Effect of Lipopolysaccharide on the Progression of Non-Alcoholic Fatty Liver Disease in High Caloric Diet-Fed Mice.

The incidence of non-alcoholic steatohepatitis (NASH) is increasing. Because gut microbiota have been highlighted as one of the key factors in the pathogenesis of metabolic syndrome, we investigated the involvement of the bacterial component in the progression of non-alcoholic fatty liver (NAFL) to NASH. C57BL/6 mice were fed with maintenance food (MF, groups A and B) or a high caloric diet (HCD, groups C and D) for 1 month. Mice were then divided into four groups: Groups A and C were inoculated with PBS, while groups B and D were inoculated with lipopolysaccharide (LPS) plus complete Freund's adjuvant (CFA). The inoculations were performed a total of 3 times over 3 months. At 6 months, while hepatic steatosis was observed in groups C and D, cellular infiltration and fibrosis were less evident in group C than in group D. Inflammatory cytokines were upregulated in groups B and D. 16S rRNA pyrosequencing of whole colon homogenates containing faeces showed that certain bacterial groups, such as Bacteroidaceae, Peptostreptococcaceae and Erysipelotrichaceae, were increased in groups C and D. Although loading of bacterial components (LPS) resulted in hepatic inflammation in both MF- and HCD-fed mice, HCD feeding was more crucial in the progression of NAFL during the triggering phase.

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits.

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.

Effect of doxazosin on insulin resistance in hypertensive patients with obesity.

We determined the effect of alpha-adrenergic blocking agent doxazosin on insulin resistance in 19 hypertensive patients (blood pressure [BP] >160/90 mmHg) with obesity (mean body mass index [BMI]: 26.7 +/- 1.9 kg/m (2)). Patients received doxazosin 4 mg/day for 12 months. Systolic and diastolic BP decreased from 169 +/- 10.8 mmHg to 147 +/- 11.9 mmHg (p < 0.0001) and from 102 +/- 8.1 mmHg to 87 +/- 5.0 mmHg (p < 0.0001), respectively. Insulin resistance and fasting immunoreactive insulin (IRI) were lower at study end vs. baseline (HOMA-R = 1.29 +/- 0.38 vs. 3.58 +/- 2.23 [p = 0.022]; IRI = 6.00 +/- 1.88 microU/ml vs 13.74 +/- 8.51 microU/ml [p = 0.046]). Total cholesterol was significantly reduced following treatment. Circulating TNF-alpha and leptin levels decreased significantly within 3 months of treatment; leptin was independently associated with insulin resistance when adjusted for BMI. We conclude that doxazosin improves insulin resistance and improves dyslipidemia in obese hypertensive patients, and has a beneficial effect on adipose endocrine activity.

Pituitary apoplexy caused by luteinizing hormone-releasing hormone in prolactin-producing adenoma.

We report a case in which pituitary apoplexy developed shortly after an intravenous (i.v.) injection of luteinizing hormone-releasing hormone (LH-RH). A 56-year-old man with prolactin-producing pituitary tumor complained of severe headache, visual field loss and facial nerve palsy shortly after LH-RH test. Magnetic resonance image (MRI) revealed a hemorrhage in the pituitary adenoma. He showed dramatic improvement in his symptoms after decompression surgery. These findings suggest a causal relationship between the i.v. injection of LH-RH and pituitary apoplexy. Possible pituitary apoplexy should be kept in mind during pituitary testing.

Effects of amlodipine on serum levels of adrenal androgens and insulin in hypertensive men with obesity.

We investigated the effects of the calcium channel blocker amlodipine besilate on serum levels of adrenal androgens and insulin in 20 men with essential hypertension and obesity (age: 51.9+/-4.7 years, body mass index: 27.7+/-1.5 kg/m2). All were treated with amlodipine besilate (Norvasc) for 3 months. Blood pressure, fasting plasma glucose (FPG), HbA1c and serum levels of insulin, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), and lipids were measured before and after a 3-month period. In 10 patients, 75 g oral glucose tolerance test (75 g-OGTT) was also performed. Amlodipine besilate treatment 1) lowered the fasting serum insulin level and total serum insulin level during 75 g-OGTT and 2) increased serum DHEA and DHEA-S levels. No changes in fasting plasma glucose, HbA1c and serum lipids were observed during treatment. We conclude that amlodipine besilate improves insulin resistance and consequently increases serum DHEA and DHEA-S levels.

Cilnidipine, the N- and L-type calcium channel antagonist, reduced on 24-h urinary catecholamines and C-peptide in hypertensive non-insulin-dependent diabetes mellitus.

To evaluate the effects of cilnidipine (CNP), L- and N-type calcium channel blocker and nilvadipine (NVP) on 24-h urinary epinephrine (U-EP), norepinephrine (U-NE), dopamine (U-DA) and C-peptide (U-CPR) in patients associated with hypertension and non-insulin-dependent diabetes mellitus (HT-NIDDM), a randomized crossover study was performed with 35 HT-NIDDM patients. The patients were given CNP (10 mg/day) and NVP (8 mg/day), separately, for 4 weeks each. After CNP treatment, U-NE, U-DA and U-CPR levels were significantly reduced compared with pre-treatment levels: 160.4 +/- 12.7 to 111.7 +/- 8.9 microg/day (mean +/- S.E., P < 0.005); 934.8 +/- 163.4 to 590.3 +/- 33.4 microg/day (P < 0.05); 86.7 +/- 9.9 to 57.6 +/- 7.4 microg/day (P < 0.05), respectively. Although no significant differences were observed in U-EP, U-NE, U-DA and U-CPR levels by NVP treatment, U-NE, U-DA and U-CPR levels after CNP treatment were significantly lower than those after NVP treatment: 111.7 +/- 8.9 versus 155.0 +/- 13.7 microg/day (P < 0.02); 590.3 + 33.4 versus 822.2 +/- 104.3 microg/day (P < 0.05); 57.6 +/- 7.4 versus 80.6 +/- 8.1 microg/day (P < 0.05), respectively. In conclusion, it was demonstrated that CNP treatment significantly reduced U-NE, U-DA and U-CPR excretion compared with NVP treatment in HT-NIDDM patients.

Analysis of the superantigen-producing ability of Yersinia pseudotuberculosis strains of various serotypes isolated from patients with systemic or gastroenteric infections, wildlife animals and natural environments.

Yersinia pseudotuberculosis is a pathogen causing gastroenteritis as well as acute and systemic infections. This organism produces a superantigenic exotoxin, designated Y. pseudotuberculosis-derived mitogen (YPM). We consider this exotoxin to be the primary pathogen of the systemic type infection. In this study, we examined 101 Y. pseudotuberculosis strains isolated from various sources, patients with the systemic or the gastroenteric type of infections, wildlife animals and natural environments for the presence of the YPM gene and the production of YPM or other related superantigens. We found that all of the strains isolated from patients with systemic type infection carried the YPM gene and produced YPM. A certain proportion of the organisms isolated from patients with the gastroenteric type infection, wildlife animals or natural environments did not carry the YPM gene nor produced superantigens. These results suggest that YPM is involved in the pathogenesis of the systemic type of Y. pseudotuberculosis infection.

18-Hydroxycortisol and 18-oxocortisol in Cushing's syndrome.

In patients with primary aldosteronism due to an aldosterone-producing adenoma and glucocorticoid-suppressible aldosteronism, 18-hydroxycortisol and 18-oxocortisol excretions are elevated. Both steroids are synthesized in the transitional zone between the zona glomerulosa and zona fasciculata. There are no reports concerning production of these steroids in Cushing's syndrome due to adrenal adenoma or hyperplasia, as far as we know. We determined the urinary excretion and serum concentration of 18-hydroxycortisol and 18-oxocortisol in eight patients with Cushing's syndrome (four due to adrenal adenoma, and four due to adrenal hyperplasia). Two of the four patients with adrenal adenoma had high levels of urinary and serum 18-hydroxycortisol and 18-oxocortisol; on the other hand all the patients with adrenal hyperplasia had normal urinary and serum levels of both steroids. Patients with high concentrations of 18-hydroxycortisol and 18-oxocortisol, however, showed no differences in clinical features, routine laboratory findings and hormonal data compared to patients with normal concentrations of 18-hydroxycortisol and 18-oxocortisol. Our data suggest that some adrenal adenomas causing Cushing's syndrome originate from transitional cells.

Serum androgen levels in hyperthyroid women.

Thyroid hormone is known to affect androgen metabolism, however, there are few studies in which alterations of androgen metabolism are simultaneously examined in patients with clinical thyroid disorders. In this study, we investigated the alterations of thyroid hormone and androgens before and during treatment in patients with hyperthyroidism. Fifteen female patients with hyperthyroidism due to Graves' disease were studied. From these patients, blood samples were obtained before treatment and at 1 month (M), 2M, 3M, 4M and 6M after beginning of treatment. Concentrations of free T4(FT4), free T3(FT3), testosterone (T), androstenedione (delta 4A), dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol(3 alpha-diol) and androsterone (AD) and sex hormone binding globulin (SHBG) were measured by radioimmunoassays (RIAs) or immunoradiometric assay (IRMA). As normal controls, ten healthy women were also studied. Before treatment concentrations of DHT (mean +/- SD: 3.35 +/- 0.78 nmol/L), 3 alpha-diol (0.78 +/- 0.11 nmol/L), AD (6.73 +/- 0.64 nmol/L) and SHBG (184.9 +/- 68.1 nmol/L) were significantly elevated compared with those of normal controls. T and delta 4A levels were not significantly different from normal values. DHT/T ratio indicating 5 alpha-reductase activity was 4.62 +/- 2.55 and significantly higher than that in normal controls. At 2 months after beginning of treatment with anti-thyroid drugs, thyroid function (FT4 and FT3) became normal and at 3 months after beginning of treatment, DHT, 3 alpha-diol, AD and DHT/T ratio decreased to normal range. SHBG level was gradually decreased, however still higher than that of the normal control group at 6 months after beginning of treatment. There was a time lag in recovery between serum androgen levels and SHBG level.

Serum androsterone levels in children.

Serum androsterone was measured by radioimmunoassay in 79 girls and 80 boys aged between six months to sixteen years. In boys, androsterone levels were found to be low until the age of 12 years and then rapidly increased between the ages of 12-13 years. This increase of serum androsterone in boys seems to be related to the rapid increase of testosterone. On the other hand, in girls the androsterone levels were found to be low until the age of 10 years. However, from this age onwards, although individual values were variable, androsterone levels did not increase as rapidly as in boys but rather gradually with age which appeared to be influenced by the progressive increase of dehydroepiandrosterone (DHEA) in girls.

Differences in down-regulation of glucocorticoid receptor mRNA by cortisol, prednisolone and dexamethasone in HeLa cells.

Glucocorticoids regulate the levels of their cognate receptors in a number of target tissues and in many different cell lines. We have compared the effect of three glucocorticoids, cortisol and its synthetic derivatives, prednisolone an dexamethasone, on the levels of glucocorticoid receptor (GR) mRNA in HeLa cells. Clinically, the synthetic derivatives are more active in hormonal action and have a longer half-life than cortisol. In the present study, the amounts of GR mRNA in HeLa cells were examined by Northern blot hybridization after treatment with cortisol, prednisolone or dexamethasone. These glucocorticoids decreased GR mRNA levels differently. After 24 h treatment with 1 x 10(-5) M cortisol, GR mRNA levels were only marginally suppressed (90% of the control), while prednisolone and dexamethasone suppressed GR mRNA levels to 67 and 57%, respectively. These differences may relate to the biological activities of these glucocorticoids. In time course studies, GR mRNA levels of the cells treated with cortisol and prednisolone decreased to the minimum levels within 4 h and then recovered gradually, while those treated with dexamethasone reached the minimum level at 8 h and remained suppressed for more than 24 h. These differences may relate to the biological half-lives of these glucocorticoids.

Exacerbation of rheumatoid arthritis after removal of adrenal adenoma in Cushing's syndrome.

A 46-year-old woman with rheumatoid arthritis had been on non-steroidal antiinflammatory agents for eighteen years until she developed cushingoid features and hypertension resistant to antihypertensive drugs. She had high plasma cortisol and 24 h urinary 17-hydroxycorticosteroids (17HCS) which were not suppressed by 8 mg dexamethasone per day for two days. The circadian rhythm of plasma cortisol was absent and plasma ACTH concentrations were suppressed before and after intravenous administration of CRH. Abdominal computed tomography demonstrated a tumor (3.0 x 3.0 x 2.3 cm) in the right adrenal gland and a 131I-6 beta-19-nor-methylcholesterol scan revealed marked uptake on the same side. The patient underwent a right adrenalectomy and the diagnosis of a cortisol secreting benign adenoma was histologically confirmed. Blood pressure declined and cushingoid features regressed, but three months after the operation and while the patient was on replacement, she complained of pain on motion, marked tenderness and swelling of fingers, wrists, elbows, knees and foot joints, and had very high rheumatoid factors. Treatment with immunosuppressive drugs and oral and intraarticular administration of glucocorticoids were necessary to relieve the clinical symptoms of rheumatoid arthritis. In summary, we report a patient with rheumatoid arthritis and Cushing's syndrome due to an adrenal adenoma, in whom rheumatoid arthritis was exacerbated after curing the Cushing's syndrome. This suggests that it is imperative to follow the development and/or course of autoimmune diseases after the treatment of Cushing's syndrome.

Enzyme-linked immunosorbent assay (ELISA) method for screening of non-classical steroid 21-hydroxylase deficiency.

Morning salivary 17-hydroxyprogesterone (17-OHP) determination is a useful method to diagnose non-classical 21-hydroxylase deficiency (NC 21-OHD). In ethnic groups with high frequency of NC 21-OHD, mass screening is thought to be necessary. A pilot screening test for NC 21-OHD was performed by measuring morning salivary 17-OHP by enzyme-linked immunosorbent assay (ELISA). Two individuals were found NC 21-OHD from 230 subjects. Results of this ELISA were well correlated with those of established radioimmunoassay. ELISA can be performed in any laboratory because of non-radioactivity and the procedure is very easy by the use of a microplate. This ELISA is suitable for large scale population studies. We conclude that morning salivary 17-OHP determination by ELISA is a useful method for NC 21-OHD screening test.

Plasma levels of C19 steroid glucuronides in pre-menopausal women with non-classical congenital adrenal hyperplasia.

Recent reports have thrown doubt on the role of measurements of plasma 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (3 alpha-diolG) as a marker of peripheral androgen metabolism in women with polycystic ovarian syndrome and idiopathic hirsutism. It has been suggested that a plasma profile of C19 steroid glucuronides may be more informative. While preliminary data indicates that both 3 alpha-diolG and androsterone G (ADTG) may arise from adrenal steroid precursors, there have been no reports of C19 steroid glucuronides in women with non-classical, or late-onset congenital adrenal hyperplasia (NC-CAH), who constitute a significant proportion of the hirsute female population. We therefore measured plasma levels of 3 alpha-diolG, ADTG and dihydrotestosterone G (DHTG) before and following a standard Cortrosyn test in 15 symptomatic and 3 asymptomatic NC-CAH patients, 5 heterozygote carriers for 21-hydroxylase deficiency (NCHETS) and 18 normal women. The effects of chronic glucocorticoid (GCR) therapy (greater than 3 months) on the C19 steroid glucuronide profile in the symptomatic patients was also investigated. Baseline plasma levels of all 3 glucuronides were significantly (P less than 0.001) higher in symptomatic patients compared with either normals or NCHETS. However, the order of discrimination was ADTG greater than 3 alpha-diolG greater than DHTG. There were no significant differences between steroid glucuronide levels for NCHET and normal women and the C19 steroid glucuronide concentrations for the asymptomatic NC-CAH patients were greater than 2 SD above the normal means. Moderate clinical improvement was observed in all patients receiving oral GCR therapy and was accompanied by approx. 80% suppression of the plasma levels of all 3 C19 steroid glucuronides. This contrasts with a mean suppression of androstenedione of only 50%. However, plasma levels of the C19 steroid glucuronides were not significantly increased in response to a short ACTH stimulation test. This may be explained by the fact that the androgen glucuronides are thought to be peripherally formed metabolites derived from unconjugated glandular secreted androgen precursors and thus their synthesis at 60 min following adrenal stimulation may lag substantially behind that of their respective precursors. There were significant linear correlations between the levels of all 3 glucuronides, but neither correlated with Ferriman-Gallway scores, body mass index or 17-hydroxyprogesterone levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum profiles of steroid hormones in patients with Cushing's syndrome determined by a new HPLC/RIA method.

We developed a method for simultaneously measuring steroid hormones in very small volumes of serum, using a combination of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). By this method, aldosterone, cortisol, 11-deoxycortisol, estrone, estradiol, androstenedione, dehydroepiandrosterone, deoxycorticosterone, 17-hydroxyprogesterone, testosterone, pregnenolone, and progesterone could be determined in a single 100-microL aliquot of serum from normal adults and patients with Cushing's syndrome. The steroid profile associated with Cushing's syndrome caused by adrenal adenoma was quite distinct from that associated with the syndrome caused by adrenal hyperplasia. Serum concentrations of androstenedione, dehydroepiandrosterone, estrone, estradiol, 17-hydroxyprogesterone, pregnenolone, and testosterone were significantly higher in patients with adrenal hyperplasia than in those with an adenoma. We compared the results of this HPLC/RIA method with those of 125I RIAs. The use of a HPLC/RIA system to obtain an accurate and sensitive profile of a range of serum steroids, as described here, obviates the need for large volumes of blood.

Aldosterone synthesis in salt-wasting congenital adrenal hyperplasia with complete absence of adrenal 21-hydroxylase.

BACKGROUND: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a disorder of cortisol and aldosterone biosynthesis that results from mutations in the CYP21 gene encoding the adrenal 21-hydroxylase P-450c21. It can cause severe salt wasting in newborns that requires long-term treatment with glucocorticoids and mineralocorticoids. We describe a spontaneous partial recovery from this disorder in a 19-year-old woman who had discontinued treatment. METHODS: We measured plasma and urinary levels of adrenal hormones, plasma renin activity, and sodium balance longitudinally in the patient and four other patients in whom adrenal hyperplasia had been diagnosed in infancy and in whom DNA analysis had predicted a complete absence of functional P-450c21. The ratio of plasma renin activity to urinary aldosterone was used as a measure of the response of the adrenal zona glomerulosa. Two patients underwent intravenous infusion of [3H]progesterone for the measurement of extraadrenal production of 21-hydroxylated precursors of aldosterone. RESULTS: The patient who had discontinued her medication excreted a normal amount of aldosterone (20.0 nmol per square meter of body-surface area per day) while following a diet low in sodium. Her ratio of plasma renin activity to urinary aldosterone-18-glucuronide excretion was 1.7 after three days of sodium restriction, as compared with a ratio of 4.7 at the age of nine years (normal range, 0.03 to 0.1). The percentage of extraadrenal conversion of progesterone to deoxycorticosterone was low. The four other patients had variable responses to sodium restriction after the neonatal period (range for plasma renin activity:urinary aldosterone-18-glucuronide, 1.9 to 19.4). CONCLUSIONS: Although patients with salt-wasting 21-hydroxylase deficiency have functionally equivalent mutations in their CYP21 genes, they may vary from one another and over time in their ability to produce mineralocorticoids. This variation may be attributable to another adrenal enzyme with 21-hydroxylase activity.

Prevalence of nonclassical steroid 21-hydroxylase deficiency based on a morning salivary 17-hydroxyprogesterone screening test: a small sample study.

Early morning salivary 17 alpha-hydroxyprogesterone (17-OHP) determination differentiates patients with non-classical 21-hydroxylase deficiency (NC21OHD) from those who are not affected. Using this test, we have conducted a trial screening study for NC21OHD and have compared the study results with previously reported figures for the frequency of this disorder. Testing was performed on 258 subjects recruited from among the medical students and employees of the New York Hospital-Cornell Medical Center. In 2 of the 249 admissible subjects, the 0700-0900 h salivary 17-OHP level was within the range for NC21OHD patients (0.72-6.7 nmol/L; n = 8). These 2 individuals were subsequently confirmed to be affected by ACTH testing. Of the subjects with morning salivary 17-OHP levels below the cut-off point of 0.72 nmol/L, 29 were recalled for ACTH testing and were confirmed to be unaffected. Prevalence of NC21OHD in the test population was determined according to ethnic group. Our study gives a prevalence by screening of 1.14% among caucasians, which agrees with values of 0.81% and 1.06% obtained by different analytical methods. Further, both affected subjects were Ashkenazi Jews, and the prevalence of 3.23% among study members from this group concurs with increased rates of 3.64% and 4.97% already reported. On the basis of a small population sample, screening so far confirms the claim that NC21OHD is the most common autosomal recessive human disorder. Using values from ACTH-proven unaffected subjects (n = 47) and NC21OHD patients (n = 10), we establish preliminary normative data for morning salivary 17-OHP levels of 0.172 nmol/L for unaffected subjects (95% confidence interval, 0.05-0.54 nmol/L) and 1.76 nmol/L for NC21OHD-affected subjects (95% confidence interval, 0.42-7.32 nmol/L).

[A method for the quantitative analysis of steroid hormones by HPLC/RIA].

To solve the problem of cross-reaction in immunoassay and determine various steroid hormones simultaneously in a small amount of sample, a method for the quantitative analysis of steroid hormones was developed. This method is a combination of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The purpose of the study is the comprehensive analysis of steroid hormones profiles in normal subjects and adreno-cortical diseases. One hundred microliters of plasma was extracted by ether and the ether layer was evaporated. The residue was redissolved and separated by HPLC. Then fractions of steroid hormones were taken and determined by RIA. In this study, cortisol (F), androstenedione (A), 17 alpha-hydroxyprogesterone (17-OHP), testosterone (T), progesterone (P), estrone (E1) and estradiol (E2) were analyzed in normal adults, congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and Cushing's syndrome. Results in normal adults were similar with those had been previously reported. In CAH, F was remarkably low and 17-OHP, A, T and P were remarkably high before treatment. During treatment some cases showed that 17-OHP, A, T and P were high, and 17-OHP and P tended to be within normal range, if F had been kept higher than about 20 micrograms/dl. In the analysis of Cushing's syndrome before treatment, there were definite differences between adenoma and hyperplasia. A, 17-OHP, T, E1 and E2 were higher in hyperplasia than those in adenoma. It is suggested that it is possible to diagnose the type of Cushing's syndrome with a small amount of plasma using this method. As a mass screening method of CAH at present, 17-OHP in dried blood on filter paper is determined, therefore the quantitative analysis of 17-OHP in dried blood on filter paper (9 mm disc) was attempted. The quantitative analysis proved to be possible, and it was considered to be applied as the secondary screening method of CAH by the use of dried blood on filter paper.