Steroid Profiling in Male Wobbler Mouse, a Model of Amyotrophic Lateral Sclerosis.

The Wobbler mouse is an animal model for human motoneuron diseases, especially amyotrophic lateral sclerosis (ALS), used in the investigation of both pathology and therapeutic treatment. ALS is a fatal neurodegenerative disease, characterized by the selective and progressive death of motoneurons, leading to progressive paralysis. Previous limited studies have reported steroidal hormone dysregulation in Wobbler mouse and in ALS patients, suggesting endocrine dysfunctions which may be involved in the pathogenesis of the disease. In this study, we established a steroid profiling in brain, spinal cord, plasma, adrenal glands, and testes in 2-month-old male Wobbler mice and their littermates by gas chromatography coupled to mass spectrometry. Our results show in Wobbler mice the following: 1) a marked up-regulation of corticosterone levels in adrenal glands, plasma, spinal cord regions (cervical, thoracic, lumbar) and brain; 2) a strong decrease in T levels in the testis, plasma, spinal cord, and brain; and 3) increased levels of progesterone and especially of its reduced metabolites 5α-dihydroprogesterone, allopregnanolone, and 20α-dihydroprogesterone in the brain, spinal cord, and adrenal glands. Furthermore, Wobbler mice showed a hypothalamic-pituitary-gonadal hypoactivity. Interestingly, plasma concentrations of corticosterone and T correlate well with their respective levels in cervical spinal cord in both control and Wobbler mice. T down-regulation is probably the consequence of adrenal hyperactivity, and the up-regulation of progesterone and its reduced metabolites may correspond to an endogenous protective mechanism in response to motoneuron degeneration. Our findings suggest that increased levels of corticosterone and decreased levels of T in plasma could be a signature of motoneuron degeneration.

Studies on neurosteroids XX. Liquid chromatography-tandem mass spectrometric method for simultaneous determination of testosterone and 5alpha-dihydrotestosterone in rat brain and serum.

Testosterone (T) and 5alpha-dihydrotestosterone (DHT) are now referred to not only as androgenic steroid hormones, but also as neuroactive steroids, because they elicit anesthetic and anxiolytic effects. Methods using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) coupled with derivatization are developed and validated to examine rat brain and serum levels of T and DHT and their stress-induced changes. The steroids are extracted with methanol-acetic acid from the brain tissue or serum, purified using solid-phase extraction cartridges, derivatized with a permanently charged reagent, 2-hydrazino-1-methylpyridine, and subjected to LC-MS-MS. [19,19,19-(2)H3]-T is used as the internal standard. The intra- and inter-assay coefficients of variation are below 10.0%, and the analytical recoveries are 98.1-103.0%. The developed methods are applied to the animal study and it was found that a fair amount of DHT is continuously and locally synthesized in the brain, and its level is not changed by the immobilization stress and depends on the brain T level.

The biochemical study of intermaxillary fixation (IMF) stress in oral surgery inpatients.

Although intermaxillary fixation (IMF) is performed to treat the patients with maxillary fracture, this procedure is very stressful to the patients. IMF has been reported to increase noradrenaline (NA) release in the brain and elevate plasma corticosterone contents in the rat. These changes were significantly attenuated by diazepam, an anxiolytic of the benzodiazepine family. These results suggest that IMF could greatly affect the pituitary-adrenal system as a stress. In the present study, in order to examine the influence of IMF on the human body function, we measured levels of 17-hydrocorticosteroids (17-OHCS) and 17-ketosteroid (17-KS), which are metabolites of the adreno-cortical hormone cortisol, in the urine of inpatients undergoing IMF. The subjects were requested to fill out a questionnaire on irritableness caused by IMF. In these patients, urinary 17-OHCS levels were significantly increased after IMF and well correlated to the results of the questionnaire. The finding suggested that urinary 17-OHCS levels reflect stress related to IMF, and that such stress mainly causes an irritated feeling. Natural killer cell activity (NK activity), which is considered to be related to stress, was measured in these patients. The relationship between 17-OHCS levels and NK activity was examined in reference to the results of the questionnaire. Questionnaire showed that most patients noted insomnia and an irritated feeling during IMF. To examine the influence of anxiolytic agents on stress related to IMF, an anxiolytic agent, ethyl loflazepate, was administered during IMF, and urinary 17-OHCS levels were measured. There was no correlation between 17-OHCS levels and NK activity in the patients. Furthermore, no correlation was observed between visual analogue scale (VSA) and NK activity. Increases in 17-OHCS levels in the group treated with ethyl loflazepate, an anxiolytic of the benzodiazepine family, were significantly lower than in the untreated group. This suggests that ethyl loflazepate reduced stress responses to IMF. It has been reported that NK activity is reduced inpatients with depression or chronic fatigue syndrome. However, NK activity may not be affected by mechanical stress such as IMF. The finding that an anxiolytic agent, ethyl loflazepate, inhibited stress responses to IMF further suggests that anxiolytic drugs are very useful for treatment of irritated feeling of the patients undergoing IMF.

Abuse-related posttraumatic stress disorder: evidence for chronic neuroendocrine activation in women.

The following study tested the hypothesis that women with post-traumatic stress disorder (PTSD) related to childhood sexual abuse would display elevated norepinephrine-to-cortisol ratios similar to that found in male combat veterans diagnosed with PTSD. Twenty-four-hour urine samples were collected from 28 women: 11 women with PTSD who experienced childhood sexual abuse (PTSD+), 8 women who experienced childhood sexual abuse without PTSD (PTSD-), and 9 nonabused controls. All urine samples were tested for creatinine, total catecholamines, free-cortisol, and 17-ketosteroid levels. Psychological testing validated that the PTSD+ group was significantly elevated on all three subscales of the Impact of Events Scale. Both abused groups (PTSD+ and PTSD-) showed a tendency for polyuria, and the PTSD+ group showed a tendency towards obesity. Thus, neuroendocrine values (micrograms/day) were adjusted by creatinine clearance rates (creatinine mg/day/kg body weight). The corrected values indicated that the PTSD+ group had significantly elevated daily levels of norepinephrine, epinephrine, dopamine, and cortisol. However, because of the parallel elevation in cortisol, the norepinephrine-to-cortisol ratio was not significantly elevated in the PTSD+ diagnosed women in contrast to the findings reported for male PTSD patients. This discrepancy may reflect an important gender difference, an interaction between gender and age at onset of the traumatic experience (childhood abuse in females vs. combat experience in young adult males), or physiological variation related to phase of the disorder.

[Diagnosis and treatment of congenital adrenal hyperplasia in women]. / Rozpoznawanie i leczenie wrodzonego przerostu nadnerczy u kobiety.

In 32 women with signs of androgenization, menstrual disturbances and distinct elevated values of 17-KS, DHEA, pregnantriole, testosterone and very reduced level of 17-OHCS congenital adrenal hyperplasia was diagnosed which established 12.5% individuals with intersexualism. The method of conservative or/and operative treatment and the prognosis were discussed.

[The characteristics of the anatomicophysiological development and hormonal activity in adolescents with tuberculosis of the respiratory organs]. / Osoblyvosti anatomo-fiziolohichnoho rozvytku ta hormonal'noï aktyvnosti u pidlitkiv, khvorykh na tuberkul'oz orhaniv dykhannia.

Parameters of physical development were evaluated using the main anthropometric tests (weight, height, chest circumference) as were those of hormonal activity (the level of somatotrophic hormone, immunoreactive insulin, the content of 17-keto-steroids) in adolescents with tuberculosis of respiratory organs. The above criteria enable one to judge of reactivity and compensatory potential of a specific process and the nature of residual alterations.

[Differential diagnosis of hirsutism in girls between 15-19 years old]. / Rozpoznanie róznicowe hirsutyzmu u dziewczat od 15-19 lat.

On the basis of clinical and hormonal investigations of 58 girls with hirsutism aged between 15-19 years there were confirmed congenital adrenal hyperplasia in 3.4%, late onset adrenal hyperplasia in 24.1% and polycystic ovary syndrome in 72.4%. The proper diagnosis were established above all on endocrinological investigations especially on determinations of 17- ketosteroids, of DHEA, of 17 OH-corticoids in urine and RIA of total and free testosterone in blood.

[UV-irradiation of the blood in the treatment of polycystic ovary syndrome]. / UF-obluchenie krovi v lechenii sindroma polikistoznykh iaichnikov.

Effect of extracorporeal UV irradiation of the blood on the clinical picture and hormonal profile of patients with the polycystic ovaries syndrome was under study. 119 women were administered 582 sessions, and in 54 of these the results were followed up for 1-25 months. A good clinical effect was achieved in 88.9% of these cases, it consisted in recovery of the cycle rhythm (70.7%), pregnancy (29.2%), reduction of hirsutism manifestations (19.04%), decrease of galactorrhea (41.7%), cessation or alleviation of headaches (86.2%), body mass reduction (40%), arterial pressure normalization (43.8%). Well-being of the majority of women was improved by therapy. 17-ketosteroids excretion normalized and persisted normal in a month after the treatment. Blood LH level and the LH/FSH ratio significantly reduced in those in whom these values were elevated in disease. Elimination of hyperandrogenism and a trend to normalization of gonadotropin secretion, resulting from extracorporeal UV irradiation of the blood, indicate that such treatment modality is pathogenetically grounded and justify its use in the treatment of women with the polycystic ovaries syndrome.

[Psycho-electroregulation in conservative treatment of patients with uterine myoma]. / Psikhoélektroreguliatsiia v konservativnoi terapii bol'nykh miomoi matki.

Basing on the concept of the uterine myoma presenting as a psychosomatic process that manifests in ischemic disease of the uterus, the author has tried psychoelectrostimulation in 76 patients with interstitial and subserous interstitial myomas as a method of psychotherapy and electrotranquilization. Indications for such treatment are the presence of psychoemotional stress syndrome (high anxiety level, pain and hemorrhagic syndromes, dysuria) and a rapid growth of the tumor. The reference group consisted of 73 patients with the same myoma forms, treated by hormonal therapy. Psychoelectroregulation has exerted a regulating effect on the central nervous, cardiovascular, endocrine and reproductive systems, it eliminated or alleviated the neurotic symptoms, and slowed down or arrested the tumor growth.

[Endocrine and ultrasound examination of hirsutism]. / Endokrinologische und ultrasonografische Untersuchung des Hirsutismus.

One hundred and fourteen nontumorous hirsute women were investigated. The degree of hirsutism, the menstrual abnormalities and the level of 17-ketosteroids, of DHEA, of 17-OH-corticoids in urine and RIA of total and free testosterone and of sex binding globulins (SHBG) in blood were performed. On the basis of endocrinological and ultrasonographic determinations 3 patients were diagnosed as congenital adrenal hyperplasia, 35 patients as late onset adrenal hyperplasia and 57 women as polycystic ovary syndrome (PCO). The maximal mean level (X + SD) of total and free testosterone above 0.5 ng/ml relatively above 5.05 pg/ml were performed in 94.3% relatively 97.1% in late onset adrenal hyperplasia and in 78.9% relatively 87.7% in PCO. The diagnosis of PCO is possible among 75% of all hirsute patients after exclusion the patients with late onset adrenal hyperplasia. The PCO is confirmed by hormonal or/and ultrasonographic examination. The diagnosis of late onset adrenal hyperplasia is allowable only by hormonal investigation.

[Functional state of the hypophysis, adrenal cortex and gonads in patients with gynecomastia]. / Funktsional'noe sostoianie gipofiza, kory nadpochechnikov i gonad u bol'nykh ginekomastiei.

Patients with gynecomastia revealed simultaneous increase in blood levels of estrogens, glucocorticoids and pituitary (first of all, gonadotropic) hormones associated with a decrease in blood-androgen concentration. Such combination of disturbances may account for hyperplasia of the male breast.

Urinary 5-ene-steroid excretion in non-classical congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency (NC-3BHSD).

The diagnosis of non-classical 3 beta-hydroxysteroid dehydrogenase deficiency (NC-3BHSD) is made either on the basis of significantly elevated serum levels of basal and post-ACTH 5-ene-steroids or by the presence of elevated urinary 5-ene-steroid metabolites. There has been only one report to date describing a single patient where the diagnosis was based on both serum and urinary 5-ene-steroid levels. We, therefore, measured both serum 5-ene-steroid responses to ACTH 1-24 (by RIA) and urinary 5-ene-steroid metabolites (GC-MS) in 42 hirsute premenopausal women. While the serum 5-ene-steroid profile was consistent with NC-3BHSD in 5 women, only 2 of them had increased excretion of 5-ene-steroid metabolites. Elevated 5-ene-steroid excretion was also observed in several patients with normal serum 5-ene-steroids. Detection of NC-3BHSD by either elevated serum 5-ene-steroids or increased urinary excretion of their metabolites in isolation may not therefore be reliable.

Determination of 17-oxosteroid sulphates in serum by ion-pair extraction, prelabelling with dansylhydrazine and high-performance liquid chromatography with fluorescence detection.

A new high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the direct determination of four serum 17-oxosteroid sulphates. Each serum sample was deproteinated with methanol, the methanol was evaporated and 17-oxosteroid sulphates in the residue were extracted with benzene as ion pairs in the presence of tetrapentylammonium ion. The ion pairs were labelled with dansylhydrazine and the hydrazones were separated by HPLC on a Capcell-Pak C8 (silicone polymer-coated silica gel modified with octyl groups) reversed-phase column using methanol-0.5% (w/v) sodium acetate-50% (v/v) acetic acid (57:42:1, v/v/v) as the mobile phase. The eluent was monitored with a fluorometric detector at an excitation wavelength of 330 nm and an emission wavelength of 540 nm.

[2 surgically treated cases of adrenocortical carcinoma producing steroid hormones without endocrinological symptoms--case report and a review of cases in the Japanese literature].

We have experienced two male cases of an adrenocortical carcinoma that have produced steroid hormones without endocrinological symptoms. Both men were admitted to hospital because of an abdominal pain and left hypochondral tumors. In each case, an abdominal CT, an ultrasonogram and an angiogram demonstrated a left adrenal tumor, and levels of blood dehydroepiandrosterone sulfate, etiocholanolone, pregnenolone sulfate and urine 17 KS were elevated. The tumors were resected and the histology revealed that they were adrenocortical carcinomas. Postoperatively, the levels of their urine hormones returned to normal values, though in one man recurrence occurred a year later. Only 8 cases of an adrenocortical carcinoma that has produced steroids without specific endocrinological symptoms have been reported in Japan, and we also report on two cases herein.

Cushing syndrome with food-dependent periodic hormonogenesis.

Periodic hormonogenesis has been described in patients with ACTH-dependent hypercorticism, and fluctuations of cortisol secretion have also been observed in patients with adrenal tumors. In this report, we studied a 41-year-old white male who presented with hypertension, central obesity, and muscle weakness of 2-years duration. His plasma cortisol was low (4.5 micrograms) in the morning and high in the evening (29.3 micrograms). Urinary free cortisol was 750 micrograms/day. A 24-hour cycle demonstrated highest values at noon and in late afternoon. This pattern was not suppressed by dexamethasone. When the patient was kept fasting, plasma cortisol remained low all day, and became elevated immediately after meal administration overnight. A left-sided adrenal mass was demonstrated and removed. In vitro, the adenylate cyclase activity of tumor tissue demonstrated more significant response to vasopressin than to ACTH; other tested peptides were inactive. We propose that a humoral factor induced by eating was responsible for the periodic hormonogenesis, directly stimulating the adrenal secretion of cortisol.

Primary aldosteronism by carcinoma of the adrenal cortex.

A 58-year-old white woman with hypertension and severe hypokalemia was found to have a carcinoma of the left adrenal gland. Plasma renin activity was constantly under the normal limit, while plasma aldosterone levels were pathologically elevated. Plasma cortisol (8:00 a.m.) and excretion rates of urinary free cortisol were within the normal range. After an adrenalectomy, relapsing excessive aldosterone secretion was successfully treated with opDDD (Lysodrene). Ten months after the diagnosis was established, the patient died from a bleeding liver metastasis.

Steroid-binding site of human and rabbit sex steroid binding protein of plasma: fluorescence characterization with equilenin.

The interaction of the estrogen d-3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one (equilenin) with the human and rabbit sex steroid binding proteins (hSBP and rSBP, respectively) has been investigated by using fluorescence and absorption spectroscopy. Equilenin competes for the binding of 5 alpha-dihydrotestosterone. The calculated binding constant of equilenin for rSBP is 1.9 X 10(7) M-1 at 4 degrees C, which can be compared with the binding constant of 5.7 X 10(7) M-1 reported for hSBP [Ross, J.B.A., Torres, R., & Petra, P.H. (1982) FEBS Lett. 149, 240]. The results of fluorescence quenching experiments with the collisional quenchers KI and acrylamide indicate that the bound steroid has limited accessibility to the bulk solvent and that there are no anionic surface groups near the steroid-binding site. The fluorescence excitation spectra of SBP-equilenin complexes are similar to the absorption spectra of equilenin in low-dielectric solvents. The fluorescence emission of the SBP-equilenin complexes, however, exhibits wavelength shifts (red shifts) opposite to those of the steroid in low-dielectric solvents or complexed with beta-cyclodextrin (blue shifts) but similar to the red shift produced by addition of the proton acceptor triethylamine to equilenin in cyclohexane. These data indicate that the steroid-binding site of hSBP and rSBP is a nonpolar cavity containing a proton acceptor that participates in a specific interaction, possibly a hydrogen bond, with the 3'-hydroxyl group of the bound steroid.

Lack of relationship of hypogonadism to muscle wasting in myotonic dystrophy.

Myotonic dystrophy is frequently associated with testicular atrophy. Since androgens may play a role in the maintenance of muscle mass, we have studied the levels of plasma testosterone and gonadotropins and of urinary 17-ketosteroids in 22 men with myotonic dystrophy, 36 normal men, and 16 men (control group) with muscle wasting. Results were correlated with muscle mass as estimated by creatinine excretion and total body potassium. Patients with myotonic dystrophy had significantly lower testosterone and higher gonadotropin levels than normal, and these changes were progressive in longitudinal studies. Testosterone levels were also lower than normal in disease control subjects. There was no correlation between low testosterone levels and diminished muscle mass in either myotonic dystrophy or disease control patients. The low plasma concentration of testosterone in men with myotonic dystrophy and other neuromuscular diseases does not appear to be directly related to their muscle wasting. This study does not exclude the possibility that an alteration in testosterone receptor or tissue effects may contribute to a loss of muscle tissue.