Paal Methlie

Effect of smoking on the serum levels of 25-hydroxyvitamin D depends on the assay employed

OBJECTIVE Because we found higher serum 25-hydroxyvitamin D (25(OH)D) levels among smokers than among non-smokers with analyses using an electrochemiluminescence immunoassay (ECLIA) from Roche, the purpose of the present study was to examine whether this difference between smokers and non-smokers was maintained using other serum 25(OH)D assays. DESIGN A cross-sectional population-based study on 6932 participants from the Tromsø study, 1994-1995, and one validation study comparing six different serum 25(OH)D assays in 53 non-smokers and 54 smokers were performed. METHODS The association between smoking, season and serum 25(OH)D as measured by ECLIA (Roche) was assessed in the population-based study using general linear models with multivariate adjustments. In the validation study, serum levels of 25(OH)D were analysed with liquid chromatography coupled with mass spectrometry assay from two different laboratories, RIA (DiaSorin), HPLC, RIA (IDS) and ECLIA (Roche). T-tests and linear mixed model analyses were performed to compare the serum 25(OH)D levels in smokers and non-smokers within and between the methods. RESULTS In the population-based study, the serum levels of 25(OH)D using the ECLIA method were 51.9, 53.2 and 72.0 nmol/l in never, former and current smokers (P<0.01). In the validation study, the serum concentration of 25(OH)D was 10.3 nmol/l higher in smokers than in non-smokers (P<0.01) using the ECLIA (Roche), while non-significantly lower serum levels of 25(OH)D were found in smokers using the other five methods. CONCLUSIONS These two studies indicate that the ECLIA (Roche) overestimates serum 25(OH)D levels in smokers by unknown mechanisms. If confirmed, this might have clinical consequences, and the issue needs further exploration.

Continuous Subcutaneous Hydrocortisone Infusion versus Oral Hydrocortisone Replacement for Treatment of Addison's Disease: A Randomized Clinical Trial

CONTEXT Conventional glucocorticoid replacement therapy fails to mimic the physiological cortisol rhythm, which may have implications for morbidity and mortality in patients with Addisons disease. OBJECTIVE The objective of the study was to compare the effects of continuous sc hydrocortisone infusion (CSHI) with conventional oral hydrocortisone (OHC) replacement therapy. DESIGN, PATIENTS, AND INTERVENTIONS This was a prospective crossover, randomized, multicenter clinical trial comparing 3 months of treatment with thrice-daily OHC vs CSHI. From Norway and Sweden, 33 patients were enrolled from registries and clinics. All patients were assessed at baseline and after 8 and 12 weeks in each treatment arm. MAIN OUTCOME MEASURES The morning ACTH level was the primary outcome measure. Secondary outcome measures were effects on metabolism, health-related quality of life (HRQoL), sleep, and safety. RESULTS CSHI yielded normalization of morning ACTH and cortisol levels, and 24-hour salivary cortisol curves resembled the normal circadian variation. Urinary concentrations of glucocorticoid metabolites displayed a normal pattern with CSHI but were clearly altered with OHC. Several HRQoL indices in the vitality domain improved over time with CSHI. No benefit was found for either treatments for any subjective (Pittsburgh Sleep Quality Index questionnaire) or objective (actigraphy) sleep parameters. CONCLUSION CSHI safely brought ACTH and cortisol toward normal circadian levels without adversely affecting glucocorticoid metabolism in the way that OHC did. Positive effects on HRQoL were noted with CSHI, indicating that physiological glucocorticoid replacement therapy may be beneficial and that CSHI might become a treatment option for patients poorly controlled on conventional therapy.

Increased Systemic Cortisol Metabolism in Patients With Schizophrenia and Bipolar Disorder: A Mechanism for Increased Stress Vulnerability?

OBJECTIVE The hypothalamic-pituitary-adrenal (HPA) axis seems dysregulated and part of the pathophysiology in bipolar disorder and schizophrenia, but the underlying mechanisms are unknown. Recent evidence indicates that systemic cortisol metabolism influences blood cortisol levels and HPA axis functioning. Our objective was to estimate systemic cortisol metabolism by means of the activity of 5α-reductase, 5β-reductase, and 11β-hydroxysteroid dehydrogenase (11β-HSD) in patients with bipolar disorder and schizophrenia spectrum disorders compared to healthy controls. METHOD Inpatients and outpatients aged 18 to 65 years with DSM-IV bipolar disorder (n = 69) or schizophrenia (n = 87) were consecutively recruited to the catchment area-based Thematically Organized Psychosis Research (TOP) study. Healthy controls (n = 169) were randomly selected from statistical records from the same catchment area and were contacted by letter inviting them to participate. Spot urine samples were collected in a cross-sectional manner from November 2006 to November 2008. Urinary free cortisol and cortisone and their metabolites were analyzed with liquid chromatography tandem mass spectrometry and used as indicators of 5α-reductase, 5β-reductase, and 11β-HSD activity. RESULTS The combined patient group had increased activity of 5α-reductase, 5β-reductase, and 11β-HSD2 (all P < .001) compared to controls. Elevated systemic cortisol metabolism was present in both schizophrenia (5α-reductase, 5β-reductase, and 11β-HSD2; all P < .001) and bipolar disorder (5α-reductase [P = .016], 5β-reductase [P = .001], and 11β-HSD2 [P = .007]). CONCLUSIONS The results indicate increased activity of cortisol metabolism in patients with bipolar disorder and schizophrenia compared to healthy controls and suggest that increased systemic cortisol metabolism is involved in the pathophysiology and stress vulnerability in these severe mental disorders. The findings should be explored further in terms of potential new drug targets, and they add to the physiologic rationale for stress coping strategies in these patient groups.

Multisteroid LC–MS/MS assay for glucocorticoids and androgens and its application in Addison's disease

Objective Liquid chromatography–tandem mass spectrometry (LC–MS/MS) offers superior analytical specificity compared with immunoassays, but it is not available in many regions and hospitals due to expensive instrumentation and tedious sample preparation. Thus, we developed an automated, high-throughput LC–MS/MS assay for simultaneous quantification of ten endogenous and synthetic steroids targeting diseases of the hypothalamic–pituitary–adrenal axis and gonads. Methods Deuterated internal standards were added to 85 μl serum and processed by liquid–liquid extraction. Cortisol, cortisone, prednisolone, prednisone, 11-deoxycortisol, dexamethasone, testosterone, androstenedione and progesterone were resolved by ultra-high-pressure chromatography on a reversed-phase column in 6.1 min and detected by triple-quadrupole mass spectrometry. The method was used to assess steroid profiles in women with Addisons disease (AD, n=156) and blood donors (BDs, n=102). Results Precisions ranged from 4.5 to 10.1% relative standard deviations (RSD), accuracies from 95 to 108% and extraction recoveries from 60 to 84%. The method was practically free of matrix effects and robust to individual differences in serum composition. Most postmenopausal AD women had extremely low androstenedione concentrations, below 0.14 nmol/l, and median testosterone concentrations of 0.15 nmol/l (interquartile range 0.00–0.41), considerably lower than those of postmenopausal BDs (1.28 nmol/l (0.96–1.64) and 0.65 nmol/l (0.56–1.10) respectively). AD women in fertile years had androstenedione concentrations of 1.18 nmol/l (0.71–1.76) and testosterone concentrations of 0.44 nmol/l (0.22–0.63), approximately half of those found in BDs of corresponding age. Conclusion This LC–MS/MS assay provides highly sensitive and specific assessments of glucocorticoids and androgens with low sample volumes and is suitable for endocrine laboratories and research. Its utility has been demonstrated in a large cohort of women with AD, and the data suggest that women with AD are particularly androgen deficient after menopause.

Grapefruit juice and licorice increase cortisol availability in patients with Addison's disease

OBJECTIVE Failure to mirror the diurnal cortisol profile could contribute to the impaired subjective health status in Addisons disease (AD). Some patients report benefit from the use of various nutritional compounds. The objective of this study was to investigate the impact of licorice and grapefruit juice (GFJ) on the absorption and metabolism of cortisone acetate (CA). DESIGN Patients (n=17) with AD on stable CA replacement therapy were recruited from the outpatient clinic at Haukeland University Hospital, Norway. They were assessed on their ordinary CA medication and following two 3-day periods of co-administration of licorice or GFJ. METHODS Time series of glucocorticoids (GCs) in serum and saliva were obtained, and GCs in 24 h urine samples were determined. The main outcome measure was the area under the curve (AUC) for serum cortisol in the first 2.6 h after orally administered CA. RESULTS Compared with the ordinary treatment, the median AUC for serum cortisol increased with licorice (53 783 vs 50 882, P<0.05) and GFJ (60 661 vs 50 882, P<0.05). Median cortisol levels in serum were also elevated 2.6 h after tablet ingestion (licorice 223 vs 186 nmol/l, P<0.05; GFJ 337 vs 186 nmol/l, P<0.01). Licorice increased the median urinary cortisol/cortisone ratio (0.43 vs 0.21, P<0.00001), whereas GFJ increased the (allo-tetrahydrocortisol+tetrahydrocortisol)/tetrahydrocortisone ratio (0.55 vs 0.43, P<0.05). CONCLUSION Licorice and in particular GFJ increased cortisol available to tissues in the hours following oral CA administration. Both patients and physicians should be aware of these interactions.

Altered systemic cortisol metabolism in bipolar disorder and schizophrenia spectrum disorders

Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is suggested as a pathophysiological factor in bipolar disorder and schizophrenia. Increased clearance of cortisol was recently indicated as a component in the HPA axis hyperdrive. The aim of the present study was to test the model of increased cortisol metabolism in a new replication sample separately and combined with a previously published sample of bipolar disorder and schizophrenia. Spot urine was sampled from 212 healthy controls (HC) and 221 patients with a schizophrenia spectrum disorder (SCZ, n = 115) and bipolar disorder (BD, n = 106). Of these, a subsample of 169 HC and 155 patients was included in a previous report. Urinary free cortisol, cortisone and their metabolites were measured, and the activities of 5α-reductase, 5β-reductase and 11β-HSD were estimated and analyzed for differences between groups. In the new sample, there was increased enzyme activity in SCZ for 5β-reductase (p = 0.024 vs HC; p = 0.027 vs BD) and 11β-HSD2 (p = 0.014 vs HC; p = 0.004 vs BD). In the combined sample, there was increased activity in SCZ for 5α-reductase (p < 0.001 vs HC; p = 0.020 vs BD), 5β-reductase (p < 0.001 vs HC; p = 0.045 vs BD) and 11β-HSD2 (p < 0.001 vs HC; p = 0.043 vs BD), and in BD for 5β-reductase (p = 0.002), 11β-HSD2 (p = 0.039) and 5α-reductase (trend, p = 0.084) (all vs HC). The findings confirm increased systemic cortisol metabolism in BD and SCZ. This is most consistent in SCZ, with BD taking an intermediate position. The design makes it impossible to determine the direction of the effect. However, the findings merit further study of cortisol metabolism as a possible component in the HPA axis dysfunction and pathophysiology of BD and SCZ.

Circadian hormone profiles and insulin sensitivity in patients with Addison's disease: a comparison of continuous subcutaneous hydrocortisone infusion with conventional glucocorticoid replacement therapy.

Conventional glucocorticoid replacement therapy in patients with Addisons disease (AD) is unphysiological with possible adverse effects on mortality, morbidity and quality of life. The diurnal cortisol profile can likely be restored by continuous subcutaneous hydrocortisone infusion (CSHI).

SRD5A2 is associated with increased cortisol metabolism in schizophrenia spectrum disorders

OBJECTIVE Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is documented in bipolar disorder and schizophrenia, but the mechanism is unclear; recently, increased activity of cortisol metabolizing enzymes was indicated in these disorders. We investigated whether five genes involved in cortisol metabolism were associated with altered activity of cortisol metabolizing enzymes in bipolar disorder (BD) and schizophrenia spectrum disorders (SCZ). METHODS A case-control sample of subjects with BD (N=213), SCZ (N=274) and healthy controls (N=370) from Oslo, Norway, were included and genotyped from 2003 to 2008. A sub-sample (healthy controls: N=151; SCZ: N=40; BD: N=39) had estimated enzyme activities based on measurements of urinary free cortisol, urinary free cortisone and metabolites. A total of 102 single nucleotide polymorphisms (SNPs) in the SRD5A1, SRD5A2, AKR1D1, HSD11B1 and HSD11B2 genes were genotyped, and significant SNPs analyzed in the sub-sample. RESULTS There was a significant association of rs6732223 in SRD5A2 (5α-reductase) with SCZ (p=0.0043, Bonferroni corrected p=0.030, T risk allele). There was a significantly increased 5α-reductase activity associated with rs6732223 (T allele) within the SCZ group (p=0.011). CONCLUSIONS The present data suggest an interaction between SCZ and SRD5A2 variants coding for the enzyme 5α-reductase, giving rise to increased 5α-reductase activity in SCZ. The findings may have implications for cortisol metabolizing enzymes as possible drug targets.

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 Cushings 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.

Effect of a pre-exercise hydrocortisone dose on short-term physical performance in female patients with primary adrenal failure

OBJECTIVE Many patients with primary adrenal insufficiency (Addisons disease) take extra doses of glucocorticoids during stressful events, but a benefit has not been demonstrated in controlled trials. Here, we investigated the effects of a pre-exercise hydrocortisone dose on cardiorespiratory, hormonal and metabolic parameters in response to short-term strenuous physical activity. DESIGN This was a randomized placebo-controlled, two-week cross-over clinical trial. PARTICIPANTS Ten women with Addisons disease and 10 age-matched healthy females participated in the study. MEASUREMENTS All women in the study underwent maximal incremental exercise testing. A stress dose of 10 mg hydrocortisone or placebo was given 1 h prior to exercise on two occasions. Blood samples were drawn before, and 0, 15 and 30 min post exercise. Oxygen uptake, maximal aerobic capacity, endocrine and metabolic responses to physical activity, as well as health status by questionnaires were evaluated. RESULTS Maximal aerobic capacity and duration of exercise were significantly lower in patients than in healthy subjects and did not improve with the treatment. After an extra hydrocortisone dose serum cortisol was significantly higher than in the healthy subjects (P<0.001). Post-exercise glucose and adrenaline levels were significantly lower and free fatty acids insignificantly higher in patients irrespective of stress dose. Stress dosing did not alter other metabolic or hormonal parameters or quality of life after the exercise. CONCLUSIONS The patients did not benefit from an extra dose of hydrocortisone in short strenuous exercise. Stress dosing may not be justified in this setting. Whether stress dosing is beneficial in other types of physical activity will have to be examined further.