Diurnal variation of steroid hormones and their reference intervals using mass spectrometric analysis.

OBJECTIVE: Accurate measurement of steroid hormones remains challenging. Mass spectrometry affords a reliable means for quantitating steroid profiles accurately. Our objective was to establish and define (1) the extent of diurnal fluctuations in steroid concentrations that potentially necessitate strict adherence to time of sample acquisition and (2) time-dependent steroid reference intervals. DESIGN: Nine steroid markers were examined in couplets in males and females. METHODS: Using isotope dilution high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis, we developed a multi-steroid profile requiring only a minimal volume of serum (0.1 mL). Couplet (AM and PM) measurements of steroid hormones for 120 healthy females (F) and 62 healthy males (M) were obtained. Patients were recruited from several participating centers. RESULTS: The following diurnal values were noted to be significantly different in both females and males: cortisone, cortisol, corticosterone, 11 deoxycortisol (11 DOC), androstenedione, 17a-hydroxyprogesterone (17 OHP) and dehydroepiandrosterone (DHEA). Testosterone was only found to have significant diurnal variance in males. Progesterone showed no significant difference in AM and PM values for either groups and thus may provide an internal control. CONCLUSIONS: When diagnosing endocrine disorders, it is imperative to acknowledge the 24-h diurnal variation of the biochemical steroid markers. We highlight the importance of standardization of collection times and appropriate implementation of reference intervals. PRECIS: We identify diurnal fluctuations in steroid concentrations with time of day and emphasize the importance of adhering to firm time of sample acquisition.

Does TSH Reliably Detect Hypothyroid Patients?

OBJECTIVES: To evaluate the reliability of normal Thyroid Stimulating Hormone (TSH) as a thyroid function test and assess the effect of Adrenocorticotropic Hormone (ACTH) on serum TSH concentration. DESIGN AND METHODS: Patients presenting to the National Institutes of Health Department of Endocrinology outpatient clinic with symptoms consistent with hypothyroidism were identified. Thyroid hormone concentrations were measured by liquid chromatography/tandem mass spectrometry and immunoassay. Patients with normal TSH concentrations were assessed for both clinical and biochemical hypothyroidism.We evaluated the effect of ACTH stimulation (performed on patients for assessment of adrenal function) on TSH concentration. RESULTS: Patients with symptoms consistent with hypothyroidism but with normal TSH values in the range of 1-4 IU/mL and normal free T4 (FT4) values by immunoassay measurements were confirmed to be biochemically hypothyroid following measurements of thyroid hormones by mass spectrometry. We present case studies of two patients, a 76-year-old male and a 58-year-old female. Improvement in the male patient's hypothyroid symptoms, including afternoon fatigue, constipation, alopecia, dry skin and high cholesterol, was documented after initiating thyroid hormone replacement.ACTH stimulation resulted in an average decrease of 17% in TSH between time 0 and 60 minutes post stimulation. CONCLUSION: Although measurement of TSH is a convenient screen for thyroid function, it is influenced by many factors which may affect its overall reliability. We believe thyroid function should be assessed by more than a single test. We recommend measurement of thyroid hormone concentrations by mass spectrometry if the patient's clinical presentation is discordant with their TSH levels.

Brain-derived neurotrophic factor Val66Met genotype and ovarian steroids interactively modulate working memory-related hippocampal function in women: a multimodal neuroimaging study.

Preclinical evidence suggests that the actions of ovarian steroid hormones and brain-derived neurotrophic factor (BDNF) are highly convergent on brain function. Studies in humanized mice document an interaction between estrus cycle-related changes in estradiol secretion and BDNF Val66Met genotype on measures of hippocampal function and anxiety-like behavior. We believe our multimodal imaging data provide the first demonstration in women that the effects of the BDNF Val/Met polymorphism on hippocampal function are selectively modulated by estradiol. In a 6-month pharmacological hormone manipulation protocol, healthy, regularly menstruating, asymptomatic women completed positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) scans while performing the n-back working memory task during three hormone conditions: ovarian suppression induced by the gonadotropin-releasing hormone agonist, leuprolide acetate; leuprolide plus estradiol; and leuprolide plus progesterone. For each of the three hormone conditions, a discovery data set was obtained with oxygen-15 water regional cerebral blood flow PET in 39 healthy women genotyped for BDNF Val66Met, and a confirmatory data set was obtained with fMRI in 27 women. Our results, in close agreement across the two imaging platforms, demonstrate an ovarian hormone-by-BDNF interaction on working memory-related hippocampal function (PET: F2,37=9.11, P=0.00026 uncorrected, P=0.05, familywise error corrected with small volume correction; fMRI: F2,25=5.43, P=0.01, uncorrected) that reflects differential hippocampal recruitment in Met carriers but only in the presence of estradiol. These findings have clinical relevance for understanding the neurobiological basis of individual differences in the cognitive and behavioral effects of ovarian steroids in women, and may provide a neurogenetic framework for understanding neuropsychiatric disorders related to reproductive hormones as well as illnesses with sex differences in disease expression and course.

Acute changes in arginine vasopressin, sweat, urine and serum sodium concentrations in exercising humans: does a coordinated homeostatic relationship exist?

The parallel response of sweat rate and urine production to changes in plasma osmolality and volume support a role for arginine vasopressin (AVP) as the main endocrine regulator of both excretions. A maximal test to exhaustion and a steady-state run on a motorised treadmill were both completed by 10 moderately trained runners, 1 week apart. Sweat, urine and serum sodium concentrations ([Na+]) were evaluated in association with the plasma concentrations of cytokines, neurohypophyseal and natriuretic peptides, and adrenal steroid hormones. When data from both the high-intensity and steady-state runs were combined, significant linear correlations were noted between: sweat [Na+] versus postexercise urine [Na+] (r=0.80; p<0.001), postexercise serum [Na+] versus both postexercise urine [Na+] (r=0.56; p<0.05) and sweat [Na+] (r=0.64; p<0.01) and postexercise urine [Na+] versus postexercise plasma arginine vasopressin concentration ([AVP](P)) (r=0.48; p<0.05). A significant positive correlation was noted between postexercise [AVP](P) and sweat [Na+] during the steady-state condition only (r=0.66; p<0.05). These correlations suggest that changes in serum [Na+] during exercise may evoke corresponding changes in sweat and urine [Na+], which are likely regulated coordinately by changes in [AVP](P) to preserve body fluid homeostasis.

Age-dependent pharmacokinetics of lamivudine in HIV-infected children.

The recommended dose of lamivudine in children is higher when compared with adults: 4 mg/kg vs approximately 2 mg/kg (150 mg) and administered twice a day. Limited data are available to demonstrate that this increased dose results in adequate exposure to lamivudine in children with human immunodeficiency virus (HIV) infection. Data were selected from children who were using lamivudine for at least 2 weeks before a full pharmacokinetic (PK) study was conducted. Lamivudine PK parameters were significantly related to age. The age of 6 years appeared to be a cutoff for a change in PK parameters of lamivudine, with children <6 years of age (n=17) having a median area under the curve 43% lower and a median peak plasma concentration 47% lower (both P<0.001) than older children (n=34). In conclusion, further investigation of the relationship between decreased lamivudine exposure and treatment outcome and long-term resistance development in younger children with HIV infection is warranted.

Trimester-specific reference intervals for thyroxine and triiodothyronine in pregnancy in iodine-sufficient women using isotope dilution tandem mass spectrometry and immunoassays.

BACKGROUND: Accurate assessment of the pregnant woman's thyroid status is critical, for both the initiation of thyroid hormone therapy and for the adjustment of thyroid hormone dose in those already receiving thyroid hormone. Trimester-specific intervals are especially important during pregnancy when thyroid insufficiency may be associated with adverse obstetric outcome and fetal neurodevelopmental deficits. We defined pregnancy-specific reference intervals for thyroxine (T4) and 3,5,3'-triiodothyronine (T3). We used a novel isotope dilution tandem mass spectrometry (LC/MS/MS) method, and compare these to reference intervals obtained by immunoassays (IAs) performed on the same samples. METHODS: Concentrations of circulating T4 and T3 were measured simultaneously during first, second and third trimesters and postpartum in iodine-sufficient, healthy, singleton pregnancies using API-3000 LC/MS/MS with deuterium-labeled internal standard (L-thyroxine-d2). Immunoassays were conducted on the same samples (T4 Dade Behring RxL, T3 DPC-Immunolite). RESULTS: Linear regression is reported for method comparisons; for T4, the slope decreased from r=0.900 in nonpregnant women to 0.802-0.820 during pregnancy. For T3, correlations between LC/MS/MS and immunoassays were weaker in all cases (r=0.407-0.574). CONCLUSION: In this longitudinal study, we established trimester-specific reference intervals for T4 and T3 by LC/MS/MS and compare these to intervals obtained by immunoassays.

Trimester-specific changes in maternal thyroid hormone, thyrotropin, and thyroglobulin concentrations during gestation: trends and associations across trimesters in iodine sufficiency.

OBJECTIVES: To describe the interrelationships of thyroid functions based on trimester-specific concentrations in healthy, iodine-sufficient pregnant women across trimesters, and postpartum. METHODS: Circulating total 3,5,3'- triidothyronine (T(3)) and thyroxine (T(4)) concentrations were determined simultaneously using liquid chromatography tandem mass-spectrometry (LC/MS/MS). Free thyroxine (FT(4)), thyroid-stimulating hormone (TSH), and thyroglobulin (Tg) were measured using immunoassay techniques. Linear mixed effects models and correlations were calculated to determine trends and associations, respectively, in concentrations. RESULTS AND CONCLUSIONS: Trimester-specific T(3), FT(4), TSH, and Tg concentrations were significantly different between the first and third trimesters (all p < 0.05); second and third trimester values were not significantly different for FT(4), TSH, and Tg (all p > 0.25) although T3 was significantly higher in the third, relative to the second trimester. T(4) was not significantly different at any trimester (all p > 0.80). With two exceptions, analyte concentrations tended not to be correlated at each trimester and at 1-year postpartum. One exception was that T(3) and T(4) tended to be associated (all p < 0.05) at all time points except the third trimester (rho = 0.239, p > 0.05). T(4) and FT(4) concentrations tended to correlate positively during pregnancy (rho 0.361-0.382, all p < 0.05) but not postpartum (rho = 0.179, p > 0.05). Trends suggest that trimester-specific measurements of T(3), FT(4), Tg, and possibly TSH are warranted.

Simultaneous simple and fast quantification of three major immunosuppressants by liquid chromatography--tandem mass-spectrometry.

OBJECTIVES: The aim of the current study was to develop a simple, fast and universal method for quantification of any combination of the three major immunosuppressants sirolimus, tacrolimus and cyclosporin in whole blood, using a LC-tandem mass spectrometer (API-2000, SCIEX, Toronto, Canada). METHODS: 250 microL whole blood was spiked with internal standard (ritonavir), and protein precipitated with 350 microL acetonitrile. The sample was centrifuged and 30 microL aliquot was injected onto the HPLC column, where it underwent an online extraction with ammonium acetate. After that the automatic switching valve was activated, changing the mobile phase to methanol and thereby eluting the analytes into the tandem mass spectrometer. The high selectivity of a tandem mass analyzer allows determination of any combination of the three drugs within a 5 min run. RESULTS: Between-day precision was between 2.4% and 9.7% for all analytes at the concentrations tested. Accuracy ranged between 98.8% and 103.2% (n = 20). The method was linear over the measuring ranges of all analytes. Within-run precision was below %CV = 6% for all analytes. Good correlation with other analytical methods was observed. CONCLUSIONS: The simplicity, universality and high throughput of the method make it suitable for application in a clinical laboratory. The method has been implemented in our laboratory for a routine use.

An evaluation of analytic goals for assays of drugs: a College of American Pathologists Therapeutic Drug Monitoring Survey Study.

OBJECTIVE: To determine if the levels of imprecision of the commonly used analytic methods for drug measurements are suitable for long-term therapeutic drug monitoring. DESIGN: In 1996, 4 identical lyophilized samples (2 in the first mailing and 2 in the second mailing 4 months later) were sent to laboratories participating in a nationwide proficiency testing program. Similarly, in 1999, replicates from a liquid pool of spiked sera were mailed 3 times, 4 months apart, to participating laboratories. For each of 11 drugs regulated under the Clinical Laboratory Improvement Amendments of 1988 and 1 metabolite, the total variance for each method was partitioned into within- and between-laboratory components. The total within-laboratory and the total survey coefficients of variation (CVs) for each method were then compared with the "acceptable" precision criteria of Glick, Burnett, and Fraser for each drug. SETTING: The first 2 mailings of the College of American Pathologists Therapeutic Drug Monitoring surveys for 1996, sets Z and ZM, and the 3 mailings of 1999, sets ZM, Z, and Z2. MAIN OUTCOME MEASURES: For each drug studied, the CV of each method was compared with the various imprecision criteria, and if greater than any of the criteria, the method was then tabulated as not meeting that specific criterion.Participants.-The approximately 5000 participants of the survey. RESULTS: The number of methods deemed as not having acceptable total long-term within-laboratory precision by the various criteria ranged from 35% to 88% in 1996 and from 22% to 77% in 1999. CONCLUSION: The number of failures possibly indicates that many of the reagent assays being utilized are not precise enough for long-term therapeutic drug monitoring of chronically administered drugs or that the published criteria used to evaluate the data in this study are too stringent.

Identification of ubiquitin as an immunophilin.

We have identified an 8.4 kDa minor immunophilin from calf thymus and Jurkat T cells as ubiquitin. It binds tacrolimus and sirolimus with K(d)'s of 0.8 and 0.08 nM, respectively. The binding of this protein to cyclosporin A is negligible. Binding of tacrolimus to two commercial sources of ubiquitin, a bovine product, and a recombinant human ubiquitin, was also demonstrated after HPLC purification of the purchased preparations.

An immunophilin-binding assay for sirolimus.

OBJECTIVE: This review examines the performance of 4 assays for sirolimus in terms of their ability to meet 6 guidelines determined by a panel of experts. BACKGROUND: Four methods have been described to date for the analysis of sirolimus concentrations in whole blood: high-performance liquid chromatography-mass spectrometry (HPLC-MS); microparticle enzyme immunoassay (MEIA); p70 S6 kinase inhibition; and an immunophilin-binding assay (IBA). METHODS: A MEDLINE search of the literature was performed to identify relevant studies. RESULTS: The HPLC methods suffer from precision problems because of the substantial specimen preparation required, and HPLC-MS methods are not practical for clinical use. Initial studies of the MEIA have found overestimation of sirolimus concentrations that may be caused by antibody cross-reactivity with sirolimus metabolites. Monitoring of sirolimus effects by p70 S6 kinase inhibition is as yet possible only theoretically, and the assay itself is not yet optimal. With the IBA, use of a T-cell protein that binds to sirolimus and that may be the intracellular target of the drug as the assay binding protein allows the assay to measure sirolimus selectively, even in the presence of structurally similar metabolites. CONCLUSION: More than 200 clinical samples have been analyzed by the IBA, and correlation with HPLC values has been good, with a regression line slope near 1.0. In addition, the assay is easier to perform and more precise than HPLC, and has the potential to be automated. Thus, the IBA appears to have certain clear advantages over the other assays.

Immunophilins: their properties and their potential role in therapeutic drug monitoring.

This article reviews recent developments in the immunophilin arena and the current state of knowledge about the biochemical properties of immunophilins. The role of immunophilin-binding assays is also explored with the conclusion that some of these immunosuppressive-drug-binding proteins may provide better assays for sirolimus and tacrolimus than current immunoassays.

Minor immunophilin binding of tacrolimus and sirolimus metabolites.

OBJECTIVES: We have previously identified three minor immunophilins of molecular weights 37 kDa, 14 kDa, and 5-8 kDa capable of binding tacrolimus and sirolimus. DESIGN AND METHODS: When tested against pure preparations of five sirolimus metabolites, the 14 kDa protein had almost no cross-reactivity, the 37 kDa protein cross-reacted from a high of 23.2% to <10% and the 5-8 kDa protein cross-reacted from <10% to 46.4%. When the 5-8 kDa immunophilin was tested in whole blood samples to assess interference in clinical samples, the demethylated sirolimus metabolites showed about 25% less cross-reactivity while the hydroxylated metabolites reacted about the same. RESULTS: Since MLC data on sirolimus metabolites to date indicates that their pharmacologic potencies are < or =10% of the parent, the 14 kDa immunophilin appears to be the best candidate for a sirolimus radioreceptor assay. The 5-8 kDa immunophilin is newly identified and its cross-reactivity with tacrolimus metabolites had not been assessed. Binding of the 5-8 kDa immunophilin to pure preparations of three tacrolimus metabolites showed virtually no binding of the protein to 13-O-demethyl and 31-O-demethyl tacrolimus and binding to 15-O-demethyl tacrolimus at 121% relative to parent tacrolimus. Cross-reactivity of 15-O-demethyl tacrolimus with the 5-8 kDa protein was then assessed in whole blood samples, and it bound at a level of 163%. MLC data indicates that 31-O-demethyl tacrolimus is equipotent to parent tacrolimus in immunosuppressive activity, while the 13-O-demethyl and 15-O-demethyl have negligible immunosuppressive activity. CONCLUSIONS: Therefore, the 5-8 kDa immunophilin would have limitations in a radioreceptor assay for tacrolimus. In addition, we have evidence that the 5-8 kDa immunophilin is a subunit of a 52 kDa immunophilin previously identified by our group, and the cross-reactivity of the 5-8 kDa immunophilin with these metabolites is similar to that found previously with the 52 kDa, indicating that the two proteins could be related.

Comparison of steady-state trough sirolimus samples by HPLC and a radioreceptor assay.

OBJECTIVES: We have previously identified a minor immunophilin of 52 kDa molecular weight capable of binding tacrolimus and sirolimus. Because immunophilins are capable of binding both parent drug and metabolites and HPLC assays are typically used to assess parent drug in clinical situations, we used this immunophilin in a radioreceptor assay (RRA) to determine if any metabolites not included in the HPLC measurement would bind to the immunophilin and be associated with thrombocytopenia in patients receiving sirolimus. DESIGN AND METHODS: We tested 51 steady-state trough whole blood samples from non-thrombocytopenic patients and 51 steady-state trough samples from thrombocytopenic patients and compared them to HPLC measurements of parent drug in the same samples. We also tested whole blood samples spiked with authentic sirolimus metabolites using RRA to ascertain the effect these metabolites have on the technique. RESULTS: We found minimal cross-reactivity in this assay for sirolimus metabolites (binding ranged from <10% to 26%), and good correlation of the radioreceptor assay with HPLC (linear regression slope 0.92, y-intercept 0.79). There was no statistically significant difference between the RRA and HPLC results in two patient groups-thrombocytopenic and non-thrombocytopenic-using the paired t-test (p<0.005) and Bland-Altman analysis. CONCLUSIONS: These findings indicate that although the RRA could be substituted for HPLC in therapeutic drug monitoring, the 52 kDa immunophilin does not offer an advantage in terms of detecting metabolites associated with thrombocytopenia. However, the RRA offers the advantages of shorter turnaround time, smaller sample volume and potential for automation.

Biochemical characterization of the minor immunophilins.

OBJECTIVE: We present biochemical characterization of the previously described 14 kDa, 37 kDa, and 52 kDa immunophilins and a newly identified 5-8 kDa immunophilin. DESIGN AND METHODS: Proteins were tested for the following enzymatic activities-rotamase, G3PDH, protein kinase C, cAMP dependent protein kinase-and for the ability to inhibit calcineurin phosphatase when complexed with tacrolimus (FK506). RESULTS: The 5-8 kDa protein, like the other minor immunophilins, lacks rotamase activity. Since the 37 kDa possesses G3PDH activity, the 5-8 kDa protein, 14 kDa protein, and 52 kDa protein were all tested and found to lack G3PDH activity. Additional work shows that none of the minor immunophilins possess protein kinase C or cyclic AMP-dependent protein kinase activity and that the 37 kDa and 5-8 kDa and probably the 52 kDa proteins are capable of inhibiting calcineurin phosphatase when bound to tacrolimus.

Pediatric reference ranges for creatine kinase, CKMB, Troponin I, iron, and cortisol.

OBJECTIVES: To determine the pediatric reference ranges for iron, cortisol, CK, CKMB, and troponin I. METHODS: Iron and CK were measured on the Vitros analyzer (Johnson and Johnson) while CKMB, troponin I, and cortisol were measured on the Immuno I (Bayer Corp.). Pediatric reference ranges were determined on hospitalized patients using the Hoffmann approach. RESULTS: Pediatric reference ranges were obtained for iron (AM and PM) and cortisol (AM and PM). Ranges were also obtained for CKMB, troponin I, and total CK. CONCLUSION: This work represents an expansion in our knowledge base on pediatric reference ranges. For iron, the 97.5th percentiles were significantly higher in the PM than in the AM. The diurnal fluctuation in 97.5th percentiles for cortisol was only 10-20%. Pediatric reference ranges for CKMB were not previously available and are important especially in the first year of life. The elevated Troponin I is found in the first year of life also represents new data.