Sex Hormones and Adrenal Steroids: Biological Variation Estimated Using Direct and Indirect Methods.

BACKGROUND: Biological variation (BV) data may be used to develop analytical performance specifications (APS), reference change values (RCV), and support the applicability of population reference intervals. This study estimates within-subject BV (CVI) for several endocrine biomarkers using 3 different methodological approaches. METHODS: For the direct method, 30 healthy volunteers were sampled weekly for 10 consecutive weeks. Samples were analyzed in duplicate for 17-hydroxyprogesterone (17-OHP), androstenedione, cortisol, cortisone, estradiol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), sex hormone-binding globulin (SHBG), and testosterone. A CV-ANOVA with outlier removal and a Bayesian model were applied to derive the CVI. For estradiol, FSH and LH, only the male subgroup was included. In the indirect method, using the same analytes and groups, pairs of sequential results were extracted from the laboratory information system. The total result variation for individual pairs was determined by identifying a central gaussian distribution in the ratios of the result pairs. The CVI was then estimated by removing the effect of analytical variation. RESULTS: The estimated CVI from the Bayesian model (µCVP(i)) in the total cohort was: 17-OHP, 23%; androstenedione, 20%; cortisol, 18%; cortisone, 11%; SHBG, 7.4%; testosterone, 16%; and for the sex hormones in men: estradiol, 14%; FSH, 8%; and LH, 26%. CVI-heterogeneity was present for most endocrine markers. Similar CVI data were estimated using the CV-ANOVA and the indirect method. CONCLUSIONS: Similar CVI data were obtained using 2 different direct and one indirect method. The indirect approach is a low-cost alternative ensuring implementation of CVI data applicable for local conditions.

An Ultrasensitive Routine LC-MS/MS Method for Estradiol and Estrone in the Clinically Relevant Sub-Picomolar Range.

BACKGROUND: Current analytical routine methods lack the sensitivity to monitor plasma estrogen levels in breast cancer patients treated with aromatase inhibitors. Such monitoring is warranted for premenopausal patients treated with an aromatase inhibitor and an LH-releasing hormone analogue in particular. Therefore, we aimed to develop a routine tandem mass spectroscopy combined with liquid chromatography (LC-MS/MS) method for estradiol (E2) and estrone (E1) for use in the sub-picomolar range. METHOD: Calibrators, quality controls (QC), or serum samples were spiked with isotope-labeled internal standard and purified by liquid-liquid extraction. The reconstituted extracts were analyzed by LC-MS/MS in negative electrospray ionization mode. QCs at 6 levels made from pooled patient sera were used to validate the accuracy, sensitivity, and precision of the method. RESULTS: We achieved limits of quantification of 0.6 pmol/L (0.16 pg/mL) for E2 and 0.3 pmol/L (0.07 pg/mL) for E1. The coefficient of variation was below 9.0% at all QC levels for E2 (range, 1.7-153 pmol/L), and below 7.8% for E1 (range, 1.7-143 pmol/L). The method is traceable to the E2 reference standard BCR576. Reference ranges for E2 and E1 in healthy, postmenopausal women were obtained, for E2: 3.8 to 36 pmol/L, for E1: 22 to 122 pmol/L. We measured and confirmed ultra-low E2 and E1 concentrations in sera from patients on the aromatase inhibitors letrozole or exemestane. CONCLUSION: This ultrasensitive LC-MS/MS method is suitable for routine assessment of serum E1 and E2 levels in breast cancer patients during estrogen suppression therapy. The method satisfies all requirements for measurement of E2 in the clinical setting as stated by the Endocrine Society in 2013. PRECIS: We report an ultrasensitive LCMS/MS routine assay that measures pretreatment and suppressed levels of estradiol/estrone during aromatase inhibitor treatment of postmenopausal breast cancer patients.

Effects of defined mixtures of POPs and endocrine disruptors on the steroid metabolome of the human H295R adrenocortical cell line.

The presence of environmental pollutants in our ecosystem may impose harmful health effects to wildlife and humans. Several of these toxic chemicals have a potential to interfere with the endocrine system. The adrenal cortex has been identified as the main target organ affected by endocrine disrupting chemicals. The aim of this work was to assess exposure effects of defined and environmentally relevant mixtures of chlorinated, brominated and perfluorinated chemicals on steroidogenesis, using the H295R adrenocortical cell line model in combination with a newly developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. By using this approach, we could simultaneously analyze 19 of the steroids in the steroid biosynthesis pathway, revealing a deeper insight into possible disruption of steroidogenesis. Our results showed a noticeable down-regulation in steroid production when cells were exposed to the highest concentration of a mixture of brominated and fluorinated compounds (10,000-times human blood values). In contrast, up-regulation was observed with estrone under the same experimental condition, as well as with some other steroids when cells were exposed to a perfluorinated mixture (1000-times human blood values), and the mixture of chlorinated and fluorinated compounds. Interestingly, the low concentration of the perfluorinated mixture alone produced a significant, albeit small, down-regulation of pregnenolone, and the total mixture a similar effect on 17-hydroxypregnenolone. Other mixtures resulted in only slight deviations from the control. Indication of synergistic effects were noted when we used a statistical model to improve data interpretation. A potential for adverse outcomes of human exposures is indicated, pointing to the need for further investigation into these mixtures.

LC-MS/MS based profiling and dynamic modelling of the steroidogenesis pathway in adrenocarcinoma H295R cells.

Endocrine disrupting chemicals have been reported to exert effects directly on enzymes involved in steroid biosynthesis. Here, we present a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for profiling the steroid metabolome of H295R human adrenocarcinoma cells. Our method can simultaneously analyse 19 precursors, intermediates and end-products, representing the adrenal steroid biosynthesis pathway. In order to obtain better insights into the processes of steroidogenesis, we investigated the dose-response relationship of forskolin, an activator of adenylate cyclase, on steroid production in H295R cells. We observed that 1.5 µM forskolin stimulated steroid production at approximately 50% of the maximum rate for most steroids. Hence, we studied the time course for steroid synthesis over 72 h in H295R cells that were stimulated with forskolin. At 24 h, we observed a peak in steroid levels for the intermediate metabolites, such as progesterone and pregnenolone, while end-products such as testosterone and cortisol continued to increase until 72 h. Finally, we show how global data provide a unique basis to develop a comprehensive, dynamic model for steroidogenesis using first order kinetics. The timeline data made it possible to estimate all reaction rate constants of the network. We propose this method as a unique and sensitive screening tool to identify effects on adrenal steroidogenesis by endocrine disrupting compounds.

Simultaneous assay of cortisol and dexamethasone improved diagnostic accuracy of the dexamethasone suppression test.

OBJECTIVES: The overnight dexamethasone (DXM) suppression test (DST) has high sensitivity, but moderate specificity, for diagnosing hypercortisolism. We have evaluated if simultaneous measurement of S-DXM may correct for variable DXM bioavailability and increase the diagnostic performance of DST, and if saliva (sa) is a feasible adjunct or alternative to serum. DESIGN AND METHODS: Prospective study of DST was carried out in patients with suspected Cushing's syndrome (CS) (n = 49), incidentaloma (n = 152) and healthy controls (n = 101). Cortisol, cortisone and DXM were assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Three hundred and two subjects underwent DST; S-cortisol was ≥50 nmol/L in 83 patients, of whom 11 had CS and 27 had autonomous cortisol secretion. The lower 2.5 percentile of S-DXM in subjects with negative DST (n = 208) was 3.3 nmol/L, which was selected as the DXM cut-off level. Nine patients had the combination of low S-DXM and positive DST. Of these, three had been misdiagnosed as having autonomous cortisol secretion. DST results were highly reproducible and confirmed in a replication cohort (n = 58). Patients with overt CS had significantly elevated post-DST sa-cortisol and sa-cortisone levels compared with controls; 23 of 25 with autonomous cortisol secretion had elevated sa-cortisone and 14 had elevated sa-cortisol. CONCLUSIONS: Simultaneous measurement of serum DXM and cortisol reduced false-positive DSTs by 20% and improved the specificity. S-DXM >3.3 nmol/L is sufficient for the suppression of cortisol <50 nmol/L. Measurement of glucocorticoids in saliva is a non-invasive and easy procedure and post-DST sa-cortisone was found particularly useful in the diagnosis of CS.

Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5.

BACKGROUND: Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. RESULTS: We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. CONCLUSION: The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene clusters in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries.

Proteomic response of human neuroblastoma cells to azaspiracid-1.

Azaspiracid-1 is a novel algal toxin, which causes an instantaneous rise of intracellular messengers, and an irreversible disarrangement of the actin cytoskeleton. Little is known regarding the molecular mechanisms that are involved in azaspiracid-1 toxicity. This study investigated global changes in protein expression by stable-isotope labelling with amino acids in culture and mass spectrometry, following exposure of human neuroblastoma cells to azaspiracid-1. The most highly upregulated proteins were involved in cellular energy metabolism, followed by cytoskeleton regulating proteins. The majority of downregulated proteins were involved in transcription, translation and protein modification. In addition, two proteins, component of oligomeric Golgi complex 5 and ras-related protein RAB1, which are involved in the maintenance of the Golgi complex and vesicle transport, respectively, were downregulated. Electron microscopy revealed a disruption of the Golgi complex by azaspiracid-1, and an accumulation of vesicles. In this study, the differential protein expression was examined prior to changes of the cytoskeleton structure in order to capture the primary effects of azaspiracid-1, however the observed changes were of unexpected complexity. Azaspiracid-1 caused a pronounced, but temporary depletion of ATP, which may be the reason for the observed complexity of cellular changes.

Identification of a saxitoxin biosynthesis gene with a history of frequent horizontal gene transfers.

The paralytic shellfish poisoning (PSP) toxins, saxitoxin, and its derivatives, are produced by a complex and unique biosynthetic pathway. It involves reactions that are rare in other metabolic pathways, however, distantly related organisms, such as dinoflagellates and cyanobacteria, produce these toxins by an identical pathway. Speculative explanations for the unusual phylogenetic distribution of this metabolic pathway have been proposed, including a polyphyletic origin, the involvement of symbiotic bacteria, and horizontal gene transfer. This study describes for the first time the identity of one gene, sxt1, that is involved in the biosynthesis of saxitoxin in cyanobacteria. It encoded an O-carbamoyltransferase (OCTASE) that was proposed to carbamoylate the hydroxymethyl side chain of saxitoxin precursor. Orthologues of sxt1 were exclusively present in PSP-toxic strains of cyanobacteria and had a high sequence similarity to each other. L. wollei had a naturally mutated sxt1 gene that encoded an inactive enzyme, and was incapable of producing carbamoylated PSP-toxin analogues, supporting the proposed function of Sxt1. Phylogenetic analysis revealed that OCATSE genes were present exclusively in prokaryotic organisms and were characterized by a high rate of horizontal gene transfer. OCTASE has most likely evolved from an ancestral O-sialoglycoprotein endopeptidase from proteobacteria, whereas the most likely phylogenetic origin of sxt1 was an ancestral alpha-proteobacterium. The phylogeny of sxt1 suggested that the entire set of genes required for saxitoxin biosynthesis may spread by horizontal gene transfer.

Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria.

Saxitoxin (STX) and its analogues cause the paralytic shellfish poisoning (PSP) syndrome, which afflicts human health and impacts coastal shellfish economies worldwide. PSP toxins are unique alkaloids, being produced by both prokaryotes and eukaryotes. Here we describe a candidate PSP toxin biosynthesis gene cluster (sxt) from Cylindrospermopsis raciborskii T3. The saxitoxin biosynthetic pathway is encoded by more than 35 kb, and comparative sequence analysis assigns 30 catalytic functions to 26 proteins. STX biosynthesis is initiated with arginine, S-adenosylmethionine, and acetate by a new type of polyketide synthase, which can putatively perform a methylation of acetate, and a Claisen condensation reaction between propionate and arginine. Further steps involve enzymes catalyzing three heterocyclizations and various tailoring reactions that result in the numerous isoforms of saxitoxin. In the absence of a gene transfer system in these microorganisms, we have revised the description of the known STX biosynthetic pathway, with in silico functional inferences based on sxt open reading frames combined with liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates. Our results indicate the evolutionary origin for the production of PSP toxins in an ancestral cyanobacterium with genetic contributions from diverse phylogenetic lineages of bacteria and provide a quantum addition to the catalytic collective available for future combinatorial biosyntheses. The distribution of these genes also supports the idea of the involvement of this gene cluster in STX production in various cyanobacteria.