RESUMO
OBJECTIVE: This study aimed to use external sleep disturbance as a model to evaluate sleep architecture in climacteric women before and after menopausal hormone therapy (MHT). METHODS: Seventeen perimenopausal and 18 postmenopausal women underwent a polysomnography protocol: an adaptation night, a reference night and a sleep disturbance night with one hand loosely tied to the bed for blood sampling. The sleep architecture of the reference and disturbance nights were compared. The 24-h urinary free cortisol concentration (UFC) was measured. The procedure was repeated after 6 months on MHT or placebo. RESULTS: Fifteen perimenopausal and 17 postmenopausal women completed the study. The perimenopausal and postmenopausal groups were combined. During external sleep disturbance, sleep was shorter and more fragmented; with less stage 2, slow-wave and rapid eye movement (REM) sleep and more wake time and awakenings, both at baseline and after the treatment period. Compared to the placebo group, sleep disturbance was minor for women on MHT: sleep was not shortened and the amount of slow-wave sleep did not decrease. Increased 24-h UFC was observed only during MHT. CONCLUSIONS: Sleep in climacteric women is easily disturbed, leading to shorter and more fragmented sleep with less deep sleep and REM sleep. Six months of MHT attenuates the observed sleep disturbance.
Assuntos
Pós-Menopausa , Transtornos do Sono-Vigília , Feminino , Humanos , Menopausa , Perimenopausa , Polissonografia/métodos , SonoRESUMO
BACKGROUND: Both pregnancy and high vitamin D concentration seem to generate a protective environment against multiple sclerosis (MS) relapses. Longitudinal case-control analysis of vitamin D concentrations during pregnancy and lactation of MS mothers is lacking. AIMS OF THE STUDY: To examine serum 25-hydroxyvitamin-D3 levels of MS patients during and after pregnancy and compare these to the levels measured in healthy controls. METHODS: Fifteen relapsing-remitting MS mothers underwent repeated testing for 25-hydroxyvitamin-D3 at 10-12, 26-28 and 35-37 gestational weeks and 1, 3 and 6 months post-partum. An identical series of samples was collected from six control mothers. RESULTS: The prevalence of vitamin D deficiency (<50 nmol/l) during pregnancy was high (73%) among MS patients. Vitamin D levels were significantly higher during pregnancy when compared to early post-partum values among MS patients. At the end of the follow-up period, the vitamin D levels returned to levels observed in early pregnancy. In healthy controls, the alterations during and after pregnancy were similar in nature, but the vitamin D concentrations were higher at all time points when compared to MS patients (P = 0.037). CONCLUSIONS: Vitamin D deficiency during the pregnancy and lactation seems to be common in mothers with MS and needs to be treated adequately.
Assuntos
Lactação/sangue , Esclerose Múltipla Recidivante-Remitente/sangue , Período Pós-Parto/sangue , Deficiência de Vitamina D/epidemiologia , Vitamina D/sangue , Adulto , Feminino , Humanos , Masculino , Gravidez , Prevalência , Recidiva , Deficiência de Vitamina D/sangue , Adulto JovemRESUMO
A positive effect of fluoxetine has been shown in some children with autism. The present study was undertaken to correlate striatal dopamine transporter (DAT) binding and cerebrospinal fluid insulin-like growth factor-1 (CSF-IGF-1) with clinical response in autistic children (n=13, age 5-16 years) after a 6-month fluoxetine treatment. Good clinical responders (n=6) had a decrease (p=0.031) in DAT binding as assessed using single-photon emission computed tomography with [123I]-nor-ß-CIT, whereas poor responders had a trend to an increase. An increase in CSF-IGF-1 (p=0.003) was detected after the treatment period, but no correlation between the clinical response and CSF-IGF-1 was found. In conclusion, fluoxetine decreases DAT binding indicating alleviation of the hyperdopaminergic state and increases CSF-IGF-1 concentration, which may also have a neuroprotective effect against dopamine-induced neurotoxicity in autistic children.
Assuntos
Transtorno Autístico/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Fluoxetina/farmacologia , Fator de Crescimento Insulin-Like I/líquido cefalorraquidiano , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Adolescente , Transtorno Autístico/líquido cefalorraquidiano , Transtorno Autístico/diagnóstico por imagem , Criança , Pré-Escolar , Corpo Estriado/diagnóstico por imagem , Feminino , Humanos , Fator de Crescimento Insulin-Like I/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Native low mol. wt (LMr) kininogen from human plasma and a kinin-free kininogen from Cohn's plasma fraction IV (HC-antigen) were isolated and studied with regard to their immunological reactivity and carbohydrate heterogeneity. Antisera were prepared against the conformational and sequence-dependent determinants of the heavy chain which is the common denominator of plasma kininogens. The molecules were characterized by single radial immunodiffusion and SDS gel electrophoresis. The effect of exoglycosidase treatment was investigated by a radioimmunoassay. It was found that the antigenic combining sites were not influenced by the partial removal of carbohydrates. These results suggest that the determinant structures of LMr kininogen reside in the peptide backbone of the protein. Carbohydrate heterogeneity was shown by different binding to concanavalin A and wheat germ lectin. With concanavalin A 52% of the immunoreactive HC-antigen was reactive compared to 76% of the native LMr kininogen. The weak reactivity (11%) of the HC-antigen towards the wheat germ lectin indicates that the N-acetylglucosamine residues are apparently blocked or missing. Immunoreactive kininogen was in all cases measured by single radial immunodiffusion or an RIA. The molecular nature of the heterogeneity is discussed in relation to the known isoelectric variations of kininogen.
Assuntos
Cininogênios/imunologia , Antígenos/análise , Sítios de Ligação de Anticorpos , Proteínas Sanguíneas/imunologia , Carboidratos/imunologia , Cromatografia de Afinidade , Concanavalina A , Eletroforese em Gel de Poliacrilamida , Epitopos , Glicosídeo Hidrolases , Humanos , Imunodifusão , Lectinas , Peso Molecular , Radioimunoensaio , Aglutininas do Germe de TrigoRESUMO
Kininogen antigen was purified from human plasma fraction IV by ion exchange chromatography, gel filtration and affinity chromatography with antibody specific immunoadsorbents. The immunologically pure glycoprotein had a mol. wt of approximately 60,000 and only one polypeptide chain by SDS-PAGE. An extensive charge heterogeneity by isoelectric focusing and gel filtration on polyacrylamide agarose could only in part depend on a comparatively high sialic acid content, but may be caused by differences in the carbohydrate structures sustained by lectin-binding heterogeneity on Con A-Sepharose. This antigen shares a dominating determinant with native plasma kininogens shown by complete patterns of identity in immunochemical analyses and with the monospecific antisera developed in rabbits against the heterogeneous components. The similar size, amino acid composition, low histidine content, lack of N-terminal amino acid and antigenic homogeneity fit all the so far known characteristics of the human kininogen heavy chain. Notably the antigenic determinant is resistant to degradation by activated kallikrein. This antigen with unimpaired immunologic activity may be a useful tool for preparation of antiserum for immunochemical determination of human plasma kininogen.
Assuntos
Cininogênios/imunologia , Alquilação , Aminoácidos/análise , Antígenos/isolamento & purificação , Humanos , Técnicas de Imunoadsorção , Ponto Isoelétrico , Substâncias Macromoleculares , Peso Molecular , Oxirredução , Ácidos Siálicos/análiseRESUMO
BACKGROUND AND PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is detected in ischemic brain cells in experimental animal models and is believed to play an important role in apoptosis. However, the natural expression of TNF-alpha during human stroke is not known. METHODS: We examined TNF-alpha immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) in brain samples of stroke victims (n=16) after variable survival (15 hours to 18 days). Systemic TNF-alpha content from a separate cohort including severe or lethal stroke cases (n=26) was also assayed. RESULTS: Neuronal TNF-alpha was demonstrated from 0.6 to 5.4 days after the onset of stroke symptoms, peaking bilaterally during days 2 and 3. Bilateral glial TNF-alpha immunoreactivity was detected during the acute phase, with the astrocytic TNF-alpha expression dominating in later phases and persisting contralaterally to the infarct in more matured phases (17 to 18 days). Invading inflammatory cells were TNF-alpha immunopositive beginning on the third day. Besides, vascular wall structures showed immunoreactivity sporadically. TNF-alpha levels were mostly nondetectable in peripheral blood. TUNEL labeling and TNF-alpha staining overlapped, although not completely, during the first days. CONCLUSIONS: The data support the hypothesis that TNF-alpha may be involved both in the acute propagation of inflammatory processes and cell death and possibly in the more delayed reconstitutive processes of human ischemic stroke.
Assuntos
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Acidente Vascular Cerebral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Encéfalo/patologia , Isquemia Encefálica/complicações , Isquemia Encefálica/patologia , Progressão da Doença , Feminino , Fluorescência , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Microcirculação/metabolismo , Microcirculação/patologia , Pessoa de Meia-Idade , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Fagócitos/metabolismo , Fagócitos/patologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/patologiaRESUMO
BACKGROUND: Light treatment through the eyes is effective in alleviating the symptoms of some psychiatric disorders. A recent report suggested that skin light exposure can affect human circadian rhythms. Bilirubin can serve as a hypothetical blood-borne mediator of skin illumination into the brain. We studied whether bright light directed to a large body area could suppress the pineal melatonin secretion or decrease serum total bilirubin in conditions that could be used for therapeutic purposes. METHODS: Seven healthy volunteers participated in two consecutive overnight sessions that were identical except for a light exposure on the chest and abdomen in the second night from 12:00 AM to 6:00 AM (10,000-lux, 32 W/m(2) cool white for six subjects and 3000-lux, 15 W/m(2) blue light for one subject). Hourly blood samples were collected from 7:00 PM to 7:00 AM for melatonin radioimmunoassays. Bilirubin was measured by a modified diazo method in blood samples taken at 12:00 AM and 6:00 AM and in urine samples collected from 7:00 PM to 11:00 PM and from 11:00 PM to 7:00 AM. RESULTS: The skin light exposure did not cause any significant changes in serum melatonin or bilirubin levels. The excretion of bilirubin in urine was also the same in both sessions. CONCLUSIONS: Significant melatonin suppression by extraocular light does not occur in humans. Robust concentration changes of serum total bilirubin do not have a role in mediating light information from the skin to the central nervous system.
Assuntos
Bilirrubina/sangue , Melatonina/sangue , Fototerapia , Fenômenos Fisiológicos da Pele , Visão Ocular , Abdome , Adulto , Análise de Variância , Bilirrubina/urina , Estudos Cross-Over , Feminino , Humanos , Imunoensaio , Masculino , TóraxRESUMO
The immunoreactivity of human low-molecular-mass kininogen from Cohn plasma fraction IV was investigated after deglycosylations and carbohydrate modifications by radioimmunoassay using the conformation-specific antiserum. Removal of all sialic acids, 44% of amino sugars and 63% of neutral sugars did not alter the immunoreactivity of the protein but the periodate-treated concanavalin A fractions showed strikingly diminished immunoreactivity. A conformational change could account for the observed effect of periodate on the decreased reactivity of the protein in radioimmunoassay. Externally added carbohydrates had no effect on immunoreactivity. The results suggest that the carbohydrate part of kininogen is not involved in the immunoreactivity although it accounts for the observed lectin-binding heterogeneity.
Assuntos
Carboidratos/fisiologia , Cininogênios/análise , Animais , Concanavalina A/farmacologia , Humanos , Cininogênios/imunologia , Peso Molecular , Conformação Proteica , Coelhos , RadioimunoensaioRESUMO
BACKGROUND: Infantile neuronal ceroid lipofuscinosis (INCL) is a progressive encephalopathy in which the patients are severely disabled by the age of 3 years. It is characterized by cerebral atrophy, selective loss of cortical neurons, and secondary loss of axons and myelin sheaths of the white matter. INCL has been shown to result from a palmitoyl protein thioesterase deficiency. The authors suggested that insulin-like growth hormones and apoptosis might play a role in the pathogenesis of INCL. METHODS: The authors measured insulin-like growth factor-1 (IGF-1) and IGF binding protein 3 (IGFBP-3) in the CSF of patients with INCL by radioimmunoassay at an early stage when myelin was starting to diminish. RESULTS: The authors found low CSF IGF-1 but normal IGFBP-3 in patients with INCL compared with control subjects. Also, they observed apoptotic cell death in biopsies of INCL patients. CONCLUSIONS: Because the IGF system seems to be important for early brain development, myelination, and neuroprotection, the authors suggest that the pathology in INCL may be associated with low CSF IGF-1.
Assuntos
Fator de Crescimento Insulin-Like I/líquido cefalorraquidiano , Lipofuscinoses Ceroides Neuronais/diagnóstico , Apoptose/fisiologia , Biópsia , Pré-Escolar , Feminino , Humanos , Lactente , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/líquido cefalorraquidiano , Masculino , Bainha de Mielina/patologia , Lipofuscinoses Ceroides Neuronais/líquido cefalorraquidiano , Lipofuscinoses Ceroides Neuronais/patologia , Neurônios/patologia , RadioimunoensaioRESUMO
We have developed two 'sandwich'-type time-resolved immunofluorometric assays (IFMA) for tumor-associated trypsin inhibitor (TATI) using monoclonal and polyclonal antibodies. In the standard assay the monoclonal antibody was immobilized onto the walls of polystyrene microstrip wells and the polyclonal reagent was labeled with a europium chelate. We tested various assay conditions in order to optimize the assay for sensitivity and measuring range. Purification of the labeled antibody by hydrophobic interaction chromatography was found to be the most important single factor affecting sensitivity. Assay sensitivity and range were also improved by acid treatment of the solid phase antibody. To improve the sensitivity further the streptavidin/biotin (SAB) system was incorporated into the IFMA technique. In this simple and fast streptavidin/biotin IFMA (SAB-IFMA) we used streptavidin-coated wells to which we added biotinylated monoclonal antibody and a serum or urine sample. After incubation for 1.5 h and washing, the polyclonal europium-labeled tracer antibody was added. After incubation for 1 h the wells were washed and the Eu fluorescence measured. The assay performance of the SAB-IFMA was compared to the standard IFMA and radioimmunoassay (RIA). The detection limit was 0.05 microgram/l and the analytical range 3000-fold. The mean analytical recovery was 101%. Other advantages of the SAB-IFMA were high sensitivity and the low amounts of monoclonal antibody required, only 1/50 of that used in the standard IFMA.
Assuntos
Fluorimunoensaio/métodos , Inibidor da Tripsina Pancreática de Kazal/análise , Anticorpos Monoclonais , Afinidade de Anticorpos , Proteínas de Bactérias , Biotina , Feminino , Humanos , Masculino , Radioimunoensaio/métodos , EstreptavidinaRESUMO
OBJECTIVE: To determine whether elevated homocysteine levels precede the development of preeclampsia. METHODS: Study subjects were selected from a population-based cohort of 1049 nulliparous women from whom serum was collected for Down syndrome screening at 16 weeks' gestation. For 34 women who developed preeclampsia, 68 control women were chosen who remained normotensive. Homocysteine was analyzed by high-performance liquid chromatography and fluorescence detection. The sample size allowed detection of a 1.25-micromol/L difference at a significance level of 0.05 and the power of 0.81. RESULTS: At 16 weeks' gestation, concentrations (mean, 95% confidence interval) of homocysteine in women who developed preeclampsia, 6.99 (6.42, 7.55) micromol/L, were similar to those who remained normotensive, 6.91 (6.45, 7.34) micromol/L. CONCLUSION: Significant changes in homocysteine metabolism did not predate the appearance of clinical preeclampsia.
Assuntos
Homocisteína/sangue , Pré-Eclâmpsia/etiologia , Adulto , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Feminino , Humanos , Pré-Eclâmpsia/sangue , Gravidez , Segundo Trimestre da GravidezRESUMO
OBJECTIVE: To study the plasma levels of homocysteine in preeclampsia and relate them to insulin sensitivity. METHODS: In association with a 3-hour intravenous glucose-tolerance test (glucose 0.3 g/kg at 0 and 0.03 IU insulin 20 minutes later), we measured plasma levels of homocysteine, vitamin B12, and folic acid in 22 women with preeclampsia and 16 controls between 29 and 39 weeks' gestation. In 14 women with preeclampsia and 11 controls, plasma samples also were collected 3 months after delivery. RESULTS: Levels of homocysteine in women with preeclampsia (6.7 +/- 0.4 micromol/L, mean +/- standard error) were higher (P < .001) than those in controls (3.8 +/- 0.2 micromol/L) and related significantly to the level of proteinuria (r = .49, P = .02). Vitamin B12 concentrations were lower in women with preeclampsia (166.0 +/- 10.4 compared with 212.4 +/- 16.4 pmol/L, P = .02), whereas levels of folic acid showed no difference between the groups. After delivery, levels of homocysteine increased to 9.1 +/- 0.6 and 8.2 +/- 0.6 micromol/L in women with preeclampsia and controls, vitamin B12 increased to 298.8 +/- 28.6 compared with 334.9 +/- 24.0 pmol/l, and folic acid decreased to 10.6 +/- 2.0 compared with 7.9 +/- 0.8 nmol/L, with no difference emerging between the groups. In women with preeclampsia but not in controls, plasma homocysteine was negatively related to insulin sensitivity (r = -.51, P = .02). The mean 2.9-fold increase in glucose or 52.5-fold increase in insulin during the insulin-sensitivity test failed to affect homocysteine levels. CONCLUSION: Women with preeclampsia have high plasma homocysteine levels that are inversely related to insulin sensitivity.
Assuntos
Homocisteína/sangue , Resistência à Insulina , Pré-Eclâmpsia/sangue , Adulto , Feminino , Humanos , GravidezRESUMO
OBJECTIVE: A high level of plasma homocysteine may be deleterious to vascular health. We therefore compared the effect of combinations of sequential oral and transdermal estradiol plus norethisterone acetate on plasma homocysteine. DESIGN: Prospective, randomized study. SETTING: Outpatient department of obstetrics and gynecology in a university hospital. PATIENT(S): Forty-two healthy, nonsmoking postmenopausal women starting hormone replacement therapy (HRT) to control climacteric symptoms. INTERVENTION(S): In a randomized order, the women started using either oral HRT (2 mg of estradiol on days 1-12, 2 mg of estradiol plus 1 mg of norethisterone acetate (NETA) on days 13-22, and 1mg of estradiol on days 23-28; n = 21) or transdermal HRT (50 microg/d of estradiol on days 1-28 and 250 microg/d of norethisterone acetate on days 15-28, n = 21) for 1 year. MAIN OUTCOME MEASURE(S): Fasting plasma levels of homocysteine were measured before the treatment and during the combined estradiol-plus-NETA phases of the sixth and 12th treatment cycles. RESULT(S): Basal homocysteine levels in the oral group (8.2 +/- 3.1 micromol/L, mean plusmn;SD) and transdermal group (8.7 plusmn; 1.8 micromol/L, mean plusmn;SD) were not affected by the estradiol-plus-NETA combination. CONCLUSION(S): Neither an oral nor a transdermal combination of sequential estradiol and NETA causes significant changes in plasma homocysteine in Finnish postmenopausal women with normal baseline homocysteine levels.
Assuntos
Estradiol/administração & dosagem , Homocisteína/sangue , Noretindrona/uso terapêutico , Administração Cutânea , Administração Oral , Esquema de Medicação , Quimioterapia Combinada , Estradiol/uso terapêutico , Feminino , Humanos , Pessoa de Meia-Idade , Noretindrona/análogos & derivados , Acetato de Noretindrona , Estudos ProspectivosRESUMO
Rett syndrome is a neurodevelopmental disease characterized by failure of somatic and brain growth. The insulin-like growth factor system mediates most actions of growth hormone. Evidence that it plays an important role in early development of the brain is increasing. The aim of the study reported was to assess the role of the insulin-like growth factor system in the pathogenesis of Rett syndrome. We measured insulin-like growth factor-I levels in serum (8 patients, mean age 9.1 years) and cerebrospinal fluid (13 patients, mean age 7 years) using a sensitive radioimmunoassay method and compared them with those in age-matched controls (13 and 26 patients, respectively). Neither serum nor cerebrospinal fluid insulin-like growth factor-I levels differed from those in controls. We also measured insulin-like growth factor binding protein-3 levels in serum (in 9 patients and 8 controls) and in cerebrospinal fluid (in 12 patients and 11 controls) and serum growth hormone levels (in 8 patients and 11 controls); the levels in patients did not differ from those in controls. We found no significant correlation between serum and cerebrospinal fluid insulin-like growth factor-I in Rett syndrome. This may indicate an independent role of insulin-like growth factor system in the central nervous system, making serum insulin-like growth factor-I measurement unreliable as an indicator of disturbed function in the central nervous system. Our results did not support the notion that a defective insulin-like growth factor-I system explains the lack of somatic and brain growth in Rett syndrome.
Assuntos
Encéfalo/crescimento & desenvolvimento , Fator de Crescimento Insulin-Like I/líquido cefalorraquidiano , Síndrome de Rett/fisiopatologia , Adolescente , Biomarcadores/análise , Encéfalo/patologia , Criança , Pré-Escolar , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/líquido cefalorraquidiano , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Sensibilidade e EspecificidadeRESUMO
The interpretation of results obtained by immunoassays is affected by many different factors and especially by assay sensitivity and specificity. The introduction of sandwich-type immunometric assays using highly sensitive non-radioactive labels and monoclonal antibodies has greatly improved sensitivity while reducing assay time and simplifying performance. Thus determination of subnormal levels of several hormones has become possible. This approach has also enabled more exact tuning of specificity but at the same time it has introduced the risk of too high selectivity. Hapten assays have also become simpler to perform, but in this case no substantial improvement in assay sensitivity has been achieved.
Assuntos
Imunoensaio/métodos , Animais , Antígenos de Neoplasias/análise , Hormônios/análise , Humanos , Imunoensaio/normas , Imunoensaio/estatística & dados numéricos , Peptídeos/análise , Ligação Proteica , Sensibilidade e EspecificidadeAssuntos
Concanavalina A/metabolismo , Cininogênios/isolamento & purificação , Lectinas/metabolismo , Lectinas de Plantas , Proteínas de Plantas/metabolismo , Carboidratos/análise , Cromatografia de Afinidade/métodos , Humanos , Focalização Isoelétrica , Cininogênios/metabolismo , Peso Molecular , Neuraminidase/metabolismoAssuntos
Biopterinas/análogos & derivados , Transplante de Medula Óssea , Corticosteroides/uso terapêutico , Adulto , Anemia Aplástica/terapia , Biopterinas/urina , Linfoma de Burkitt/terapia , Creatinina/urina , Infecções por Citomegalovirus/urina , Doença Enxerto-Hospedeiro/urina , Humanos , Infecções/urina , Leucemia/terapia , NeopterinaAssuntos
Síndrome de Down/diagnóstico , Segundo Trimestre da Gravidez/sangue , Diagnóstico Pré-Natal/métodos , Biomarcadores/sangue , Gonadotropina Coriônica/sangue , Estudos Transversais , Síndrome de Down/epidemiologia , Estriol/sangue , Feminino , Finlândia/epidemiologia , Humanos , Satisfação do Paciente , Gravidez , alfa-Fetoproteínas/análiseRESUMO
Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stable-isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether-ethyl acetate from 250 microL or 500 microL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode after separation on a reversed-phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d(2) testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2-100 nmol/L. Interassay CVs were 4.2-7.6 % at mean concentrations of testosterone of 3.3-45 nmol/L. Total measurement uncertainty (U, k = 2) was 12.9 % and 13.4 % at testosterone levels of 2.0 nmol/L and 20 nmol/L, respectively. The limit of detection was 0.05 nmol/L (signal-to-noise ratio = 3) and the overall method recovery of testosterone was 95 %. Correlation (r) with our in-house extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18-30, 31-50, 51-70 and over 70 years were established. Sensitivity and specificity of the LC-MS/MS method offer advantages over immunoassay and make it suitable for use as a high-throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.