Circulating glucocorticoid bioactivity in the preterm newborn after antenatal betamethasone treatment

Antenatal glucocorticoid treatment of mothers at risk of premature delivery is highly cost-effective in reducing neonatal mortality and morbidity. However, there is only limited information on the actual glucocorticoid bioactivity (GBA) reaching the fetus. By employing a recently developed recombinant cell bioassay, we studied circulating GBA in preterm newborns exposed to the standard antenatal betamethasone regimen (12 mg betamethasone twice, 24-h interval, for the mother; repeated in 7-10 d if required). Plasma GBA and cortisol concentrations were measured in cord blood of 71 infants (mean gestational age, 28.9 wk; range, 24.6-32 wk; mean birth weight, 1208 g; range, 480-2010 g). The median time between the last administered betamethasone dose and birth was 2.0 d. Cord GBA ranged from less than 15.6 to 170 nmol/liter cortisol equivalents. The level was highly dependent on the time between the last betamethasone dose and birth, i.e. infants born shortly (<12 h) after the last steroid dose displayed on average 4-fold higher GBA than that in the reference group (infants with >7 d since the last betamethasone dose before birth or without treatment; 74 vs. 21 nmol/liter cortisol equivalents; P < 0.0001). By contrast, if more than 72 h had elapsed between the last steroid dose and birth, circulating GBA was strongly dependent on cord cortisol (r = 0.85; P < 0.0001; n = 30). In multiple regression analysis adjusted for cord cortisol concentration and the time since the last steroid dose, increased umbilical artery resistance, a sign of severe fetal distress, was associated with lower cord GBA (P = 0.01). In conclusion, antenatal exposure of preterm fetuses to betamethasone causes a sizeable, but brief, peak of supraphysiological GBA, and approximately 3 d after the last betamethasone dose, circulating GBA derives from cord cortisol concentration.     

Quality of glucocorticoid replacement in adrenal insufficiency: clinical assessment vs. timed serum cortisol measurements

OBJECTIVE: Evaluation of glucocorticoid replacement quality in adrenal insufficiency (AI) relies primarily on clinical judgement and thus largely depends on the physician's expertise. It is a matter of debate whether cortisol day curves are of value in assessing glucocorticoid replacement quality. Here we compared the results of a structured clinical assessment to the outcome of repeated, timed serum cortisol measurements. DESIGN: Cross-sectional study in the outpatient department of a university teaching hospital. PATIENTS: Forty-six patients (19 men, 27 women, age range 16-76 years) with primary (n = 23) and secondary (n = 23) AI on stable replacement with a median dose of 37.5 mg cortisone acetate (range 25-50 mg) since 10 +/- 7 years (range 1-31 years). MEASUREMENTS: Clinical performance was scored by structured assessment of signs and symptoms, physical examination and routine biochemical tests. Serum cortisol was measured on two to three separate occasions in three timed samples after the morning glucocorticoid dose. Bone mineral density was measured in 15 patients with long-standing glucocorticoid replacement. RESULTS: Thirty-seven patients were considered well replaced, whereas clinical scores suggested over- or under-replacement in five and four, respectively. There was no correlation of the clinical score with total or body weight-adjusted glucocorticoid dose. The mean z score of serum cortisol differed significantly between under- and over-replaced patients (P < 0.05) but neither group differed significantly from well-replaced patients. Bone mineral density was normal in all patients studied. CONCLUSIONS: Our results suggest that serum cortisol day curves are of limited value in the monitoring of glucocorticoid replacement. Bone mineral density in AI is generally normal and does not require routine follow-up.     

Transactivation assay for determination of glucocorticoid bioactivity in human serum

We have developed a mammalian cell (COS-1) bioassay, which measures glucocorticoid bioactivity (GBA) directly from a small amount of human serum. The assay is based on the expression of human glucocorticoid receptor (GR) together with a coactivator protein and reporter plasmid containing GR response elements upstream of the luciferase gene. Ten microliters of human serum, in duplicate, are added directly to the cell culture medium, and GBA is derived from reporter gene activity. The assay differentiates between biopotencies of synthetic steroids, and importantly, mifepristone (RU486) is able to block glucocorticoid-induced response. The assay is sensitive (<15.6 nM cortisol in fetal calf serum) and precise, with the within- and between-assay coefficients of variation less than 8% and 10%, respectively. We measured serum GBA (bioassay) and cortisol (RIA) levels in 34 asthmatic children (age range, 5.7-14.2 yr) at baseline and after treatment with either inhaled budesonide (800 microg/d, n = 14), fluticasone propionate (500 microg/d, n = 14), or cromones (control group, n = 6). Pretreatment serum GBA and cortisol levels correlated strongly (r = 0.90, P < 0.0001, n = 34). Two months of treatment with inhaled budesonide resulted in excess GBA in circulation, which was not attributable to endogenous cortisol (P < 0.001). In the fluticasone propionate group, the presence of serum excess GBA was at the borderline of statistical significance (P < 0.08) after 2 months of inhalation therapy, and no excess GBA was detected in the cromone group. In conclusion, our bioassay enables measurement of mammalian cell response to bioactive glucocorticoids in circulation and provides a novel means to investigate patients receiving drugs acting through the GR.     

Changes in serum cortisol levels during community-acquired pneumonia: the influence of dexamethasone

In community-acquired pneumonia (CAP), the cortisol level on admission can be a useful biomarker for prognosis. Serial cortisol measurements during the clinical course of disease and their association with disease outcome have never been reported. Furthermore, the time to recovery of the hypothalamic-pituitary-adrenal axis after a short course of dexamethasone during infection is unclear. We analyzed data from 270 hospitalized patients with CAP. Total serum cortisol was measured on presentation, day 1, 2, 4, and on control visit (day 30). Intensive care unit (ICU) admission and mortality were assessed. Additionally, to study the influence of dexamethasone on the kinetics of the cortisol response, we analyzed serial cortisol values of 43 patients treated with a four-day regimen of dexamethasone 5 mg. During hospital stay, 26/270 patients (9.6%) were admitted to the ICU and 15/270 patients (5.6%) died. Compared to patients with an uneventful recovery, cortisol on presentation was significantly higher in patients with an adverse outcome (360 μg/L, IQR 209-597 vs. 238 μg/L, IQR 151-374) (p:0.01), and also remained significantly higher throughout the course of disease. Dexamethasone treatment resulted in nearly complete suppression of the endogenous cortisol production after the first dose, but cortisol production was fully recovered on control visit. In conclusion, we showed that an adverse outcome of CAP was associated with persisting higher total serum cortisol throughout the course of disease. Delta-cortisol could be another meaningful biomarker in CAP. Next, our data indicate that a four-day dexamethasone regimen during CAP does not lead to prolonged secondary adrenal insufficiency.     

Confirmation of the inhibitory influence of exogenous oxytocin on cortisol and ACTH in man: evidence of reproducibility

The influence of low-dose oxytocin perfusion (32 mIU/min) on ACTH and cortisol plasma levels was tested in 8 normal male volunteers (age 18-26). The 1-h oxytocin perfusion periods were preceded and followed by two 1-h saline control periods. During the first period, there was a slight ACTH concentration decrease in 4 individuals. Oxytocin perfusion induced a clear-cut plasma ACTH decrease in 7 out of the volunteers, and a slight decrease in the remaining one. During the second saline infusion, there was a marked ACTH increase in 7 out of the volunteers, and a weak increase in one (P less than 0.0001). A similar pattern was observed in the plasma cortisol levels (P less than 0.00001). Furthermore, a control saline perfusion was performed in 4 of the 8 volunteers and compared to the 4 corresponding oxytocin perfusion tests: the differences between the 2 sets of tests was highly significant both for ACTH (P less than 0.003) and for cortisol (P less than 0.007). Lastly, the reproducibility of this inhibitory influence was retested in 4 volunteers: the tests were repeated under the same conditions 7 days later. There were no global differences between the 2 sets of tests either for ACTH (NS) or for cortisol (NS). ACTH and cortisol concentration fluctuations during the period between each set of tests were not significant. The following variations were measured for ACTH (NS) and for cortisol (NS). The present results confirm the inhibitory influence of low-dose oxytocin perfusion on ACTH and cortisol levels in normal males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of myostatin concentrations in human serum: Circulating concentrations in young and older men and effects of testosterone administration

Methodological problems, including binding of myostatin to plasma proteins and cross-reactivity of assay reagents with other proteins, have confounded myostatin measurements. Here we describe development of an accurate assay for measuring myostatin concentrations in humans. Monoclonal antibodies that bind to distinct regions of myostatin served as capture and detector antibodies in a sandwich ELISA that used acid treatment to dissociate myostatin from binding proteins. Serum from myostatin-deficient Belgian Blue cattle was used as matrix and recombinant human myostatin as standard. The quantitative range was 0.15–37.50 ng/mL. Intra- and inter-assay CVs in low, mid, and high range were 4.1%, 4.7%, and 7.2%, and 3.9%, 1.6%, and 5.2%, respectively. Myostatin protein was undetectable in sera of Belgian Blue cattle and myostatin knockout mice. Recovery in spiked sera approximated 100%. ActRIIB-Fc or anti-myostatin antibody MYO-029 had no effect on myostatin measurements when assayed at pH 2.5. Myostatin levels were higher in young than older men (mean ± S.E.M. 8.0 ± 0.3 ng/mL vs. 7.0 ± 0.4 ng/mL, P = 0.03). In men treated with graded doses of testosterone, myostatin levels were significantly higher on day 56 than baseline in both young and older men; changes in myostatin levels were significantly correlated with changes in total and free testosterone in young men. Myostatin levels were not significantly associated with lean body mass in either young or older men.     

Meal timing,fasting and glucocorticoids interplay in serum leptin concentrations and diurnal profile

BACKGROUND: In a previous study, we reported a 4-fold increase in serum leptin following total parenteral nutrition given after surgery. We hypothesised that the perioperative fasting and stress contributed to this, possibly mediated by increased serum insulin and cortisol. OBJECTIVE: To test the hypothesis that fasting, in combination with glucocorticoids, sensitises the leptin response to subsequent energy intake. DESIGN: Healthy volunteers were randomised into two groups, one group received dexamethasone (DEX), 0.1 mg twice daily for 3 days, while the other group served as a control. Each group was then subdivided into two feeding protocols. Protocol 1, where a standard meal was given at 0, 24, 36 and 48 h and protocol 2 where the same meal was given at 0 and 48 h. Blood samples were drawn before, as well as every other hour during the study period for determination of serum leptin, insulin, glucose and cortisol concentrations. RESULTS: In all groups serum leptin increased significantly following each meal (P<0.01). The rise in serum leptin in response to the standard meal was higher when the meal was taken in the evening (P<0.001) or following longer duration of fasting (P<0.02). In those fasting for 48 h, leptin decreased by 60% and showed no diurnal variation. DEX intake increased leptin concentrations in those fasting for short periods (P<0.02) but not for 48 h. CONCLUSIONS: Long durations of fasting sensitise the response of leptin to subsequent energy intake and abolish the DEX-induced upregulation of leptin. Meal timing is an important factor determining the leptin diurnal rhythm, but other factors must contribute since the leptin response to a standard meal taken in the evening was greater than to the same meal taken in the morning.     

Role of glucocorticoids and glucocorticoid receptor in priming of macrophages caused by glucocorticoid receptor blockade

We previously reported that glucocorticoid receptor (GR) blockade (injected with GR antagonist RU486) primed the host responses to lipopolysaccharide. Since decrease of GR and elevated glucocorticoids (GCs) have been always reported as parallel responses, we hypothesize that both GCs and GR play important roles in GR blockade induced priming. We first confirm that the production of nitric oxide (NO), superoxide (O₂⁻), and PKCα expression are all increased in peritoneal macrophages from GR blockade rats, indicating that macrophages are primed by GR blockade. Furthermore, using unilateral adrenalectomy rats, we find that the elevated GCs caused by a feedback mechanism following GR blockade may be involved in the process of priming. In vitro experiments in RAW264.7 cells show the inhibitory effect of GCs on NO production, which can be thoroughly blocked by RU486, indicating the increase of NO production in GR blockade rats is due to the elimination of GCs’s anti-inflammatory function. In contrast, 10⁻⁷ M corticosterone induces significant increases in O₂⁻ release, PKCα expression and phosphorylation, which cannot be reversed by RU486, demonstrating a previously unrecognized pro-inflammatory role of GCs in enhancing PM activation through a GR-independent pathway. The effect of GCs on PKCα expression even exists in GR deficient COS-7 cells as well as in GR knock-down RAW264.7 cells. In conclusion, both GR impairment and elevation of GCs are involved in the priming of macrophages caused by GR blockade. The findings of the divergent roles of GCs in modulation of inflammation may change therapeutic strategy for inflammatory diseases with GCs.     

92例早产儿血糖紊乱高危因素分析

目的 探讨早产儿血糖紊乱的高危因素及临床特点.方法 采用微量法对92例早产儿进行连续血糖监测.结果 血糖持续正常28例(30.5%),异常64例(69.5%);胎龄< 31周的早产儿(10.7%)无一例血糖持续正常;8例低体重早产儿(1 200～1 500 g)空腹血糖全部异常;高低血糖交替发生18例(19.6%);恢复正常血糖水平时间平均4.5 d,最长达45 d;重度窒息组全部异常,其中8例为高血糖,3例高血糖持续不能纠正死亡.结论 低出生体重、胎龄、1分钟Apgar 评分是早产儿发生血糖紊乱的高危因素,胎龄越小、体重越低、1分钟Apgar 评分越低血糖紊乱发生率越高.持续高血糖不能纠正往往预示病情危重预后差.     

Diurnal rhythm of serum melatonin, ACTH and cortisol in asthma patients with long term glucocorticoid treatment

Forty one patients (21 women and 20 men aged 35 to 45 years), with bronchial asthma were divided into two groups: those treated with glucocorticoids (with an equivalent daily dose of 8 mg prednisone) and those receiving no glucocorticoid preparations. The control group consisted of 27 healthy volunteers. The diurnal rhythm of serum melatonin, ACTH and cortisol was evaluated in all subjects. It was found that the dysfunction and reduced reactivity of the pituitary-adrenal axis in asthmatics receiving chronic glucocorticotherapy was accompanied by suppressed melatonin rhythm.     

A pilot study examining the relationship between stress and serum cortisol concentrations in women with asthma

The mechanism (s) by which stress exacerbates asthma is unknown. One explanation could be a reduction in endogenous serum cortisol concentrations as a result of stress. Our objective was to determine if a reduction in morning serum cortisol concentrations is associated with higher levels of stress in women with asthma. In this pilot study, seven women with a history of allergic-asthma were prospectively assigned to either low, moderate, or high stress groups based on a combination of their level of current stress and their resources to cope with the stress. After stress group assignment, women donated a morning blood sample, which was analyzed for serum cortisol concentration by an independent laboratory whose personnel were blinded to the subjects' stress status. Three women were assigned to the low stress group, two to the moderate stress group and two to the high stress group. Serum cortisol concentrations ranged from 8 to 23 microg/dl, averaging 14 +/- 6 microg/dl. A Spearman rank correlation indicated that serum cortisol concentrations were significantly inversely related to the stress groupings (r(s) = -0.915; P = 0.025). These results suggest that a reduction in morning serum cortisol concentration may be associated with higher levels of stress and lower resources to cope with the stress in women with allergic-asthma.     

力竭大鼠血清睾酮、皮质醇的变化及对心脏的可能影响

力竭性运动损伤多见于军人及运动员，心脏损伤尤为常见。与力竭性心脏损伤可能相关的机制是多方面的，其机体生理病理变化亦是包括诸多方面，其中包括内分泌的改变，如肾上腺素、睾酮和皮质醇等。有研究表明睾酮具有保护心脏的作用，而不恰当升高的皮质醇则可导致心脏损伤。本文旨在通过大鼠力竭性游泳试验，研究大鼠血清睾酮、皮质醇的变化及其对心脏的可能保护机制。     

Reduced serum inhibin concentrations during ovulatory cycles of estrogen-treated rhesus monkeys: an indicator of FSH bioactivity

Female rhesus monkeys treated with exogenous estrone initially were anovulatory. Although estrone and estradiol concentrations were maintained 1.5- to 2.5-fold elevated, i.e. in the midfollicular range, ovulatory cycles resumed in three of four animals after 6-15 months of anovulation. During the ovulatory cycles the serum bio LH concentrations were the same in estrone-treated animals as during ovulatory cycles of control monkeys, but the daily basal serum FSH concentrations detectable by RIA were significantly reduced during the ovulatory cycles of the estrone-treated animals compared to the cycles of the controls. In the present study serum inhibin concentrations were measured to determine whether or not they were increased and the cause of the selective decrease in FSH concentrations in the estrogen-treated monkeys. Serum LH, FSH, progesterone, and inhibin concentrations were measured by RIA in blood samples collected during the third year of continuous estrogen treatment. The lack of an effect of elevated estrogen on LH concentrations and a significant estrogen-induced decrease in serum FSH concentrations during ovulatory cycles was confirmed (FSH, control: 5.6 +/- 0.68 ng/ml; estrogen-treated: 2.5 +/- 0.09 ng/ml; P = 0.01). There was also a significant decrease in the serum inhibin concentrations detectable by RIA during the follicular phase of the estrone-treated monkeys compared to the follicular phase of the control animals (119 +/- 17 vs. 462 +/- 105 microliter eq/ml; P = 0.04). These results indicate that the lower serum concentrations of FSH in the estrogen-treated monkeys were not a result of an increase in ovarian secretion of inhibin. The lower inhibin concentrations suggest that FSH bioactivity, as well as immunoreactive FSH, is significantly reduced during the ovulatory cycles of the estrone-treated monkeys. Even though the estrogen treatment decreased the FSH bioactivity, sufficient FSH secretion occurs in the presence of the elevated estrone and estradiol concentrations to induce and support apparently normal ovulatory cycles.     

危重症早产儿胃肠外营养相关胆汁淤积的影响因素分析

目的探讨危重症早产儿胃肠外营养(PN)相关胆汁淤积(PNAC)的影响因素。方法选取2017-01～2017-12该院新生儿重症监护室采用静脉营养时间超过14 d早产儿300例作为研究对象,按照是否发生PNAC分为非PNAC组215例和PNAC组85例。比较两组早产儿的临床和营养因素情况,采用非条件二分类Logistic回归分析PNAC的危险因素。结果两组在新生儿感染、贫血及机械通气等方面比较差异有统计学意义(P0.05)。两组在禁食时间、脂肪乳总量、奶总量及热卡总量等方面比较差异有统计学意义(P0.05)。多因素分析结果显示,新生儿感染(OR=2.352)、禁食时间长(OR=1.263)、PN持续时间长(OR=1.854)、氨基酸热卡比例高(OR=2.865)、脂肪乳热卡比例高(OR=1.854)是PNAC的危险因素,经口摄入高热卡为保护因素(OR=0.025)。结论新生儿感染、PN持续时间及禁食时间长等因素是早产儿发生PNAC的危险因素。     

Dose-response effects of exogenous pulsatile human corticotropin-releasing hormone on adrenocorticotropin, cortisol, and gonadotropin concentrations in agonadal women

Acute activation of the hypothalamic-pituitary axis with CRH has been reported to suppress gonadotropin secretion in women of reproductive age. In this study we specifically examined the effects of increasing doses of human CRH (hCRH) on circulating concentrations of ACTH, cortisol, and gonadotropins in five agonadal women, aged 46-65 (mean, 51.2) yr. The subjects had undergone either natural menopause or surgical removal of their ovaries at least 1 yr before study. Each woman was studied on four separate occasions and received either saline or hCRH at a dose of 0.5, 1.0, or 2.0 micrograms/kg BW through an indwelling iv catheter in a randomized, single blind fashion. During each experiment, five sequential iv injections of the same dose of hCRH or saline were administered at 90-min intervals over an 8-h period, followed by a 10-micrograms iv bolus of GnRH to test for pituitary gonadotropin responsiveness. Blood samples for measurement of LH, FSH, PRL, ACTH, and cortisol were obtained at 15-min intervals through an indwelling iv in the contralateral arm. Episodic pulses of LH secretion were analyzed using the Cluster computer program. Transverse mean LH, FSH, and PRL levels did not change with increasing hCRH doses. Mean (+/- SEM) LH pulse frequency [saline, 5.2 +/- 0.4/8 h; hCRH, (0.5 micrograms/kg), 4.8 +/- 0.2; hCRH (1 microgram/kg), 5.2 +/- 0.2; hCRH (2 micrograms/kg), 5.4 +/- 0.2] and amplitude [saline, 14.4 +/- 4.2 IU/L; hCRH (0.5 microgram/kg), 14.0 +/- 2.4; hCRH (1 microgram/kg), 15.8 +/- 2.5; hCRH (2 micrograms/kg), 17.2 +/- 2.9] did not differ among groups. Although the transverse mean levels of ACTH [saline, 8.7 +/- 0.2 pmol/L; hCRH (0.5 microgram/kg), 12.4 +/- 0.3; hCRH (1 microgram/kg), 11.5 +/- 0.4; hCRH (2 micrograms/kg), 12.8 +/- 0.4] did not change with increasing doses of hCRH, the duration of cortisol peaks after hCRH was longer and accounted for the increased transverse mean at each dose [saline, 152.8 +/- 4.1 nmol/L; hCRH (0.5 microgram/kg), 265.4 +/- 10.5; hCRH (1 microgram/kg), 329.7 +/- 14.3; hCRH (2 micrograms/kg), 348.2 +/- 12.1]. These findings suggest that ever larger doses of pulsatile hCRH continue to increase adrenal output of cortisol secondary to more sustained ACTH responses. However, hCRH-induced acute hypercortisolism does not alter gonadotropin secretion in agonadal women.