Persistent urinary incontinence in female Golden Retrievers following laser ablation of intramural ectopic ureters may be associated with the presence of historical urinary tract infection.

OBJECTIVE: To identify predictive factors for postoperative continence in female Golden Retrievers following cystoscopic-guided laser ablation of intramural ectopic ureters (CLA-EU). ANIMALS: 41 client-owned female entire Golden Retrievers with uni- or bilateral intramural ectopic ureter(s) were retrospectively enrolled. METHODS: Patients were diagnosed with ectopic ureters with a combination of ultrasonography and cystoscopy. CLA-EU was performed for all dogs so that each ureteral opening was considered to be in an appropriate position by a single operator. All dogs had short-term follow-up 4 weeks and long-term follow up > 10 weeks after the procedure via telephone, which included urinary continence scoring. Clinical factors and ultrasonographic and cystoscopic findings from initial presentation were evaluated to identify predictive factors for postoperative continence. RESULTS: Short-term urinary continence was achieved in 46.3% of dogs with no additional medical therapies. Presence of historical urinary tract infections prior to CLA-EU (OR, 0.130; 95% CI, 0.020 to 0.621; P = .018) was negatively correlated and ureteral dilatation (OR, 34.260; 95% CI, 1.813 to 2,143; P = .043) was positively correlated with likelihood of urinary continence. Long-term urinary continence was achieved in 63.4% of dogs, and presence of historical urinary tract infections was negatively prognostic (OR, 0.173; 95% CI, 0.023 to 0.856; P = .048). CLINICAL RELEVANCE: Female Golden Retrievers undergoing CLA-EU have similar outcomes to those reported for other mixed-breed cohorts with > 30% of dogs failing to regain urinary continence. Historical urinary tract infections were significantly associated with both short- and long-term urinary continence in our population.

Serum cortisol assay performance following the 1 mg overnight dexamethasone suppression test.

BACKGROUND: The 1 mg overnight dexamethasone suppression test (ONDST) is recommended for the differential diagnosis of Cushing's syndrome and the investigation of adrenal incidentalomas. Despite documented variation in serum cortisol immunoassay performance, little has been published regarding its effect on the ONDST. AIMS: Assess the performance of three immunoassay platforms (Roche Elecsys II, Abbott Alinity & Siemens Centaur) when compared to a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. METHODS: Samples (n = 77) sent to the laboratory as part of an ONDST were retrieved prior to disposal, anonymized, and analysed on all platforms. Samples with factors impacting immunoassay analysis quality were excluded. Results were statistically compared to an LC-MS/MS method that previously demonstrated excellent comparability to a candidate reference method. RESULTS: The Roche gen II showed a mean bias of -2.4 nmol/L and a Passing-Bablok fit of y = -0.9 + 0.97x. This was not affected by sex. The Abbott showed a mean bias -18.8 nmol/L, and a fit of y = -11.3 + 0.88x. This bias was -20.7 nmol/L in females versus -17.2 nmol/L in males. The Siemens had a mean bias of 2.3 nmol/L and a fit of y = 1.4 + 1.07x. This bias was 5.7 nmol/L in males versus -1.0 nmol/L in females. CONCLUSIONS: Clinicians should be aware of the method-dependent variation that exists within serum cortisol analysis during the ONDSTs. Roche and Siemens aligned more closely with LC-MS/MS while the Abbot may cause a reduction in ONDST sensitivity. This data supports assay-specific cut-offs for the ONDST.

Development of a total serum testosterone, androstenedione, 17-hydroxyprogesterone, 11ß-hydroxyandrostenedione and 11-ketotestosterone LC-MS/MS assay and its application to evaluate pre-analytical sample stability.

Background Classically, serum testosterone (T) and androstenedione (A4) have been the mainstay for the biochemical assessment of hyperandrogenism. However, recent evidence suggests 11ß-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) may also be important. Here, we describe the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitation of total serum T, A4, 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we applied the method to assess pre-analytical stability. Methods An isotopically labelled internal standard was added to samples prior to supported liquid extraction (SLE). Extracts were analysed using LC-MS/MS to detect T/A4/17OHP/11OHA4 and 11KT along with their corresponding internal standards. Samples (n = 7) were collected from healthy volunteers (n = 14) and left incubated at 20 °C for up to 72 h. Tubes were retrieved at select time points, centrifuged, separated and frozen prior to analysis. Results The total run time was 4 min. For all analytes, intra- and inter-assay imprecision did not exceed 7.9% and 5.3%, respectively; matrix effects were negligible and mean recoveries ranged from 95.3 to 111.6%. The limits of quantitation (LOQs) were 0.25 nmol/L for T, A4 and 11OHA4, 0.50 nmol/L for 17OHP, and 0.24 nmol/L for 11KT. No significant change was observed in pre-centrifugation A4 or female T concentrations over 72 h. Significant increases (p < 0.01) in concentrations of 11KT, 17OHP, 11OHA4 and male T were observed after 2, 8, 12 and 24 h, respectively. Conclusions We developed a robust LC-MS/MS assay for the quantitation of total serum T/A4/17OHP/11OHA4 and 11KT. Applying the method to determine pre-analytical stability suggests samples requiring 11KT need separating from the cells within 2 h.

Oestradiol measurement during fulvestrant treatment for breast cancer.

Biochemical evaluation of menopausal status is used to inform treatment decisions, including clinical trial eligibility in women with oestrogen receptor positive breast cancer. However, fulvestrant may interfere with oestradiol immunoassays and confound accurate assessment in this context. We conducted a service evaluation of two immunoassays and an LC-MS/MS assay to determine the extent of the interference. Serum oestradiol levels were analysed by two immunoassays (Siemens Centaur XP and Abbott Architect) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). Immunoassay gave higher serum oestradiol results than LC-MS/MS at low concentrations, with improved analytical sensitivity demonstrated by LC-MS/MS. Cross-reactivity of fulvestrant was observed for each immunoassay. We have shown that two commonly used immunoassays do not demonstrate the required sensitivity or specificity for the measurement of oestradiol in a breast cancer population. For patients receiving fulvestrant, spurious results may be generated that could impact treatment decisions. LC-MS/MS is recommended in this setting.

Development of a rapid liquid chromatography tandem mass spectrometry method for the quantitation of serum dexamethasone and its clinical verification.

Background Measurement of serum dexamethasone during the overnight dexamethasone-suppression test has been recommended to reduce false-positive results when investigating Cushing's syndrome or increasingly commonly found adrenal incidentalomas. Despite this, there remains a paucity of well-validated dexamethasone methods currently available. Here, we describe the development of a rapid and sensitive liquid chromatography tandem mass spectrometry serum dexamethasone assay and verify its utility in a cohort of postmenopausal females. Method Isotopically labelled internal standard was added to samples prior to supported liquid extraction. Extracts were analysed using liquid chromatography tandem mass spectrometry in the positive electrospray ionization mode. Multiple reaction monitoring was used to detect dexamethasone and its corresponding internal standard transitions. Normal healthy postmenopausal women ( n = 95) were recruited and underwent an overnight dexamethasone suppression test, with serum dexamethasone and cortisol measurements at 09:00 after administration of oral dexamethasone 1 mg at 23:00 the night before. Results Mean intra- and inter-assay imprecision were 4.1% and 2.9%, respectively, for dexamethasone concentrations of 1.5, 6.0 and 12.0 nmol/L. Matrix effects were found to be negligible at 106-109% with recovery ranging from 96 to 100%. The limit of quantitation was 0.25 nmol/L, and structural analogue analysis proved the method to be robust against interferences. Applying a serum dexamethasone cut-off of >3.3 nmol/L was associated with a serum cortisol ≤50 nmol/L in 84/95 individuals. Conclusion We have developed a sensitive and robust liquid chromatography tandem mass spectrometry method for the quantitation of serum dexamethasone. The method can be used to identify false-positive results during the overnight dexamethasone suppression test or for pharmacokinetic studies.

Evaluation of an Axial Pattern Flap Based on the Cranial Cutaneous Branch of the Saphenous Artery: A Cadaveric Perfusion Study.

OBJECTIVES: To confirm the anatomic location of the cranial cutaneous branch of the saphenous artery (CCSA), delineate the angiosome of the CCSA, and provide guidelines for clinical use of an axial pattern flap based on the CCSA. STUDY DESIGN: Anatomic study. ANIMALS: Greyhound cadavers (n=10). METHODS: Shortly after euthanasia, the CCSA was identified and isolated in each hindlimb. Methylene blue and radiographic perfusion studies were performed. The skin was freed from the thigh for photographic and radiographic images. The dimensions of the skin area suitable for use as an axial pattern flap were related to anatomical landmarks. Mock surgical elevation and transposition of the flap in 2 dogs allowed assessment of flap mobility and ease of donor site closure for clinical use. RESULTS: The CCSA was reliably identified in all dogs branching from the saphenous artery as it became superficial to the sartorius muscle, immediately distal to the caudal cutaneous branch and proximal to the genicular branches. Genicular branches were variably paired or singular. Perfusion studies defined the CCSA angiosome as the area cranial to the saphenous artery and caudal to the cranial border of the thigh, extending proximally from the level of the medial tibial condyle to two-thirds of the distance to the inguinal ring. CONCLUSION: An axial pattern flap based on the CCSA could be expected to cover skin defects of the cranial aspect of the distal thigh and stifle, the popliteal region caudal to the stifle, and the proximal medial crus.

Serum Cortisol: An Up-To-Date Assessment of Routine Assay Performance.

BACKGROUND: Accurate serum cortisol quantification is required for the correct diagnosis and management of adrenal pathologies. Presently, most laboratories use immunoassay to measure serum cortisol with proficiency schemes demonstrating a wide dispersion of results. Here, we investigate the effects of sex, matrix, and antibody specificity on serum cortisol quantification in 6 routine assays. METHODS: Surplus serum was obtained before disposal and the following cohorts were created: males, nonpregnant females, pregnant patients, and patients prescribed either metyrapone or prednisolone. Samples were anonymized and distributed to collaborating laboratories for cortisol analysis by 6 routine assays. Cortisol was also measured in all samples using an LC-MS/MS candidate reference measurement procedure (cRMP); cortisol-binding globulin (CBG) was measured in the nonpregnant and pregnant female cohorts. RESULTS: Considerable inter- and intraassay variation was observed across the male and nonpregnant female cohorts relative to the cRMP. Four immunoassays underrecovered cortisol in the pregnancy cohort, and CBG was found to be significantly higher in this cohort than in the nonpregnant females. In the metyrapone and prednisolone cohorts, all immunoassays overestimated cortisol. The first generation Roche E170 and Siemens Centaur XP were particularly prone to overestimation. In all cohorts the routine LC-MS/MS assay aligned extremely well with the cRMP. CONCLUSIONS: Despite the clinical importance of serum cortisol, the performance of routine immunoassays remains highly variable. Accurate quantification is compromised by both matrix effects and antibody specificity. Underpinning this study with a cRMP has highlighted the deficiencies in standardization across routine cortisol immunoassays.

A direct assay for the routine measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone by liquid chromatography tandem mass spectrometry.

BACKGROUND: The measurement of androgens in many laboratories is often limited to testosterone. To more accurately determine the androgen status in both sexes, the measurement of other androgens such as dihydrotestosterone, the more potent metabolite of testosterone, and androstenendione and dehydroepiandrosterone, the most abundant circulating androgens in women would be informative. We report a combined liquid chromatography tandem mass spectrometry method for the measurement of these androgens. METHODS: Internal standards in methanol (10 µL) were added to 100 µL serum followed by the addition of zinc sulphate (100 µL). After mixing, 100 µL of acetonitrile was added and was further mixed. The samples were centrifuged and the steroids extracted using an automated online solid phase extraction on a C18 cartridge by a Waters Acquity with online sample manager coupled to a TQS mass spectrometer. RESULTS: Separation of the androgens was achieved by liquid chromatography. The run time was 6.5 min per sample. The lower limit of quantitation was 0.1 nmol/L for testosterone, androstenedione and dihydrotestosterone and 1 nmol/L for dehydroepiandrosterone. The coefficient of variation of the assay in serum for testosterone was <6%, androstenedione <8% and dihydrotestosterone and dehydroepiandrosterone <10%. DISCUSSION: We have developed a rapid assay for the liquid chromatography tandem mass spectrometry measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone in a routine clinical laboratory. The assay requires a small volume of serum, and all analytes are measured simultaneously. The assay is rapid and simple to execute offering the potential for routine clinical application.

Candidate Reference Measurement Procedure for the Quantification of Total Serum Cortisol with LC-MS/MS.

BACKGROUND: Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Although certified reference materials (CRMs) are available to standardize cortisol measurements, External Quality Assessment (EQA) schemes still demonstrate a wide dispersion of results. We present a serum cortisol candidate reference measurement procedure that, through analysis of a Joint Committee for Traceability in Laboratory Medicine-listed panel of higher-order CRMs, provides metrologically traceable results. METHOD: Isotope-labeled internal standard was added to samples before supported liquid extraction. Extracts were analyzed with LC-MS/MS in positive electrospray ionization mode. Multiple reaction monitoring was used to detect cortisol and its corresponding internal standard transitions. We measured samples in triplicate over 3 days and calculated the mean result. RESULTS: Mean intra- and interassay imprecision were 1.3% and 1.5%, respectively, for concentrations of 154, 510, and 769 nmol/L. Ionization efficiency studies and structural analog analysis proved the method to be robust against interferences. Through analysis of 34 CRMs (83-764 nmol/L), expanded measurement uncertainty was calculated to be 5% (95% CI). The mean bias between the measured and target CRM concentrations was statistically insignificant at -0.08%. CONCLUSIONS: The accuracy and low measurement uncertainty of this method qualify it as a CRM procedure. Metrological traceability has been achieved through the analysis of higher-order CRMs. This method could be used to underpin serum cortisol EQA schemes to provide samples with a traceable target value, enabling participating laboratories to determine the accuracy and measurement uncertainty of their assays.

Liquid chromatography tandem mass spectrometry in the clinical laboratory.

Clinical laboratory medicine has seen the introduction and evolution of liquid chromatography tandem mass spectrometry in routine clinical laboratories over the last 10-15 years. There still exists a wide diversity of assays from very esoteric and highly specialist manual assays to more simplified kit-based assays. The technology is not static as manufacturers are continually making improvements. Mass spectrometry is now commonly used in several areas of diagnostics including therapeutic drug monitoring, toxicology, endocrinology, paediatrics and microbiology. Some of the most high throughput analyses or common analytes include vitamin D, immunosuppressant monitoring, androgen measurement and newborn screening. It also offers flexibility for the measurement of analytes in a variety of different matrices which would prove difficult with immunoassays. Unlike immunoassays or high-pressure liquid chromatography assays using ultraviolet or fluorescence detection, mass spectrometry offers better specificity and reduced interferences if attention is paid to potential isobaric compounds. Furthermore, multiplexing, which enables multiple analytes to be measured with the same volume of serum is advantageous, and the requirement for large sample volumes is decreasing as instrument sensitivity increases. There are many emerging applications in the literature. Using mass spectrometry to identify novel isoforms or modified peptides is possible as is quantification of proteins and peptides, with or without protein digests. Future developments by the manufacturers may also include mechanisms to improve the throughput of samples and strategies to decrease the level of skill required by the operators.

A widely applicable plasma renin activity assay by LC-MS/MS with offline solid phase extraction.

BACKGROUND: The measurement of plasma renin activity using LC-MS/MS is an attractive alternative to radioimmunoassay techniques. A published method for renin activity by LC-MS/MS uses equipment that is not available in all LC-MS/MS laboratories. Here, we present an offline modification to allow users of LC-MS/MS to perform this analysis without specialist equipment. METHODS: Samples were prepared in duplicate and incubated for 6.5 h and 24 h. Solid phase extraction was performed offline using Waters Oasis MAX ion-exchange 96-well plate. A method comparison was performed between this assay and a previously published assay using on-line solid phase extraction. RESULTS: The offline method for plasma rennin activity gave similar results to the on-line method across the concentration range. Analytical performance was also found to be comparable and the use of a 24 h incubation for low samples was also deemed unnecessary. DISCUSSION: The method described is an acceptable alternative to a previously published assay which does not require the use of highly specialist equipment and will be applicable to the majority of LC-MS/MS users.

A rapid direct assay for the routine measurement of oestradiol and oestrone by liquid chromatography tandem mass spectrometry.

BACKGROUND: Liquid chromatography tandem mass spectrometry (LC-MS/MS) is rapidly becoming the technology of choice for measuring steroid hormones. We have developed a rapid LC-MS/MS assay for the routine analysis of serum oestradiol and oestrone. The assay uses a relatively small volume and has a rapid run time. METHODS: Supported liquid extraction was performed on 250 µL of sample using methyl tertiary butyl ether. The extract was dried and reconstituted with 100 µL of 40% methanol. Online automated solid phase extraction was performed on 75 µL of extract using C18 cartridges on a Waters OSM coupled to a Waters TQS mass spectrometer. Serum samples (n = 197) were analysed by LC-MS/MS and a commercial immunoassay. RESULTS: The lower limit of quantitation for oestradiol and oestrone was 10 and 6 pmol/L, respectively. The coefficient of variation (CV) of the assay for oestradiol and oestrone concentrations of 125 pmol/L was <7%. The assay had a CV of 10% at 22 pmol/L for oestradiol and 5% at 16 pmol/L for oestrone. The average recovery for oestradiol was 102% and oestrone was 106%. The comparison with a commercial immunoassay gave the following equation: Immunoassay = 0.94 × LC-MS/MS + 21 pmol/L. The run time was 4.5 min per sample. DISCUSSION: We have developed a rapid assay for the LC-MS/MS measurement of oestradiol and oestrone which does not require derivatization in the sample preparation. The assay is suitable for routine clinical use or for clinical trials. The assay demonstrated superior performance compared to immunoassays at lower concentrations making it more suitable for use in males and patients on aromatase inhibitors.

Supported liquid extraction as an alternative to solid phase extraction for LC-MS/MS aldosterone analysis?

BACKGROUND: Supported liquid extraction (SLE) techniques are relatively new compared to other sample preparation approaches such as solid phase extraction (SPE), liquid-liquid extraction (LLE) and protein precipitation (PPE). We investigated the use of SLE as an alternative to SPE for the liquid chromatography tandem mass spectrometry (LC-MS/MS) measurement of aldosterone. METHODS: Samples (n = 83) were analysed by the routine method using SPE. The same samples were subsequently analysed using two different SLE 96-well plate devices (Thermo and Biotage) with methyl-tertiary butyl ether extraction. A direct comparison of the three extraction techniques on two different mass spectrometers was also performed. RESULTS: Both results using SLE plates gave excellent agreement with the results from the SPE analysis. The area counts obtained with the Biotage plates were considerably higher than those obtained using the Thermo plate. CONCLUSIONS: SLE is an acceptable alternative to SPE for the LC-MS/MS analysis of aldosterone. Using SLE reduces the time required for sample preparation.

Development of a rapid assay for the analysis of serum cortisol and its implementation into a routine service laboratory.

BACKGROUND: LC-MS/MS is rapidly becoming the technology of choice for measuring steroid hormones. We have developed a rapid LC-MS/MS assay for the routine analysis of serum cortisol. We have used this assay to investigate the effects of gender and exogenous steroid interference on the immunoassay measurement of serum cortisol. METHODS: Zinc sulphate (40 µL) was added to 20 µL of sample. This was vortexed for 10 s followed by the addition of 100 µL of internal standard in methanol. Following mixing and centrifugation, 10 µL of sample was injected into an Acquity LC system coupled to a Quattro Premier tandem mass spectrometer. Serum samples (n = 149) were analysed by LC-MS/MS and two commercial immunoassays. Results were then compared for all samples and for gender differences. A further set of serum samples (n = 171) was analysed by the LC-MS/MS assay and a GC-MS assay. RESULTS: Cortisol had a retention time of 0.98 min and the assay had an injection-to-injection time of 2.6 min per sample. Mean recovery was 99% and mean CV was 8%. The immunoassays gave comparisons of: Roche = 1.23 × LC-MS/MS -1.12 nmol/L and Abbott = 0.94 × LC-MS/MS + 11.97. The comparison with GC-MS showed LC-MS/MS = 1.11 × GC-MS - 22.90. DISCUSSION: We have developed an LC-MS/MS assay for serum cortisol analysis that is suitable for routine clinical use and has been in use in our laboratory for 12 months. The availability of this assay will give more reliable results in patients receiving exogenous steroid therapy.

Quantitation of aldosterone in human plasma by ultra high performance liquid chromatography tandem mass spectrometry.

Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC-MS/MS. Standards, quality controls and samples (250µL) were extracted using Oasis(®) HLB 96-well plates. Extract (30µL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5µm guard column, coupled to a Kinetex PFP, 100mm×2.1mm, 2.6µm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters(®) Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0>189.1 for aldosterone and 366.0>194.1 for d7-aldosterone respectively, which co-eluted at 2.65min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26pmol/L and 30pmol/L respectively. The assay was linear up to 3200pmol/L (r(2)=0.9999). Mean intra- and inter-assay imprecision and bias were all <10%. Comparison of the UPLC-MS/MS method with an immunoassay in routine clinical use in the UK yielded the equation UPLC-MS/MS=0.789(RIA)-41.7, linear regression r(2)=0.88, n=54. We have developed a sensitive and specific method for the extraction and measurement of aldosterone from human plasma. The method features a simple 96-well plate solid phase extraction procedure, highly selective column chemistry and short chromatographic run times.

Ranolazine stabilizes cardiac ryanodine receptors: a novel mechanism for the suppression of early afterdepolarization and torsades de pointes in long QT type 2.

BACKGROUND: Ranolazine (Ran) is known to inhibit multiple targets, including the late Na(+)current, the rapid delayed rectifying K(+)current, the L-type Ca(2+)current, and fatty acid metabolism. Functionally, Ran suppresses early afterdepolarization (EADs) and torsades de pointes (TdP) in drug-induced long QT type 2 (LQT2) presumably by decreasing intracellular [Na(+)](i) and Ca(2+)overload. However, simulations of EADs in LQT2 failed to predict their suppression by Ran. OBJECTIVE: To elucidate the mechanism(s) whereby Ran alters cardiac action potentials (APs) and cytosolic Ca(2+)transients and suppresses EADs and TdP in LQT2. METHODS: The known effects of Ran were included in simulations (Shannon and Mahajan models) of rabbit ventricular APs and Ca(2+)transients in control and LQT2 models and compared with experimental optical mapping data from Langendorff rabbit hearts treated with E4031 (0.5 µM) to block the rapid delayed rectifying K(+)current. Direct effects of Ran on cardiac ryanodine receptors (RyR2) were investigated in single channels and changes in Ca(2+)-dependent high-affinity ryanodine binding. RESULTS: Ran (10 µM) alone prolonged action potential durations (206 ± 4.6 to 240 ± 7.8 ms; P <0.05); E4031 prolonged action potential durations (204 ± 6 to 546 ± 35 ms; P <0.05) and elicited EADs and TdP that were suppressed by Ran (10 µM; n = 7 of 7 hearts). Simulations (Shannon but not Mahajan model) closely reproduced experimental data except for EAD suppression by Ran. Ran reduced open probability (P(o)) of RyR2 (half maximal inhibitory concentration = 10 ± 3 µM; n = 7) in bilayers and shifted half maximal effective concentration for Ca(2+)-dependent ryanodine binding from 0.42 ± 0.02 to 0.64 ± 0.02 µM with 30 µM Ran. CONCLUSIONS: Ran reduces P(o) of RyR2, desensitizes Ca(2+)-dependent RyR2 activation, and inhibits Ca(i) oscillations, which represents a novel mechanism for its suppression of EADs and TdP.

Designing calcium release channel inhibitors with enhanced electron donor properties: stabilizing the closed state of ryanodine receptor type 1.

New drugs with enhanced electron donor properties that target the ryanodine receptor from skeletal muscle sarcoplasmic reticulum (RyR1) are shown to be potent inhibitors of single-channel activity. In this article, we synthesize derivatives of the channel activator 4-chloro-3-methyl phenol (4-CmC) and the 1,4-benzothiazepine channel inhibitor 4-[-3{1-(4-benzyl) piperidinyl}propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (K201, JTV519) with enhanced electron donor properties. Instead of activating channel activity (~100 µM), the 4-methoxy analog of 4-CmC [4-methoxy-3-methyl phenol (4-MmC)] inhibits channel activity at submicromolar concentrations (IC(50) = 0.34 ± 0.08 µM). Increasing the electron donor characteristics of K201 by synthesizing its dioxole congener results in an approximately 16 times more potent RyR1 inhibitor (IC(50) = 0.24 ± 0.05 µM) compared with K201 (IC(50) = 3.98 ± 0.79 µM). Inhibition is not caused by an increased closed time of the channel but seems to be caused by an open state block of RyR1. These alterations to chemical structure do not influence the ability of these drugs to affect Ca(2+)-dependent ATPase activity of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase type 1. Moreover, the FKBP12 protein, which stabilizes RyR1 in a closed configuration, is shown to be a strong electron donor. It seems as if FKBP12, K201, its dioxole derivative, and 4-MmC inhibit RyR1 channel activity by virtue of their electron donor characteristics. These results embody strong evidence that designing new drugs to target RyR1 with enhanced electron donor characteristics results in more potent channel inhibitors. This is a novel approach to the design of new, more potent drugs with the aim of functionally modifying RyR1 single-channel activity.

Simultaneous analysis of cortisol and cortisone in saliva using XLC-MS/MS for fully automated online solid phase extraction.

Salivary cortisol measurements are increasingly being used in the investigation of disorders of the hypothalamic-pituitary-adrenal axis. In the salivary gland, cortisol is metabolised to cortisone by the action of 11ß-hydroxysteroid dehydrogenase type 2, and cortisone is partly responsible for the variable interference observed in current salivary cortisol immunoassays. The aim of this study was to validate an assay for the simultaneous analysis of salivary cortisol and cortisone using the Spark Holland Symbiosis™ in eXtraction liquid chromatography-tandem mass spectrometry (XLC-MS/MS) mode for fully automated online solid phase extraction (SPE). Saliva samples were diluted in water with the addition of internal standard (d4-cortisol and d7-cortisone). Online SPE was performed using the Spark Holland Symbiosis™ with HySphere™ C18 SPE cartridges and compounds were eluted onto a Phenomenex® C18 guard column attached to a Phenomenex® Onyx monolithic C18 column for chromatography. Mass spectrometry used the Waters® Xevo™ TQ MS in electrospray positive mode. Cortisol and cortisone eluted with their internal standards at 1.95 and 2.17 min, respectively, with a total run time of four minutes. No evidence of ion-suppression was observed. The assay was linear up to 3393 nmol/L for cortisol and 3676 nmol/L for cortisone, with lower limits of quantitation of 0.75 nmol/L and 0.50 nmol/L, respectively. Intra- and inter-assay imprecision was <8.9% for cortisol and <6.5% for cortisone across three levels of internal quality control, with accuracy and recovery within accepted limits. High specificity was demonstrated following interference studies which assessed 29 structurally-related steroids at supra-physiological concentrations. We have successfully validated an assay for the simultaneous analysis of salivary cortisol and cortisone using XLC-MS/MS and fully automated online SPE. The assay benefits from increased specificity compared to immunoassay and minimal sample preparation which allows high sample throughput and is thus suitable for use in a routine clinical laboratory.

Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11ß-hydroxylase inhibitor metyrapone.

BACKGROUND: The accurate measurement of cortisol by immunoassay is compromised by the potential for cross-reactivity of reagent antibodies with structurally related steroids present in serum. This susceptibility is potentiated when normal steroid metabolism is altered pharmaceutically by antisteroidogenic drugs utilized in the management of Cushing's syndrome to moderate cortisol production. The clinical implications of falsely elevated cortisol results include over-treatment and unrecognized hypoadrenalism. To investigate the effect of the 11ß-hydroxylase inhibitor metyrapone on serum cortisol assay, a comparison of measurement by immunoassay versus liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted. METHODS: Cortisol was measured in serum from three patient groups: (1) patients receiving metyrapone (n = 112 samples from 10 patients); (2) control group of patients diagnosed with Cushing's syndrome currently receiving no treatment (n = 31); and (3) control group of patients with no adrenal pathology and not receiving medication known to interfere in cortisol immunoassay (n = 67). RESULTS: Bland-Altman plots showed agreement between methods for the control group (bias = 1.1% [-4.3 nmol/L]) and Cushing's control group (bias = 1.3% [-3.7 nmol/L]). This was in stark contrast to the metyrapone therapy group (bias = 23% [59 nmol/L]). The difference between LC-MS/MS versus immunoassay in the metyrapone therapy group positively correlated with metyrapone dose and serum 11-deoxycortisol concentration (Pearson's correlation coefficient r = 0.47, 95% CI 0.32-0.61; P < 0.0001). CONCLUSIONS: These data show that liability of immunoassay measurement of serum cortisol to interference in patients receiving metyrapone may lead to erroneous clinical decisions concerning dose titration.