Proficiency testing of 25-hydroxyvitamin D (25-OHD) assays.

The Vitamin D External Quality Assessment Scheme (DEQAS) has been monitoring 25-OHD assay performance since 1989. The scheme has expanded rapidly in recent years and has 670 participants in 35 countries (July 2009). Five samples of human serum are distributed quarterly and the results analyzed to give an All-Laboratory Trimmed Mean (ALTM) and SD. Each participant has internet access to a preliminary report after submission of results and, following the results deadline, a final report is e-mailed to designated staff in each laboratory. The last 15 years has seen an improvement in mean inter-laboratory imprecision (CV), from 32.0% (1994) to 15.3% (2009) and most major methods are now giving results within plus or minus 7.4% of the ALTM (2009). DEQAS has regularly conducted and reported on a number of investigations into the performance of 25-OHD methods. A gas chromatography-mass spectrometry (GC-MS) reference method for 25-OHD is under development and will be used to assess whether the ALTM remains the most appropriate target for DEQAS samples.

Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation.

The International Quality Assessment Scheme for Vitamin D metabolites (DEQAS) was introduced in 1989. Initially, the aim was to improve the reliability of 25-hydroxyvitamin D (25-OHD) assays but the scheme was extended in 1997 to include 1,25-dihydroxyvitamin D (1,25(OH)(2)D). DEQAS has 95 members in 18 countries (January 2003). Five serum samples are distributed quarterly and participants are given up to 6 weeks to return their results for statistical analysis. The majority of participants use commercial kits for both analytes. A performance target was set by an advisory panel in 1997 and, at present, requires participants to get 80% or more of their results within +/-30% of the All-Laboratory Trimmed Mean (ALTM). The performance targets are under continual review. In 2003, 59% of participants met the target (cf. 52% in 2000). A questionnaire, distributed in January 2003, requested information on methods and the interpretation of results. Reference ranges varied but there was reasonable agreement on the 25-OHD concentrations below which Vitamin D supplementation was advised. A minority (22%) of respondents was unsure whether Vitamin D(3) or Vitamin D(2) was used to treat patients in their locality. The majority (52%) of assays for 1,25(OH)(2)D were done 'on demand' and others for apparently spurious reasons. Most respondents thought participation in DEQAS extremely important and the planned introduction of on-line reporting should enhance its value.

Feasibility of using paper impregnated with urine instead of liquid urine for assessing ovarian activity.

BACKGROUND: Ovarian activity should ideally be assessed by serial non-invasive methods that require simple procedures for sample collection and storage. Measurement of urinary oestrone-3-glucuronide and pregnanediol-3 alpha-glucuronide is a non-invasive method available for assessment of ovarian activity, but transport of large numbers of urine samples is cumbersome and samples need to be stored frozen. An alternative sample collection, transport and storage procedure that is easier to handle and requires no or minimal cold storage facilities will particularly benefit studies in which ovulatory activity needs to be assessed in field settings. OBJECTIVE: To evaluate the feasibility of using paper impregnated with urine as an alternative to liquid urine for the measurement of oestrone-3-glucuronide and pregnanediol-3 alpha-glucuronide concentrations in the assessment of ovarian activity. METHODS: Urine samples collected daily throughout regular menstrual cycles were stored as liquid urine at -20 degrees C, and as paper impregnated with urine, in the refrigerator for 3 to 12 months or at room temperature for 1 to 6 months. Oestrone-3-glucuronide and pregnanediol-3 alpha-glucuronide concentrations were measured in these urine samples by enzyme immunoassay. Values obtained were correlated using Spearman's correlation test. RESULTS: The pattern of oestrone-3 glucuronide and pregnanediol-3 alpha-glucuronide concentrations estimated using paper impregnated with urine followed that of liquid urine in all storage conditions used. Values obtained by two methods correlated significantly (p < 0.001 to 0.0001) though the paper impregnated with urine gave slightly higher values. CONCLUSIONS: Paper impregnated with urine can be used to facilitate sample collection, transport and storage of urine when oestrone-3-glucuronide and pregnanediol-3 alpha-glucuronide measurements are required in a large number of serial samples to assess ovarian activity.

Blood flow velocities in the basal vein after subarachnoid haemorrhage. A prospective study using transcranial duplex sonography.

BACKGROUND: Early recognition of emerging delayed neurological deficits (DND) in patients after subarachnoid haemorrhage (SAH) is not always possible by transcranial Doppler sonography. Aim of this study was to investigate a) whether determination of blood flow velocities in deep cerebral basal veins can predict DND in these patients b) the correlation of venous flow velocity to cerebral blood flow (CBF). METHODS: a) We prospectively investigated the mean flow velocity in the basal vein (VBVR), in the middle cerebral artery (VMCA) and in the extracranial internal carotid artery (VICA) in 66 patients after spontaneous SAH. Examinations were performed daily during the first 10 days, using transcranial duplex sonography. Thirty-seven patients had VMCA exceeding 120 cm/s. They were categorised in three groups: 1: no delayed neurological deficit; II: transient DND; III: permanent DND or death associated with vasospasm. b) In another group of 14 patients, interdiane variations in global cerebral blood flow (CBF) measured by the Kety-Schmidt-method were correlated with variations in VBVR, VMCA, and VICA. FINDINGS: a) In patients without deficit, VBVR was significantly elevated above normal values the first day (p < 0.05), and days 5 and 6 (p < 0.1) after VMCA exceeding 120 cm/s. In group III (permanent deficit), flow velocities in the BVR were significantly below normal on day 5 (p < 0.05) and 9 (p < 0.1). b) The correlation between changes in VBVR to changes in CBF (r = 0.78, p < 0.001) was closer than between changes in VMCA to the changes in CBF (r = 0.54, p < 0.05). INTERPRETATION: In case of elevated VMCA, patients with higher VBVR seem to have a better outcome. Changes in CBF correlate better with VBVR than with arterial flow velocities.

Circulating sex hormones and endometrial stromelysin-1 (matrix metalloproteinase-3) at the start of bleeding episodes in levonorgestrel-implant users.

Unpredictable endometrial bleeding is the major side-effect of levonorgestrel-releasing s.c. implants (Norplant), otherwise a method of choice for long-term contraception. The mechanisms responsible for bleeding are still unknown and no reliable treatment is available. Several matrix metalloproteinases (MMP) are expressed and activated in human endometrium only at menstruation and specific synthetic inhibitors of MMP fully prevent the tissue breakdown that occurs in menstrual-like endometrial explants. To investigate whether MMP are inappropriately expressed and activated in Norplant-treated endometria during bleeding episodes, volunteers were recruited to provide blood and endometrial biopsies at the start of bleeding episodes and during non-bleeding intervals. Whereas serum concentrations of levonorgestrel and sex hormones showed no change at bleeding, except for a slight decrease of oestradiol concentration, the expression and activation of stromelysin-1 released by explants cultured for 1 day were consistently increased at the start of bleeding episodes. Furthermore, stromelysin-1 was immunolocalized in stromal cells within breakdown areas of several bleeding endometria, but not in non-bleeding endometria. These observations suggest that the expression and activation of stromelysin-1 participate in the initiation of bleeding episodes upon Norplant contraception. New strategies in the prevention and treatment of abnormal bleeding based on MMP control should be envisaged.

Effect of sequential transdermal progesterone cream on endometrium, bleeding pattern, and plasma progesterone and salivary progesterone levels in postmenopausal women.

BACKGROUND: Transdermal progesterone is being used in some countries as a purported treatment for menopausal symptoms, either alone or prescribed in conjunction with estrogen, but little information exists regarding the biological activity and effectiveness of this method of delivery of progesterone in protecting the endometrium from excess proliferation. This study was designed to evaluate the use of sequential transdermal progesterone. End-points evaluated included endometrial cellular response and bleeding pattern as well as plasma hormone levels and salivary progesterone estimations. METHOD: Twenty-seven postmenopausal women were treated with continuous transdermal estrogen (28-day cycle) and a cream containing 16, 32 or 64 mg of progesterone in each 4-cm extrusion from a tube of Pro-Feme administered daily in a sequential (days 15-28 of cycle) regimen. Blood and endometrial samples were analyzed for progesterone response prior to therapy, after the first 14 days of unopposed transdermal estrogen and following 14 days of transdermal progesterone. Saliva samples were taken during the last 14 days of the 84-day study, when the final progesterone cream therapy was being applied. RESULTS: Hormone assay indicated that physiological levels of estradiol were achieved, but progesterone levels were insufficient to induce any detectable change in the endometrium. Only one patient experienced bleeding during the study period. Levels of salivary progesterone were so variable as to be considered completely unreliable in determining the potential influence on biological activity. INTERPRETATION: Pro-Feme transdermal progesterone administered in a 16-, 32- or 64-mg daily dose for 14 days in a sequential regimen does not appear to be effective in inducing a secretory change in a proliferative endometrium. Salivary progesterone levels were not of value in managing the therapy of postmenopausal women.

Serum binding of steroid tracers and its possible effects on direct steroid immunoassay.

We studied the serum protein binding of 3H-labelled progesterone, oestradiol and testosterone, and five 125I-labelled analogues of these steroids. All tracers investigated appeared to be bound by proteins in every serum sample tested. The addition of blocking agents caused a substantial reduction in serum protein binding of 3H-labelled steroids, but had relatively little effect on the binding of analogue steroid tracers. Use of analogue steroid tracers in conventional direct immunoassays for oestradiol and progesterone produced anomalous results for some patient samples when compared to extraction radioimmunoassays, but assays where tracer binding to serum constituents was prevented by adoption of two-step procedures appeared to avoid anomalous results. The results suggest that serum protein binding of steroid analogue tracers may be a source of interference in some direct steroid immunoassays.

Immunometric assay for lutropin (hLH) based on the use of universal reagents for enzymatic labelling and magnetic separation and monitored by enhanced chemiluminescence.

A solid-phase immunometric assay of human lutropin (hLH) is described. Two different anti-hLH antibodies were utilized as capture antibodies, and anti-IgG antibodies covalently coupled to magnetic particles and horseradish peroxidase, respectively, served as 'universal' detection reagents. An anti-hLH antibody raised in rabbits was incubated with a goat anti-rabbit IgG covalently bound to magnetic particles. The resulting complex was added to a separately incubated mixture of hLH and monoclonal anti-hLH antibody. Following incubation, the immunocomplex was sedimented in a magnetic field and the supernatant discarded. Finally a sheep anti-mouse antibody (F(ab')2 fragment) conjugated to horseradish peroxidase as label was added. Following a further incubation, the particles were sedimented in the magnetic field and washed. The hLH content of the sample was quantitated by measuring 'enhanced chemiluminescence'. The sensitivity of the assay was 2.5 +/- 0.9 IU/l (mean +/- SD), the within-run variation ranged from 7.9 to 11%, the between-run variation from 12.9 to 19.8%. Cross-reaction with hFSH or hTSH could not be detected, but was approximately 0.1% with hCG. The results correlated well with those obtained by radioimmunoassay (r = 0.84).

Design of immunoassays for use in developing countries.

PIP: The applicability of both radioimmunoassay and non-radiometric assay methods for use in research and clinical laboratories in developing countries are reviewed. The WHO Special Program of Research in Human Reproduction has distributed radioimmunoassay reagents to laboratories collaborating on fertility research for 10 years, but it is predicted that non-radiometric methods will be used in the near future. Some of the qualities required by sites in developing countries are stability at high temperatures, low cost, simple procedures, simple and non-dedicated instrumentation or none at all, ease of local manufacture or practical distribution and supply, and small, practical storage requirements. Biological samples should not require invasive procedures. Assay time must be brief if patients are waiting, but it is usually not possible to handle large sample volumes to same time. Radioisotopes have the advantage of affecting the kinetics of the test reaction to a lesser extent than non-radiometric assays, but they do required separation of bound from free hormone. Radioimmunoassays also can be repeated, while many non-radiometric assays are destroyed in the assay procedure. Time-resolved fluorescence assays are a good choice for the medium term.^ieng

Qual o valor dos constituintes da saliva para predizer ovulaçäo? / What the value of saliva constituents to predict ovulation?

Foram relatados diferenças na concentraçäo de alguns constituintes da saliva (esteroides, eletrólitos, glicose e enzimas) que ocorrem durante o ciclo menstrual e que talvez poderiam ser usados para predizer a ovulaçäo. Nos medimos estradiol, progesterona, glicose, sódio, potássio e cálcio em amostras da mistura salivar colhidas diariamente durante todo o ciclo menstrual. O dia da ovulaçäo foi determinado por ultra-sonografia e medidas de LH em urina. O perfil de estradiol e progesterona em saliva refletiu aquele classicamente encontrado em sangue. Os níveis dos eletrólitos em saliva näo mostraram padräo ciclíco definido mesmo após a correçäo da taxa de produçäo da saliva. Foram observadas alteraçöes cíclicas na concentraçäo de glicose em saliva, sendo que os valores máximos (picos) foram encontrados no dia ou no dia após a aovulaçäo, ou seja, durante o período periovulatório. Pequenos picos foram observados durante as fases folicular e lútea sem, entretanto, mostrarem um padräo reprodutivel. Estas alteraçöes foram melhor vistas nas amostras de jejum. Amostras sem o jejum tiveram concentraçöes altas de glicose o que tornou difícil a identificaçäo dos picos. Este estudo confirma o valor da medida dos esteróides em saliva, mas entretanto, sugere que medidas de glicose e eletrólitos em saliva têm, provavelmente, pouco valor como teste preditivo para ovulaçäo

Inhibition by gossypol of cyclic AMP production in mouse Leydig cells.

Gossypol has been shown to inhibit steroidogenesis in leydig cells. This study examined the mechanism of this action by investigating the effect of gossypol on leydig cell hCG receptor binding, adenylate cyclase activity and cyclic AMP production in leydig cells. Gossypol had no effect on hCG binding to cell membranes but inhibited LH-stimulated cyclic AMP production in whole purified leydig cells. It also reduced the stimulation caused by LH, Gpp(NH)p, forskolin and fluoride in membranes from leydig cells and also from liver. It is therefore possible that gossypol affects cyclic AMP production at the level of ATP conversion to cyclic AMP, although effects on the G-protein and catalytic subunit cannot be ruled out.

Site of gossypol inhibition of steroidogenesis in purified mouse Leydig cells.

Gossypol, a phenolic compound that has been studied as a potential male contraceptive, inhibits basal and LH-stimulated testosterone release from Leydig cells in vitro. The present study investigates the mechanism of this inhibition using preparations of purified mouse Leydig cells. Gossypol inhibited LH-stimulated progesterone, 17-hydroxyprogesterone and androstenedione production by mouse Leydig cells incubated in vitro. It also inhibited dibutyryl cyclic AMP-stimulated production of testosterone but was without effect in the presence of added pregnenolone or 25-hydroxycholesterol. These results suggest gossypol exerts its major inhibitory effect on Leydig cell function at a point between LH-dependent stimulation of cyclic AMP dependent protein kinase activity and increased availability of cholesterol for side chain cleavage.

The viability of mouse Leydig cells in vitro following incubation with gossypol.

Gossypol, a phenolic compound that has been studied as a potential male contraceptive, has also been shown to inhibit basal and LH-stimulated testosterone production by mouse Leydig cells in vitro. This inhibition persists even when cells treated with gossypol are washed prior to reincubation with LH. While gossypol has been reported to be cytotoxic, its effect on steroidogenesis does not appear to be a consequence of cytotoxicity as there was no reduction in the number of viable cells after incubations in which inhibition of testosterone production was observed.

Studies on the nature of prolactin-like immunoreactivity in bromelin-treated cervical mucus.

Prolactin has been reported to be present in cervical mucus at concentrations higher than those found in blood. Our initial findings appeared to confirm this and the material fulfilled criteria of validity generally applied when an immunoassay is employed on a new biological matrix, i.e. parallelism and chromatographic identity. Further experiments demonstrated that prolactin concentrations in cervical mucus were less than 40 mU/L and the prolactin-like immunoreactivity originally detected was due to the action of the enzyme bromelin which was used to liquefy the mucus. Bromelin has a similar molecular weight to prolactin and appeared to digest prolactin tracer and reduce its ability to bind antiserum in a manner paralleling the effect of adding pituitary prolactin.

Inhibition by (+) and (-) isomers of gossypol of testosterone release from mouse Leydig cells in vitro.

Racemic (+/-) mixtures of gossypol isomers exert an antifertility effect by inhibiting sperm motility and spermatogenesis. Purified (+) gossypol has been shown to be without these actions. In this study pure preparations of both (+) and (-) gossypol were found to inhibit, in a similar manner, both basal and LH-stimulated release of testosterone by isolated Leydig cells at concentrations down to 21 microM. It is possible that use of low doses of pure (-) gossypol could inhibit fertility with less endocrine side effects.

Inhibition by gossypol of testosterone production by mouse Leydig cells in vitro.

Gossypol, a polyphenolic compound prepared from cotton seed, has potential uses as a male contraceptive. It inhibits spermatogenesis and sperm motility but has toxic side effects and furthermore may have effects on endocrine function though evidence for this is contradictory. In this study, gossypol had a dose-dependent inhibitory action on release of testosterone from dispersed mouse Leydig cells. Doses down to 20 microM and 10 microM significantly inhibited unstimulated and LH-stimulated release, respectively.

Multicenter evaluation of assays for estradiol and progesterone in saliva.

Blood samples can be difficult to obtain in studies involving serial sampling, especially in developing countries where there may also be logistic, ethical, and cultural constraints that make frequent blood collection impractical. Assays for steroids in saliva may avoid some of these difficulties. A multicenter study involving laboratories in five countries was carried out to compare the results of assays for salivary estradiol and progesterone performed with centrally provided reagents and assay protocols. Concentrations of salivary steroid as obtained by all but one center were comparable with those reported in the literature. We conclude that assays of hormones in saliva are useful adjuncts to those performed on other body fluids.