• Corticosteroid-binding globulin regulates cortisol pharmacokinetics.

      Perogamvros, Ilias; Aarons, Leon; Miller, A G; Trainer, Peter J; Ray, David W; Department of Endocrinology, Christie Hospital and Endocrine Sciences Research Group, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK . School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, UK . Department of Biochemistry, University Hospital of South Manchester, Wythenshawe, UK. (2010-11-05)
      Objective:  Corticosteroid-binding globulin (CBG) is the principal carrier for cortisol in the circulation. Variations in CBG binding capacity are predicted to alter total serum cortisol disposition, but free serum cortisol is believed to be unaffected. Unbound cortisol pharmacokinetics (PK) have not been studied in the context of CBG changes. We aimed to assess the regulation of cortisol PK by CBG. Design and subjects:  Women on oestrogens (OCP), patients homozygous for a non-functioning CBG variant (CBG null) and healthy controls (HV) were studied before and after IV and oral administration of hydrocortisone 20mg. Measurements:  PK parameters were studied for total serum cortisol (SerF), free serum cortisol (FreeF) and cortisone (FreeE), and salivary cortisol (SalF) and cortisone (SalE): area under the curve (AUC), clearance (CL), half-life, and volume of distribution (Vd). Results:  Following IV hydrocortisone, AUC and half-life of SerF were significantly higher in the OCP group and lower in the CBG null. SerF CL and Vd were significantly lower in the OCP group and increased in the CBG null, compared to HV. PK parameters for FreeF and the salivary biomarkers were not different between the CBG null and HV, although OCP patients still had higher AUC compared to HV and prolonged half-life. These findings were confirmed following oral hydrocortisone, but concentration-time profiles were highly heterogeneous and SalF interpretation was problematic due to oral contamination. Conclusions:  We have demonstrated that CBG has a distinct effect on cortisol PK. When CBG binding is disrupted, FreeF retains normal PK characteristics, although CBG null patients lack a CBG-bound pool of readily releasable cortisol. Women on oestrogens may have altered free serum cortisol kinetics and thus may be potentially overexposed to glucocorticoids.
    • Measurement of salivary cortisol with liquid chromatography-tandem mass spectrometry in patients undergoing dynamic endocrine testing.

      Perogamvros, Ilias; Owen, Laura J; Keevil, Brian G; Brabant, Georg E; Trainer, Peter J; Department of Endocrinology, Christie Hospital, Manchester, UK. (2009-03-19)
      Objective: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) eliminates cross-reactivity, which is a major limitation of immunoassays used for the measurement of salivary cortisol (SalC). We aimed to evaluate the potential of SalC measured by LC-MS/MS in patients undergoing assessment of the HPA axis. Design and patients: Cross-sectional study of 78 patients admitted for routine testing in a specialized endocrine unit. Measurements: Matched serum and saliva samples were collected from 68 patients who had a Short Synacthen Test (SST, 250 mcg IM) and 10 patients who had an Insulin Tolerance Test (ITT, insulin 0.15 U/kg IV). Serum cortisol (SerC) was measured with an automated immunoassay and SalC with LC-MS/MS. Adequate SerC responses were >500 nmol/l. Results: In all patients with adequate responses the relative increase in SalC was significantly higher than in SerC [6.4(0.3-26.1) vs 1.0(0.3-4.9), P<0.0001)]. The SerC-SalC relationship was better explained by an exponential rather than a linear model (R(2)=0.83 vs R(2)=0.65, both P<0.0001). Based on 59 patients with adequate SerC responses to an SST, an adequate SalC response was defined as 8.3 nmol/l. 7 patients following an SST and 3 patients following an ITT showed inadequate responses in both SerC and SalC, but two patients with CBG deficiency showed a low SerC with normal SalC. Conclusions: We have shown an excellent diagnostic sensitivity and specificity of LC-MS/MS SalC in the assessment of the HPA axis and superiority over SerC when CBG levels are altered. The exponential relationship between SerC and SalC supports the concept of CBG binding capacity saturation.
    • Novel corticosteroid-binding globulin variant that lacks steroid binding activity.

      Perogamvros, Ilias; Underhill, C; Henley, D E; Hadfield, K D; Newman, W G; Ray, David W; Lightman, S L; Hammond, G L; Trainer, Peter J; Department of Endocrinology, Christie Hospital, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom. (2010-10)
      BACKGROUND: Corticosteroid-binding globulin (CBG) is the principal carrier for glucocorticoids in the circulation and a regulator of their bioavailability. Inherited CBG deficiencies are rarely reported, and only three causative mutations in four families have been described. PATIENTS, METHODS, AND RESULTS: In a 26-yr-old female with hypotension, fatigue, and undetectable total serum cortisol at presentation, we have identified a novel homozygous c.776g>t transversion in exon 3 of the CBG (SERPINA6) gene. This results in a p.Gly237Val substitution that is predicted to influence the positioning of two β-sheets that constitute part of the CBG steroid-binding site. Two siblings were also homozygous for the variant, whereas her mother and an unaffected sibling were heterozygous. No other symptomatic family members were identified apart from the proband. Individuals homozygous for the variant had serum CBG levels below the reference range when measured by RIA, but CBG was unmeasurable in cortisol-binding capacity assays. In the same individuals, we observed very low baseline and stimulated total serum cortisol levels but normal free serum and salivary cortisol and plasma ACTH. In a study of ultradian cortisol pulsatility, increased pulse frequency was only observed in the proband. CONCLUSION: We describe a novel CBG variant that lacks steroid binding activity. All mutant homozygotes have very low total serum cortisol, but normal free serum cortisol levels. The only biochemical feature to distinguish the symptomatic subject was increased cortisol pulsatility, and we suggest that this may influence glucocorticoid signaling and contribute to symptoms previously associated with CBG deficiency.
    • Serum regulates cortisol bioactivity by corticosteroid-binding globulin dependent and independent mechanisms, as revealed by combined bioassay and physicochemical assay approaches.

      Perogamvros, Ilias; Kayahara, M; Trainer, Peter J; Ray, D W; Department of Endocrinology, Christie Hospital, Manchester, UK Endocrine Sciences Research Group, University of Manchester, Manchester, UK. (2011-02-21)
      Context:  Corticosteroid-binding globulin (CBG) is the principal carrier of natural glucocorticoids in the circulation and we hypothesized that it modulates glucocorticoid bioactivity. Alterations in CBG, the presence of non-cortisol, naturally occurring glucocorticoids and the use of potent, synthetic glucocorticoids, all make it difficult to assess adrenal activity in-vivo; these problems can be addressed by a glucocorticoid bioassay. Design and subjects:  A bioassay was developed for serum glucocorticoid bioactivity (GBA) and a physicochemical ultrafiltration-liquid chromatography-tandem mass spectrometry assay for free serum cortisol (FreeF). We studied individuals homozygous and heterozygous for a non-functioning CBG variant (CBG G237V) and healthy controls. Results:  FreeF concentrations were similar in healthy controls, and those with absent functional CBG, but surprisingly we found low GBA in CBG null individuals. This may suggest that CBG delivers cortisol to target cells. However, further experiments revealed that dilution of serum in the bioassay caused release of cortisol from CBG, resulting in elevated GBA measurements in all but the CBG G237V homozygotes. Furthermore, we identified a specific and potent inhibitory effect of high concentration serum on glucocorticoid sensitivity of the recipient cells used in the bioassay. Analysis of inflammatory synovial fluid, a filtrate of serum with lower CBG concentration, revealed elevated free cortisol compared to non-inflammatory synovial fluid, a change not attributable to interconversion between cortisol and cortisone. Conclusions:  Our findings reveal that dilution of CBG enhances cortisol release, and so bioactivity, and also that serum potently induces glucocorticoid resistance in target cells.
    • Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography-tandem mass spectrometry: application in basal and stimulated conditions.

      Perogamvros, Ilias; Owen, Laura J; Newell-Price, John; Ray, David W; Trainer, Peter J; Keevil, Brian G; Department of Endocrinology, The Christie, Manchester, UK. ilias.perogamvros@nhs.net (2009-11-01)
      Immunoassays used for the measurement of salivary cortisol are limited by variable interference from cortisone. Salivary cortisone is a consequence of the salivary glands expressing 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) which converts cortisol to cortisone. We report a combined salivary cortisol and cortisone (SalF and SalE respectively) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to address the cortisone cross-reactivity in cortisol immunoassays and as a tool to study 11beta-HSD2 activity. The method was linear up to 400 nmol/L for SalF and 200 nmol/L for SalE and the lower limits of quantitation were 0.39 nmol/L (SalF) and 0.78 nmol/L (SalE). No evidence of ion suppression was found and precision, accuracy and recovery were within internationally accepted limits. No interference was identified from 13 structurally related steroids. SalF, SalE and SalF/SalE were significantly greater in the morning than at bed-time and following stimulation of the adrenal glands. As serum cortisol increased, an exponential rise was observed in SalF and a linear increase in SalE which reached a plateau at higher SalF concentrations. We have developed a novel, robust LC-MS/MS assay for the combined measurement of SalF and SalE. Our results confirm the 11beta-HSD2 activity of the salivary glands resulting in high SalE concentrations and the enzyme saturation at high substrate concentrations. This method can be used as a simple, non-invasive and highly specific tool to assess the value of salivary cortisol as a surrogate for free serum cortisol and as a potential novel way to assess 11beta-HSD2 activity.