A confirmatory method for detection of a banned substance: the validation experience of a routine EU laboratory.

The Commission Decision 2002/657/EC is a fundamental reference document for the UE laboratories involved in residue analysis although its implementation has caused some difficulties in the requirements interpretation. In this work a pragmatic validation approach of a quantitative confirmatory method for the detection of 17-alpha-(alpha-NT) and 17-beta-19-nortestosterone (beta-NT) in bovine urine by gas chromatography mass spectrometry is proposed. The 19-nortestosterone is a banned anabolic steroid for which no minimum required performance limit (MRPL) has been laid down, therefore the limit reported in Italian Residue Monitoring Plan (2 microg L(-1)) has been considered the reference level to evaluate the method performances. The decision limit (CCalpha) and the detection capability (CCbeta) were obtained by the calibration curve procedure. The minimum required performance level (mrpl), which represents the starting concentration of the calibration curves, was preliminary fixed estimating the results dispersion of blank urine samples fortified at 2 microg L(-1) for each isomer. The found CCalpha and CCbeta were 1.5 and 1.9 microg L(-1) for alpha-NT and 1.2 and 1.4 microg L(-1) for beta-NT. The precision (repeatability and within-laboratory reproducibility) and recoveries were suitable for the investigated concentration range (1-3 microg L(-1)). Finally, the method ruggedness (minor and major changes) has been also demonstrated.

Platelet serotonin pathway in menstrual migraine.

In order to understand the possible 5-hydroxytryptamine (5HT) anomalies in migraine, particularly in the period before the headache attack, we compared the levels of 5HT, its stable metabolite 5-hydroxyindoleacetic acid (5HIAA) and platelet monoaminoxidase (MAO) activity in patients with menstrual migraine with those of healthy female controls. In every subject, blood samples were drawn during both follicular and late luteal phases of the menstrual cycle. In controls, platelet 5HT levels remained stable, whereas 5HIAA levels and MAO activity were higher in the luteal than in the follicular phase, suggesting an increased catabolism of 5HT which occurs physiologically just before menses. In menstrual migraine 5HIAA levels and MAO activity showed similar changes with higher values in the luteal than in the follicular phase. The luteal phase values were significantly higher than those of controls. Also, and in contrast to controls, 5HT levels decreased in the luteal phase. These data suggest that 5HT availability is reduced in menstrual migraine, possibly due to an increased catabolism and/or to a reduced synthesis, and hence predisposes patients to migraine attacks.

Changes of neuroendocrine axes in patients with menstrual migraine.

Menstrual migraine (MM) is a menstrually related disorder (MRD) characterized by several symptoms in common with premenstrual syndrome (PMS). It has been hypothesized that in both MM and PMS hormonal cyclicity could change the balance of neurotransmitters and neuromodulators like monoamine and opioid. In this article we analyze all the data collected by our group on the central opioid tonus and the adrenergic and serotonergic systems in patients affected by menstrual migraine.

Changes of opioid modulation of the hypothalamo-pituitary-adrenal axis in patients with severe premenstrual syndrome.

To assess the function of the hypothalamus-pituitary-adrenal axis in patients with severe premenstrual syndrome (PMS) 28 patients and 14 asymptomatic controls were studied during the mid- to late-luteal phase of the menstrual cycle. The response of plasma cortisol to both high-dose naloxone and corticotropin-releasing hormone (CRH) was assessed. Naloxone stimulated a significant cortisol release in controls whereas it was otherwise almost absent in patients. CRH stimulated a greater release of cortisol in patients than in controls. Fifteen patients met criteria for either current anxiety and/or mood disorders. The cortisol secretion after both naloxone and CRH stimulations was similar for PMS patients with or without psychiatric disorders. These data indicate that endogenous opioids modulate the activity of the hypothalamus-pituitary-adrenal axis. Irrespective of the concomitant presence of menstrual migraine or psychiatric disorder, such control is altered in patients with severe PMS because of the possible hyposensitivity of opiate receptors. The hyperresponsiveness to CRH may be the consequence of the reduced inhibition that endogenous opioids tonically exert on HPA axis.

Life events impact in patients with secondary amenorrhoea.

To evaluate the relationship between stressful life events and the onset of secondary amenorrhoea Paykel's semi-structured interview for Recent Life Events was administered to patients affected by secondary amenorrhea and also to healthy volunteers. The number, quality, and objective negative impact of life events were compared among different hormonal subtypes of secondary amenorrhoea and healthy normally menstruating women, as a control group. The number of life events in amenorrhoeic patients (N = 131) was significantly greater than those observed in the control group (N = 64) (45.9 vs 32.8%). Moreover, where only hypothalamic hypogonadotrophic amenorrhoea was considered, the occurrence of life events was significantly higher (59.8%) than in hyperandrogenic (26.6%) or in normogonadotrophic (20.4%) patients. The most prevalent events among hypothalamic hypogonadotrophic amenorrhoeic patients were those classified as 'undesirable', 'uncontrolled' and with 'Objective Negative Impact'. The present study supports the hypothesis of a cause-effect relationship between stressful personal life events and the onset of secondary amenorrhoea of hypogonadotrophic subtype.

The neuroendocrine effects of sumatriptan, a specific ligand for 5-HT1-like receptors.

OBJECTIVE: A relationship between the serotoninergic and the opiatergic system in the pathogenesis of head pain is supported by several data. This study was carried out to investigate the neuroendocrine effects of sumatriptan, a specific serotonin agonist used in the treatment of migraine, on hypothalamic-pituitary-adrenal axis (PHA) hormones. DESIGN: Two consecutive studies were performed. In study A, eight subjects received a subcutaneous (s.c.) injection of sumatriptan (6 mg). In study B, a further six subjects were randomized to receive either sumatriptan or placebo. SUBJECTS: Healthy volunteers recruited within the staff (eight males and six females) were studied. MEASUREMENT: In study A, plasma cortisol and PRL were measured by direct RIA and beta-endorphin after extraction and chromatography. Samples were collected from 60 minutes before to 120 minutes after the administration of the drug, at 15-minute intervals. According to the data of the first study, in study B, in addition to cortisol and beta-endorphin, ACTH was also measured. RESULTS: Significant increases in the mean beta-endorphin and cortisol concentrations were found in every subject receiving sumatriptan, while no significant changes were observed in prolactin plasma levels. Study B confirmed the activation of the pituitary-adrenal axis, additionally demonstrating the release of ACTH, and indicated that placebo has no effects. CONCLUSION: Acute s.c. stimulation with sumatriptan activates the pituitary-adrenal axis.

The association of menstrual migraine with the premenstrual syndrome.

To investigate the comorbidity of premenstrual syndrome (PMS) and menstrual migraine, the Menstrual Distress Questionnaire (MDQ) was prospectively administered for two consecutive menstrual cycles to 22 patients with menstrual migraine, 12 cases with migraine without aura and 15 patients with PMS. MDQ scores varied throughout the menstrual cycle in each patient group, the wider changes being shown by patients with PMS. Fourteen menstrual migraine patients and 4 migraine without aura patients achieved diagnostic criteria for PMS over two menstrual cycles. In these patients MDQ scores did not differ from PMS sufferers at any stage of the menstrual cycle. The premenstrual increase of each cluster of PMS symptoms was identical in menstrual migraine and PMS subjects with the exception of negative affect. We suggest that PMS symptoms should be taken into account in the IHS diagnostic criteria for menstrual migraine.

Neuroendocrine changes in luteal function in patients with premenstrual syndrome.

The present study evaluates the luteal progesterone (P) and LH secretions in 14 patients affected by premenstrual syndrome (PMS) and in 14 asymptomatic controls through the evaluation of their episodic release. PMS was prospectively confirmed in two consecutive menstrual cycles using Moos' Menstrual Distress Questionnaire. A pulsatility study was performed during the luteal phase. Blood samples were drawn every 10 min for 12 h, beginning at 0800 h. Statistically significant pulses were detected using the Detect program, and the degree of concordance of LH and P pulses was estimated. Similar mean 12-h P levels were found in controls (mean +/- SD, 13.9 +/- 9.3 nmol/L) and patients (14.2 +/- 10.1). LH levels were also similar in the two groups. Patients showed a higher P pulse frequency (13.4 +/- 1.8 vs. 11.4 +/- 2.3; P < 0.02) and a reduced amplitude of secretory episodes (126.5 +/- 61.6% vs. 187.1 +/- 126.7%; P < 0.03) than controls. Similarly, PMS patients showed pulsatile LH release of increased frequency and reduced amplitude than controls. A significant degree of concordance between LH and P pulses was observed in both groups, with a time lag of 0-10 min; that is, P secretory episodes follow LH with a delay of 0-10 min. These findings demonstrate that despite the fact that integrated P levels in PMS patients are similar to those in control subjects, the episodic secretion of the hormone is characterized by pulses of increased frequency and reduced amplitude. This phenomenon is temporally related to LH secretion, thus reinforcing the concept of PMS as a neuroendocrine disorder.

Stressful life events and affective disorders inhibit pulsatile LH secretion in hypothalamic amenorrhea.

The aim of this study was to evaluate relationships between emotional state and hypothalamic activity in patients with hypothalamic secondary amenorrhea. Sixty-seven normal weight patients with hypothalamic amenorrhea were submitted to concomitant psychological and LH pulsatility evaluation. Structured clinical interview for anxiety and depressive disorders (DSM III-R) as well as life events investigations (Paykel test) were performed. LH pulses (blood sampling every 10 min for 4 hr) were analyzed through DETECT program and Instantaneous Secretory Rate were computed. Twenty-one patients reporting life events associated to the onset of amenorrhea had LH pulse frequency (2.28 +/- 1.10 pulses/4 hr) lower than those without life events (3.40 +/- 1.46 p = .007). LH pulses amplitude was lower in patients meeting a DSM III-R (21 cases: 1.22 +/- 0.96 mIU/ml) diagnosis than in those without (1.99 +/- 1.20 p = .04) diagnosis. Plasma estradiol and FSH levels as well as duration of amenorrhea and Body Mass Index were similar among groups. It is concluded that psychogenic factors (namely the presence of life events related to the onset of menstrual disorder) are associated with significant and specific changes of hypothalamic activity which could be involved in determining hypogonadism.

Psychosomatic disorders related to gynecology.

In this paper current issues on four psychosomatic disorders related to gynecology will be reviewed. The history, nosological problems and psychoneuroendocrine correlates of psychogenic amenorrhea have been summarized taking into account the important role of psychological factors in inducing loss of menses. Definition and diagnostic problems in assessing menstrually related disorders (formerly premenstrual syndrome) have been reviewed, looking at this syndrome as a disorder of adaptation to the cyclical changes of neuroendocrine functions. The impact of stress on fertility and, on the other hand, the effects of infertility on psychological well-being have been pointed out trying to ascertain the pathways involved in these mutual relationships. Finally, the issue of mood changes at menopause and the effects of steroid replacement on affective state have been discussed. As a whole, these evidences indicate the importance of a close cooperation between gynecologist and psychiatrist in the management of gynecological disorders.

Oral magnesium successfully relieves premenstrual mood changes.

Reduced magnesium (Mg) levels have been reported in women affected by premenstrual syndrome (PMS). To evaluate the effects of an oral Mg preparation on premenstrual symptoms, we studied, by a double-blind, randomized design, 32 women (24-39 years old) with PMS confirmed by the Moos Menstrual Distress Questionnaire. After 2 months of baseline recording, the subjects were randomly assigned to placebo or Mg for two cycles. In the next two cycles, both groups received Mg. Magnesium pyrrolidone carboxylic acid (360 mg Mg) or placebo was administered three times a day, from the 15th day of the menstrual cycle to the onset of menstrual flow. Blood samples for Mg measurement were drawn premenstrually, during the baseline period, and in the second and fourth months of treatment. The Menstrual Distress Questionnaire score of the cluster "pain" was significantly reduced during the second month in both groups, whereas Mg treatment significantly affected both the total Menstrual Distress Questionnaire score and the cluster "negative affect." In the second month, the women assigned to treatment showed a significant increase in Mg in lymphocytes and polymorphonuclear cells, whereas no changes were observed in plasma and erythrocytes. These data indicate that Mg supplementation could represent an effective treatment of premenstrual symptoms related to mood changes.

Naproxen sodium in menstrual migraine prophylaxis: a double-blind placebo controlled study.

In this study, the efficacy of Naproxen sodium (Nxs) in the prophylaxis of Menstrual Migraine (MM) was tested, versus Placebo (PL). Forty women suffering from MM were admitted to a double-blind treatment protocol with Nxs 550 mg twice each day by mouth or Placebo (PL), for 3 months; in the next 3 months all the women were treated with the active drug in an open study. The headache intensity and duration, as well as the number of days of headache and the analgesic consumption, were significantly reduced with Nxs compared to PL. The efficacy of Nxs, shown also in improving premenstrual pain, and its good tolerability, support the use of this drug in the prophylactic therapy of MM.

Opioid control of the hypothalamus-pituitary-adrenal axis cyclically fails in menstrual migraine.

To assess the biological correlates of the precipitation of migraine attacks in the perimenstrual period, plasma beta-endorphin (beta-EP) and cortisol responses to naloxone (8 mg iv) and corticotropin releasing hormone (100 micrograms iv) were evaluated in both the follicular phase and the premenstrual period in 7 patients suffering from menstrual migraine and in 7 healthy, asymptomatic control volunteers. In the controls, naloxone evoked a significant release of both beta-EP (F = 5.86, p less than 0.002) and cortisol (F = 4.43, p less than 0.008), independently of the menstrual cycle phase (F = 0.31 and 1.04, for beta-EP and cortisol, respectively). Menstrual migraine patients, on the other hand, showed a significant hormone response only in the follicular phase, not in the premenstrual period. Corticotropin releasing hormone significantly increased beta-EP and cortisol in both the controls and the menstrual migraine patients, independently of the menstrual cycle phase. In both the naloxone and corticotropin releasing hormone testings, the basal beta-EP levels measured in the premenstrual period were lower than those observed in the follicular phase (p less than 0.02). These data demonstrate a cyclical, premenstrual dysfunction of the hypothalamic control exerted by opioids on the hypothalamus-pituitary-adrenal axis. Impairment of this fundamental adaptive mechanism (involved in stress responses and in pain control) could establish a causal relationship between menstrual-related migraine attacks and premenstrual opioid hyposensitivity.

Neuroendocrine correlates of premenstrual syndrome: changes in the pulsatile pattern of plasma LH.

Some studies suggest that patients suffering from premenstrual syndrome (PMS) may be affected by an endogenous opioid dysfunction. Since opioids are the main modulators of the pulsatile LH secretion, we evaluated plasma LH pulsatility in 13 patients with PMS (aged 33.1 yr) and in six asymptomatic control volunteers (aged 31.5 yr), in the late luteal phase (-7, -5 days before their next menses). The patients were prospectively evaluated for two menstrual cycles with the Menstrual Distress Questionnaire; the main symptoms which worsened during the premenstrual period were mood swings and water retention. The pulsatility of plasma LH secretion was studied by collecting blood samples every 10 min for 12 hr, starting at 0800h. The presence of LH pulses was estimated using the program DETECT on the raw data. This program also allows the computation of the instantaneous secretory rate (ISR). Ovulation was ascertained in all the controls and in nine PMS patients by means of urinary LH assay and luteal progesterone (P) determination. The remaining four patients did not ovulate. Both the ovulatory and the anovulatory PMS patients had an increased number of LH pulses/12 hr (10.3 +/- 2.4 and 11.5 +/- 4.4, mean +/- SD, respectively) in comparison with the controls (7.0 +/- 1.3 pulses, p less than 0.01), together with a reduced amplitude and duration. Similar findings were obtained with the ISR computation. Plasma P levels were similar in both the ovulatory patients and controls. The increased frequency and reduced amplitude of LH pulses in the PMS patients most likely reflect a dysfunction of hypothalamic Gn-RH release, possibly linked to a reduction of opioid inhibition.

Premenstrual failure of alpha-adrenergic stimulation on hypothalamus-pituitary responses in menstrual migraine.

In nine women suffering from menstrual migraine (MM), and in six healthy asymptomatic volunteers, plasma beta-endorphin (beta-EP), growth hormone (GH), norepinephrine (NE), and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) concentrations were measured in response to clonidine (0.075 mg, i.v.) stimulation. In MM patients clonidine testing was performed in both the early and the late luteal phases of the menstrual cycle. Premenstrual symptoms were prospectively evaluated in the actual cycle using the Moos Menstrual Distress Questionnaire. beta-EP (after gel chromatography) and GH were measured using radioimmunoassay. NE and MHPG were evaluated by HPLC using electrochemical detection. In both phases of the menstrual cycle clonidine significantly reduced NE and MHPG levels in MM patients and controls in a similar way. In MM patients beta-EP and GH plasma levels were stimulated by clonidine only in the early luteal phase, whereas they remained unchanged when they were stimulated in the premenstrual period. In controls the response of both hormones was not affected by the menstrual cycle. The lack of hormonal response to clonidine in MM may suggest a postsynaptic alpha 2-adrenoreceptor hyposensitivity during the premenstrual period. This demonstrates a transient vulnerability of the neuroendocrine/neurovegetative systems, and could thus be a factor facilitating the precipitation of both behavioral changes and migraine attacks.

Naproxen sodium in the treatment of premenstrual symptoms. A placebo-controlled study.

The prostaglandin system is thought to play a role in the etiology of the premenstrual syndrome. Many authors describe that prostaglandins are involved in both central and peripheral symptoms. To test this hypothesis, we studied the effects of naproxen sodium treatment (550 mg twice daily, from day -7, in relation to next menses, to the 4th day of the cycle) in 34 patients suffering from premenstrual syndrome. Six cases dropped out. Fourteen women were given placebo for the first three cycles of the trial, followed by active drug. The other 14 patients were given naproxen sodium, beginning from the first cycle. In order to evaluate premenstrual symptoms, the Moos menstrual distress questionnaire was prospectively applied during the 2-month run-in period and at the 3rd and 6th cycles of treatment. During our double-blind naproxen sodium study, both menstrual and premenstrual 'pain' decreased during active drug treatment, while placebo was ineffective. We also obtained a significant improvement of premenstrual 'behavioral changes' which is probably related to the relief of painful symptomatology. In conclusion, this study indicates that naproxen sodium is a useful and safe drug in the treatment of premenstrual and menstrual pain related symptoms.

Premenstrual increase of intracellular magnesium levels in women with ovulatory, asymptomatic menstrual cycles.

Intracellular magnesium (Mg) levels regulate several enzymatic reactions and the hypoactivity of Mg has been involved in different pathological states. In addition to other factors, gonadal hormones, too, have been found to interfere in Mg balance. This study evaluates the changes in Mg in women throughout the normal menstrual cycle and those measured at weekly intervals in males. Magnesium and potassium (K) levels were measured in the plasma, red blood cells (RBC), lymphocytes (LC) and polymorphonucleated cells (PMN) of 11 normal menstruating women, in different periods of their menstrual cycle. Blood samples were collected every 4th day. According to the time and LH, progesterone and estradiol levels, they were classed as follicular (Foll), periovulatory (OV), luteal (Lut) or premenstrual (PM). The Menstrual Distress Questionnaire completed by the subjects revealed that no significant symptomatology was present. Four blood samples were also collected from 4 normal males, at weekly intervals. Mg and K were determined by atomic absorption spectrophotometry. LC and PMN were purified by centrifugation on a Ficoll discontinuous gradient. No differences were found in males versus females in the Mg or K contents in the different compartments. In the 4 periods tested, the K levels were constant, in both females and males. The same applies for Mg contents in males. In females, Mg contents, as well as the Mg/K ratio in LC and PMN, showed a significant increase in the premenstrual period compared with the other periods of the cycle. On the contrary, plasma and RBC Mg levels were constant throughout the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)