalance in acute intermittent porphyria patients: results of a case control study

Background: Acute Intermittent Porphyria (AIP) is a rare disease that results from a deficiency of hydroxymethylbilane synthase, the third enzyme of the heme biosynthetic pathway. AIP carriers are at risk of presenting acute life-threatening neurovisceral attacks. The disease induces overproduction of heme precursors in the liver and long-lasting deregulation of metabolic networks. The clinical history of AIP suggests a strong endocrine influence, being neurovisceral attacks more common in women than in men and very rare before puberty. To asses the hypothesis that steroidogenesis may be modified in AIP patients with biochemically active disease, we undertook a comprehensive analysis of the urinary steroid metabolome. Methods: A case–control study was performed by collecting spot morning urine from 24 AIP patients and 24 healthy controls. Steroids in urine were quantified by liquid chromatography-tandem mass spectrometry. Parent steroids (17-hydroxyprogesterone; deoxycorticosterone; corticoesterone; 11-dehydrocorticosterone; cortisol and cortisone) and a large number of metabolites (N = 55) were investigated. Correlations between the different steroids analyzed and biomarkers of porphyria biochemical status (urinary heme precursors) were also evaluated. The Mann–Whitney U test and Spearman’s correlation with a two tailed test were used for statistical analyses. Results: Forty-one steroids were found to be decreased in the urine of AIP patients (P  0.51, P < 0.01). Conclusions: Comprehensive study of the urinary steroid metabolome showed that AIP patients present an imbalance in adrenal steroidogenesis, affecting the biosynthesis of cortisol and resulting in decreased out-put of cortisol and metabolites. This may result from alterations of central origin and/or may originate in specific decreased enzymatic activity in the adrenal gland. An imbalance in steroidogenesis may be related to the maintenance of an active disease state among AIP patients.This work was supported by grants from Instituto de Salud Carlos III FEDER, (CP/10/00576) and the Spanish “Fondo de Investigación Sanitaria” (PI11/00767) to Jordi To-Figuera