Steroidogenesis in patients with adrenal incidentalomas: Extended steroid profile measured by liquid chromatography-mass spectrometry after ACTH stimulation and dexamethasone suppression.

OBJECTIVE: Describe the secretion and profile of adrenal steroids in patients with adrenal incidentalomas compared to control subjects. DESIGN, SETTING AND PARTICIPANTS: A prospective study, 73 patients with adrenal incidentalomas, 21 bilateral and 52 unilateral and 34 matched controls in University Hospital. METHODS: Collect fasting blood sample before and 60 min after ACTH test (250 µg IV). One week later, perform overnight 1 mg dexamethasone test. The following steroids were measured by liquid chromatography-mass spectrometry (LC-MS): pregnenolone, 17-OH pregnenolone, 17-OH progesterone, 11-deoxycorticosterone, 11-deoxyortisol, 21-deoxycortisol, corticosterone, cortisol, androstenedione and aldosterone. RESULTS: Mean baseline serum cortisol was higher in incidentalomas, bilateral 361 ± 124, (range 143-665) nmol/L,(p < .0001), unilateral 268 ± 89 3.2 (range 98-507) nmol/L (p < .019) compared to controls 207 ± 100 (range 72-502) nmol/L. ACTH stimulation showed significantly higher levels in bilateral and unilateral cases compared to controls. After dexamethasone, mean serum cortisol levels suppressed in bilaterals 89 ± 69 (range 30-3) nmol/L (p < .0001), 58 ± 52 (range 16-323) nmol/L in unilateral (p < .01) compared to 26 ± 9 (range 7-46) nmol/L in controls. Mean baseline serum corticosterone was higher in bilateral 9.3 ± 4.8 (range 2.4-18.4) nmol/L (p < .005) and unilateral 7.3 ± 5.7 (range 0.1-30.3) nmol/L (p < .01) compared to controls 4.2 ± 2.4 (range 1.1-10.2) nmol/L, after ACTH stimulation significantly increased to higher levels in bilateral (p < .0002) and unilateral cases (p < .044) compared to controls. After dexamethasone, mean levels were 2.5 ± 2.6 (range 0.5-12.5) nmol/L in bilateral (p < .0006), 1.5 ± 1.6 (range 0.3-9.3) nmol/L in unilateral (p < .09) and 0.75 ± 0.46 (range 0.1-2.1) nmol/L in controls. Mean baseline serum 11-deoxycorticosterone (DOC) was higher in bilaterals 0.32 ± 0.23 (range 0.08-1.1) nmol/L (p < .03) compared to controls 0.15 ± 0.21 (range 0.08-1.1) nmol/L. ACTH stimulation increased levels to 3.27 ± 1.72 (range 0.5-7.4) nmol/L in bilateral cases compared to controls 1.369 ± 1.53 (range 0.1-7.1) nmol/L (p < .0001). Dexamethasone decreased levels to baseline (p ns). There were significant differences in serum 21-deoxycortisol (p < .0002) and serum pregnenolone (p < .004) only after ACTH stimulation. CONCLUSIONS: There is increased activity in several steroid biosynthesis pathways and higher steroid levels in bilateral compared to unilateral cases and evidence of hypercortisolism in 30% unilateral and 62% of bilateral incidentalomas.

Laboratory monitoring of patients with hereditary tyrosinemia type I.

BACKGROUND: The prognosis of patients with Hereditary Tyrosinemia Type 1 (HT-1) has greatly improved with early detection through newborn screening and the introduction of nitisinone (NTBC) therapy. A recent guideline calls for periodic monitoring of biochemical markers and NTBC levels to tailor treatment; however, this is currently only achieved through a combination of clinical laboratory tests. We developed a multiplexed assay measuring relevant amino acids, succinylacetone (SUAC), and NTBC in dried blood spots (DBS) to facilitate treatment monitoring. METHODS: Tyrosine, phenylalanine, methionine, NTBC and SUAC were eluted from DBS with methanol containing internal standards for each analyte and analyzed by liquid chromatography tandem mass spectrometry over 6.5 min in the multiple reaction monitoring positive mode. RESULTS: Pre-analytical and analytical factors were studied and demonstrated a reliable assay. Chromatography resolved an unknown substance that falsely elevates SUAC concentrations and was present in all samples. To establish control and disease ranges, the method was applied to DBS collected from controls (n = 284) and affected patients before (n = 2) and after initiation of treatment (n = 29). In the treated patients SUAC concentrations were within the normal range over a wide range of NTBC levels. CONCLUSIONS: This assay enables combined, accurate measurement of revelevant metabolites and NTBC in order to simplify treatment monitoring of patients with HT-1. In addition, the use of DBS allows for specimen collection at home to facilitate more standardization in relation to drug and dietary treatment.

Correlation Between Size and Function of Unilateral and Bilateral Adrenocortical Nodules: An Observational Study.

OBJECTIVE. Adrenal incidentalomas occur in 5% of adults and can produce autonomous cortisol secretion that increases the risk of metabolic syndrome and cardiovascular disease. The objective of our study was to evaluate the relationship between adrenal nodule size measured on CT and autonomous cortisol secretion. SUBJECTS AND METHODS. In a prospective study of 73 patients 22-87 years old with incidentalomas, unilateral in 52 patients and bilateral in 21 patients, we measured maximum nodule diameter on CT and serum cortisol levels at 8:00 am, 60 minutes after the adrenocorticotropic hormone stimulation test, and after the dexamethasone suppression test. We also studied 34 age-, sex-, and body mass index-matched control subjects. Statistics used were Spearman correlation coefficients, t tests, ANOVA test, and multivariate analysis. RESULTS. The mean maximum diameter of unilateral nodules measured on CT was larger on the right (2.47 ± 0.98 [SD] cm) than on the left (2.04 ± 0.86 cm) (p = 0.01). In the bilateral cases, the mean diameter of the right nodules was 2.69 ± 0.93 cm compared with 2.13 ± 0.89 cm on the left (p = 0.06). Mean baseline serum cortisol level was significantly higher in the patients with incidentalomas (bilateral, 13.1 ± 4.5 mcg/dL [p < 0.001]; unilateral, 9.7 ± 3.2 mcg/dL [p = 0.019]) than in the control subjects (7.5 ± 3.6 mcg/dL). After dexamethasone suppression test, serum cortisol levels were suppressed to less than 1.8 mcg/dL in 100% of control subjects, 33% of patients with bilateral incidentalomas, and 62% of patients with unilateral incidentalomas (p < 0.001). There were significant correlations between maximum nodule diameter on CT and serum cortisol levels after the dexamethasone suppression test (ρ = 0.500; p < 0.001) and at baseline (ρ = 0.373; p = 0.003). CONCLUSION. Increasing size of adrenal nodules is associated with more severe hyper-cortisolism and less dexamethasone suppression; these cases need further evaluation and possibly surgery because of increased risks of metabolic syndrome and cardiovascular mortality.

Urinary fluticasone propionate-17beta-carboxylic acid to assess asthma therapy adherence.

Although the National Asthma Education and Prevention Program Expert Panel Report 3 recommends referral to specialists to address adherence, guidelines do not provide a tool to determine nonadherence. This study was designed to prospectively evaluate the characteristics of urinary analysis of fluticasone propionate-17beta-carboxylic acid (FP17betaCA) as a test to verify if a specific patient has not taken fluticasone propionate (FP) within 16-24 hours. Urine of asthmatic subjects was prospectively analyzed 16-24 hours after witnessed administration of orally inhaled FP using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis; limit of quantitation was 10.3 pg/mL. Results were compared with those from asthmatic subjects not receiving inhaled FP. Thirty asthmatic subjects receiving inhaled FP (2 oral inhalations of FP at 110 micrograms each or 1 oral inhalation twice daily of fluticasone and salmeterol in fixed combination at 250/50 micrograms for 1 week) were compared with 30 asthmatic subjects not receiving FP. FP17betaCA was detected in the urine of 30 of 30 asthmatic subjects receiving FP (median, interquartile range [IQR; 413.5, 212.8-1230.0] range 12.4-3290.0 pg/mL [corrected for urine creatinine: median, IQR {576.2, 188.1-1306.6} range 6.3-5425.9 ng/g Cr]) and was undetectable in 30 of 30 subjects not receiving inhaled FP. The sensitivity and specificity of LC-MS/MS to detect FP17betaCA in urine were 100% (95% exact binomial confidence interval, 88-100) and 100% (95% exact binomial confidence interval, 88-100), respectively. Analysis of FP17betaCA in urine provides a sensitive method that may be used to verify that a specific patient may not have administered FP within a 16- to 24-hour window before testing.

Increasing liquid chromatography-tandem mass spectrometry throughput by mass tagging: a sample-multiplexed high-throughput assay for 25-hydroxyvitamin D2 and D3.

BACKGROUND: The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography-tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D2 and D3 [25(OH)D2 and 25(OH)D3], which increased sample throughput 5-fold. METHODS: After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D2 and 25(OH)D3 for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay. RESULTS: TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 µg/L, 25(OH)D2], 2.2 nmol/L [0.9 µg/L, 25(OH)D3], and 10 nmol/L [4 µg/L, 25(OH)D2], 5 nmol/L [2 µg/L, 25(OH)D3], respectively. The assay was linear to 250 nmol/L (100 µg/L). Interassay CVs across the reportable range were 3.7%-15.2%. Spiked recoveries were 94%-119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D2 (n=1733) and 25(OH)D3 (n=7614) (R2=0.96 and 0.97), respectively. CONCLUSIONS: Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D2 and 25(OH)D3 allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.

Self-assembly of hydroxy(phenyl)iodonium ions in acidic aqueous solution: preparation, and X-ray crystal structures of oligomeric phenyliodine(III) sulfates.

The treatment of [(diacetoxy)iodo]benzene with sodium bisulfate leads to the formation of oligomeric cationic species resulting from self-assembly of hydroxy(phenyl)iodonium ions, [PhIOH](+), in an aqueous acidic media. Depending on the PhI(OAc)(2):NaHSO(4) ratio, three new oligomeric products have been isolated and characterized by X-ray crystallography. The treatment of 5 equiv of PhI(OAc)(2) with 1 equiv of NaHSO(4).H(2)O affords the previously unknown mu-oxo-[bis(acetoxy)iodo]benzene, PhI(OAc)OI(OAc)Ph, which was structurally characterized by X-ray crystallography. In the solid-state, this mu-oxo product forms an isolated diamond-core dimeric structure with pentagonal-planar iodine centers. The interaction of PhI(OAc)(2) with 1 equiv of NaHSO(4).H(2)O affords a solid-state polymeric phenyliodine(III) sulfate, [(PhIO)(3).SO(3)](n), in which bis(mu-oxo)triiodanyl dication repeat units are linked by sulfate anions. Three of these neighboring polymeric chains form pseudo triple-helix supramolecular structure, which translates along crystallographic b-axis. The reaction of PhI(OAc)(2) with 3 equiv of NaHSO(4).H(2)O results in the formation of a tetrameric phenyliodine(III) sulfate, PhI(OH)OSO(2)O(Ph)IOI(Ph)OSO(2)OI(OH)Ph, the solid state structure of which consists of mu-oxodiiodanyl dicationic species linked by sulfate anions to the terminal hydroxy(phenyl)iodonium groups. The pseudo double-helix structure of this compound formed by the strong intermolecular interactions between two neighbor mu-oxodiiodanyl dicationic fragments and four (per pseudo helix dimer) areas of sulfate anions to the terminal hydroxy(phenyl)iodonium groups interactions.

Renal medullary 11 beta-hydroxysteroid dehydrogenase type 1 in Dahl salt-sensitive hypertension.

The Dahl salt-sensitive rat is a widely used model of human salt-sensitive forms of hypertension. The kidney plays an important role in the pathogenesis of Dahl salt-sensitive hypertension, but the molecular mechanisms involved remain a subject of intensive investigation. Gene expression profiling studies suggested that 11 beta-hydroxysteroid dehydrogenase type 1 might be dysregulated in the renal medulla of Dahl salt-sensitive rats. Additional analysis confirmed that renal medullary expression of 11 beta-hydroxysteroid dehydrogenase type 1 was downregulated by a high-salt diet in SS-13BN rats, a consomic rat strain with reduced blood pressure salt sensitivity, but not in Dahl salt-sensitive rats. 11 beta-Hydroxysteroid dehydrogenase type 1 is known to convert inactive 11-dehydrocorticosterone to active corticosterone. The urinary corticosterone/11-dehydrocorticosterone ratio as well as urinary excretion of corticosterone was higher in Dahl salt-sensitive rats than in SS-13BN rats. Knockdown of renal medullary 11 beta-hydroxysteroid dehydrogenase type 1 with small-interfering RNA attenuated the early phase of salt-induced hypertension in Dahl salt-sensitive rats and reduced urinary excretion of corticosterone. Knockdown of 11 beta-hydroxysteroid dehydrogenase type 1 did not affect blood pressure in SS-13BN rats. Long-term attenuation of salt-induced hypertension was achieved with small hairpin RNA targeting renal medullary 11 beta-hydroxysteroid dehydrogenase type 1. In summary, we have demonstrated that suppression of 11 beta-hydroxysteroid dehydrogenase type 1 expression in the renal medulla attenuates salt-induced hypertension in Dahl salt-sensitive rats.

Suppression of 11beta-hydroxysteroid dehydrogenase type 1 with RNA interference substantially attenuates 3T3-L1 adipogenesis.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which regulates the local level of glucocorticoids, has been suggested to be involved in the development of obesity. A definitive functional role for 11beta-HSD1 in adipogenesis, however, remains to be established. We developed 3T3-L1 cell lines stably transfected with a small hairpin RNA (shRNA) targeting 11beta-HSD1. A shRNA containing two nucleotide substitutions was used as a control. Silencing of 11beta-HSD1 substantially attenuated the accumulation of lipid droplets and the expression of adipogenesis marker genes, which was induced by a mixture containing either corticosterone or dexamethasone. Silencing of 11beta-HSD1 increased the concentration of 11-dehydrocorticosterone in the culture supernatant but did not significantly affect the levels of corticosterone or dexamethasone. Translocation of glucocorticoid receptors to the nucleus in response to glucocorticoids was significantly attenuated by silencing 11beta-HSD1. The number of cells entering the S phase of the cell cycle following the induction of adipogenesis was significantly reduced by silencing 11beta-HSD1. 11beta-HSD1 shRNA delivered by lentiviral vectors after the induction of differentiation, however, did not affect the progression of adipogenesis. These results indicate that 11beta-HSD1 plays a significant functional role in the initiation of 3T3-L1 adipogenesis and provide new mechanistic insights into the role of 11beta-HSD1 in the development of obesity and related diseases.

Serum Cortisol Levels via Radioimmunoassay vs Liquid Chromatography Mass Spectrophotometry in Healthy Control Subjects and Patients With Adrenal Incidentalomas.

BACKGROUND: Adrenal incidentalomas (AIs) are present in 4% of adults. As many as 30% may secrete cortisol autonomously in the absence of specific signs of overt hypercortisolism, in a phenomenon called subclinical hypercortisolism (SH). Diagnosis of SH is established by serum cortisol resistance to dexamethasone suppression. METHODS: We compared serum cortisol concentrations, as determined by radioimmunoassay (RIA) and liquid chromatography/tandem mass spectronomy (LC/MS-MS), in 73 patients with AI group (52 with unilateral AI) and 34 control subjects in 3 scenarios: basal; after 1-mg dexamethasone suppression; and after 0.25-mg stimulation with cosyntropin, a synthetic derivative of adrenocorticotropic hormone (ACTH). To bolster evidence for the diagnosis of SH, we also measured salivary cortisol levels at 11 PM and after DST, as well as plasma ACTH and serum dehydroepiandrosterone sulfate (DHEA-S) levels. RESULTS: We observed significant positive correlation (r = 0.9345, P <.001) for all 318 pairs of serum cortisol values, as measured by both methods. CONCLUSIONS: Serum cortisol concentrations in patients with AI and in control subjects were very similar, as measured by RIA and LC/MS-MS.

Methodology, Criteria, and Characterization of Patient-Matched Thyroid Cell Lines and Patient-Derived Tumor Xenografts.

Objective: To investigate the molecular underpinnings of thyroid cancer, preclinical cell line models are crucial; however, ∼40% of these have been proven to be either duplicates of existing thyroid lines or even nonthyroid-derived lines or are not derived from humans at all. Therefore, we set out to establish procedures and guidelines that should proactively avoid these problems, which facilitated the creation of criteria to make valid preclinical models for thyroid cancer research. Design: Based on our recommendations, we systematically characterized all new cell lines that we generated by a standardized approach that included (1) determination of human origin, (2) exclusion of lymphoma, (3) DNA fingerprinting and histological comparisons to establish linkage to presumed tissue of origin, (4) examining thyroid differentiation by screening two to three thyroid markers, (5) examination of biological behavior (growth rate, tumorigenicity), and (6) presence of common thyroid cancer genetic changes (TP53, BRAF, PTEN, PIK3CA, RAS, TERT promoter, RET/PTC, PAX8/PPARγ, NF1, and EIF1AX). Results: We established seven new thyroid cell lines (LAM136, EAM306, SDAR1, SDAR2, JEM493, THJ529, and THJ560) out of 294 primary culture attempts, and 10 patient-derived tumor xenografts (PDTXs; MC-Th-95, MC-Th-374, MC-Th-467, MC-Th-491, MC-Th-493, MC-Th-504, MC-Th-524, MC-Th-529, MC-Th-560, and MC-Th-562) out of 67 attempts. All were successfully validated by our protocols. Conclusions: This standardized approach for cell line and PDTX characterization should prevent (or detect) future cross-contamination and ensure that only valid preclinical models are used for thyroid cancer research.

Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and Correlation of Results With Clinical Outcomes.

CONTEXT: Measurement of thyroglobulin (Tg) by mass spectrometry (Tg-MS) is emerging as a tool for accurate Tg quantification in patients with anti-Tg autoantibodies (TgAbs). OBJECTIVE: The objective of the study was to perform analytical and clinical evaluations of two Tg-MS assays in comparison with immunometric Tg assays (Tg-IAs) and Tg RIAs (Tg-RIAs) in a cohort of thyroid cancer patients. METHODS: A total of 589 samples from 495 patients, 243 TgAb-/252 TgAb+, were tested by Beckman, Roche, Siemens-Immulite, and Thermo-Brahms Tg and TgAb assays, two Tg-RIAs, and two Tg-MS assays. RESULTS: The frequency of TgAb+ was 58%, 41%, 27%, and 39% for Roche, Beckman, Siemens-Immulite, and Thermo-Brahms, respectively. In TgAb- samples, clinical sensitivities and specificities of 100% and 74%-100%, respectively, were observed across all assays. In TgAb+ samples, all Tg-IAs demonstrated assay-dependent Tg underestimation, ranging from 41% to 86%. In TgAb+ samples, the use of a common cutoff (0.5 ng/mL) for the Tg-MS, three Tg-IAs, and the USC-RIA improved the sensitivity for the Tg-MSs and Tg-RIAs when compared with the Tg-IAs. In up to 20% of TgAb+ cases, Tg-IAs failed to detect Tg that was detectable by Tg-MS. In Tg-RIAs false-high biases were observed in TgAb+ samples containing low Tg concentrations. CONCLUSIONS: Tg-IAs remain the method of choice for Tg quantitation in TgAb- patients. In TgAb+ patients with undetectable Tg by immunometric assay, the Tg-MS will detect Tg in up to 20% additional cases. The Tg-RIA will detect Tg in approximately 35% cases, but a significant proportion of these will be clinical false-positive results. The undetectable Tg-MS seen in approximately 40% of TgAb+ cases in patients with disease need further evaluation.

Synthesis and structure of amino acid-derived benziodazoles: new hypervalent iodine heterocycles.

New N-functionalized benziodazoles were prepared by the peracetic oxidation of 2-iodobenzamides derived from alanine or valine. X-ray crystal structural analysis of two benziodazole-based phenyliodonium derivatives provides insight into facile interchange between benziodazoles and iminium benziodoxoles under acidic or basic conditions. [reaction: see text]

Development and characterization of a differentiated thyroid cancer cell line resistant to VEGFR-targeted kinase inhibitors.

BACKGROUND: Vascular endothelial growth factor-targeted kinase inhibitors have emerged as highly promising therapies for radioiodine-refractory metastatic differentiated thyroid cancer. Unfortunately, drug resistance uniformly develops, limiting their therapeutic efficacies and thereby constituting a major clinical problem. APPROACH AND METHODS: To study acquired drug resistance and elucidate underlying mechanisms in this setting, BHP2-7 human differentiated thyroid cancer cells were subjected to prolonged continuous in vitro selection with 18 µM pazopanib, a clinically relevant concentration; acquisition of pazopanib resistance was serially assessed, with the resulting resistant cells thereafter subcloned and characterized to assess potential mechanisms of acquired pazopanib resistance. RESULTS: Stable 2- to 4-fold in vitro pazopanib resistance emerged in response to pazopanib selection associated with similar in vitro growth characteristics but with markedly more aggressive in vivo xenograft growth. Selected cells were cross-resistant to sunitinib and to a lesser extent sorafenib but not to MAPK kinase (MEK1/2) inhibition by GSK1120212. Genotyping demonstrated acquisition of a novel activating KRAS codon 13 GGC to GTT (glycine to valine) mutation, consistent with the observed resistance to upstream vascular endothelial growth factor receptor inhibition yet sensitivity to downstream MAPK kinase (MEK1/2) inhibition. CONCLUSIONS: Selection of thyroid cancer cells with clinically utilized therapeutics can lead to acquired drug resistance and altered in vivo xenograft behavior that can recapitulate analogous drug resistance observed in patients. This approach has the potential to lead to insights into acquired treatment-related drug resistance in thyroid cancers that can be subjected to subsequent validation in serially collected patient samples and that has the potential to yield preemptive and responsive approaches to dealing with this important clinical problem.

Companion-diagnostic testing limited to KRAS codons 12 and 13 misses 17% of potentially relevant RAS mutations in colorectal cancer.

BACKGROUND: KRAS codons 12 and 13 mutations are commonly used to identify colorectal carcinoma (CRC) patients who are unlikely to benefit from anti-EGFR therapy. However, humans have four different homologous RAS proteins and no routine screening is performed for the other mutation sites. Non-screened mutations may still be present in a significant subset of patients without KRAS codon 12 and 13 mutations. METHODS: We developed a LightCycler screening assay that encompasses codons 12, 13 and 61 of all RAS genes. Screen-positive specimens were characterized by Sanger sequencing. 130 CRC specimens were screened for all RAS genes. The results for KRAS codons 12 and 13 were compared with an FDA approved method (Qiagen). RESULTS: Twenty-nine of 130 specimens (22.3%) were positive for KRAS codons 12 and 13, with 100% congruence with the Qiagen method. Six additional specimens were identified to have mutations. One mutation in HRAS codon 61, two in KRAS codon 61, and three in NRAS codon 61. CONCLUSION: Limiting RAS testing to only KRAS codons 12 and 13 in companion diagnostic testing of CRC results in nearly 1/5 of patients with RAS mutations not being excluded from costly EGFR antagonist treatment, despite likely futility. Inclusion of all RAS genes in companion diagnostic screening is warranted.

Comparison of multiple steroid concentrations in serum and dried blood spots throughout the day of patients with congenital adrenal hyperplasia.

BACKGROUND/AIM: periodic measurement of plasma concentrations of cortisol precursors on a clinic visit may be of limited value in patients with congenital adrenal hyperplasia because it does not reflect a patient's circadian patterns of adrenal steroid secretion. Steroid profiling in dried blood spots (DBS) may allow for more frequent and sensitive monitoring. METHODS: we compared the agreement between 17α-hydroxyprogesterone (17-OHP) and androstenedione (D4A) levels determined from DBS samples and concurrently collected serum samples. Blood was drawn from 9 congenital adrenal hyperplasia patients every 4 h over a 24-hour period. Serum and DBS steroid levels were measured by liquid chromatography tandem mass spectrometry. RESULTS: DBS determinations of 17-OHP overestimated corresponding serum levels (mean difference 1.67 ng/ml), and underestimated D4A serum levels (mean difference 0.84 ng/ml). However, the DBS assay yielded excellent agreement (97%) with serum 17-OHP, but did considerably poorer for D4A (31%). CONCLUSIONS: our results indicate an excellent agreement between DBS and serum 17-OHP measurements to identify the peaks and troughs associated with an individual's circadian pattern. Larger-scale studies are required to evaluate the utility of DBS for home monitoring and to determine if more frequent monitoring leads to improved clinical outcomes.

Liquid chromatography-tandem mass spectrometry analysis of urinary fluticasone propionate-17beta-carboxylic acid for monitoring compliance with inhaled-fluticasone propionate therapy.

BACKGROUND: Inhaled corticosteroids including fluticasone propionate (FP) are the most effective treatment for persistent-asthma. Noncompliance ranging from 20% to 80% of treated patients is associated with substantial health care costs, morbidity and fatalities. A noninvasive test to assess FP treatment compliance is needed. The major metabolite of FP is FP-17beta-carboxylic acid (FP17betaCA) and is excreted in urine. This study demonstrates the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure FP17betaCA in urine and evaluation of FP17betaCA urinary elimination. EXPERIMENTAL: Fluorometholone was used as the internal standard. After acetonitrile precipitation, samples were extracted with dichloromethane, washed and dried. Reconstituted extract (60 microL) was subjected to reversed-phase chromatography and positive-ion mode LC-MS/MS analysis. Assay precision, linearity, recovery and sample stability were determined. Elimination evaluation included measurement of FP17betaCA in urine collected daily from human subjects before (day 1), during treatment (days 2-5; dose FP-110 microg 2 puffs/day), and following cessation of FP therapy (days 6-14; n=4). RESULTS: Linear range of the FP17betaCA assay was 10.3-9510pg/mL. Limit of quantitation (LOQ) was 10.3 pg/mL and recovery ranged from 85.8% to 111.9%. Inter-assay CVs were 7.4-12.0% for FP17betaCA concentrations of 11.1-5117 pg/mL. Urine FP17betaCA was absent in subjects prior to FP therapy, detectable (180-1991 ng FP17betaCA/g creatinine) throughout the dosing period and reached below the LOQ at 6 days after therapy cessation. CONCLUSIONS: Measurement of FP17betaCA by LC-MS/MS has acceptable analytical performance for clinical use. These data support the clinical utility of measuring FP17betaCA in urine to monitor patient compliance with FP therapy.

Renal interstitial corticosterone and 11-dehydrocorticosterone in conscious rats.

Deficiencies in the conversion between active and inactive glucocorticoids in the kidney can lead to hypertension. However, the significance of glucocorticoid metabolism in specific kidney regions in vivo is not clear, possibly in part due to the difficulty in measuring glucocorticoid levels in kidney regions in vivo. We used microdialysis techniques to sample renal interstitial fluid from conscious rats. The levels of corticosterone (active) and 11-dehydrocorticosterone (inactive) were analyzed by liquid chromatography-tandem mass spectrometry. Direct infusion of the 11beta-hydroxysteroid dehydrogenase (11beta-HSD) inhibitor carbenoxolone into the renal medulla induced hypertension, and significantly increased corticosterone levels and the corticosterone/11-dehydrocorticosterone ratio, an index of 11beta-HSD activity, in the renal medullary microdialysate, but not in urine or the plasma. Further characterization of conscious, untreated rats (n = 13-16) indicated that corticosterone concentrations (ng/ml) were 0.8 +/- 0.1, 1.0 +/- 0.1, 66.7 +/- 8.1, and 7.9 +/- 1.1 in cortical microdialysate, medullary microdialysate, the plasma, and urine, respectively. The corticosterone/11-dehydrocorticosterone ratios were 0.8 +/- 0.1, 0.6 +/- 0.1, 10.6 +/- 1.4, and 1.7 +/- 0.1, respectively, in these 4 types of sample. The expression level of 11beta-HSD1 was higher in the medulla than in the cortex, whereas 11beta-HSD2 was most enriched in the outer medulla. Microdialysate levels of corticosterone were approximately 1.6-fold higher in afternoons than in mornings, whereas plasma levels differed by 2.8-fold. These results demonstrated that corticosterone excess in the renal medulla might be sufficient to cause hypertension and provided the first characterization of renal interstitial glucocorticoids.