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Biomedical subjects

D D Brandon

Publications and source records attributed to D D Brandon.

At least 19 recordsLinked to original sources
PubMed2000

Cortisol production rates in subjects with suspected Cushing's syndrome: assessment by stable isotope dilution methodology and comparison to other diagnostic methods.

It can be difficult to establish the diagnosis of Cushing's Syndrome (CS) in patients with mild or nonspecific clinical and biochemical findings, because available diagnostic tests have limited predictive values. We hypothesized that measurement of 24-h cortisol production rates (CPRs) might be a more sensitive indicator of CS in such patients. We measured CPRs in 28 patients with suspected CS (but equivocal biochemical findings) and in 22 healthy control subjects, by infusing tracer amounts of deuterated cortisol, with simultaneous measurements of 24-h urine free cortisol (UFC) levels; and we frequently sampled serum cortisol levels. CPRs were calculated from the ratio of isotopic enrichment to isotopic dilution of cortisol measured by gas chromatography-negative ion chemical ionization mass spectrometry. Nine of the patients proved to have CS by surgery (CS-Yes), whereas 19 patients were determined not to have CS by biochemical testing (CS-No). Mean 24-h UFCs, nocturnal serum cortisol levels, and CPRs were higher in CS-Yes, compared with CS-No and normal subjects. However, one CS-Yes patient had a normal 24-h UFC, two had normal nocturnal serum cortisol levels, and two had normal 24-h CPRs. There was extensive overlap in all of the biochemical parameters between the CS-Yes and the CS-No groups. Thus, measurement of CPR does not seem to offer any diagnostic advantage over available tests for the diagnosis of CS. Patients with proven CS can have normal UFC levels, normal CPRs, or normal nocturnal cortisol levels, whereas patients not thought to have CS may have elevated levels of any one or more these parameters.

Adult↗
PubMed1999

Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry.

Presented here is a stable isotope dilution technique for determining cortisol production rate (CPR). The method involves extraction and derivatization of cortisol isoforms from serum (0.5 ml), separation of derivatives by gas chromatography, and detection by using negative ion chemical ionization mass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol production rate with the use of small amounts of pooled serum, even in the presence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d0, m/z 782) and deuterated cortisol (d3, m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) standards: d0/d3 ratios ranging from 1 to 8%; 2) controls: d3-cortisol added to serum with known cortisol concentration; 3) subjects: 24-h pooled serum samples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d3-cortisol at 2-4% of their estimated CPR. Recovery after the solid phase extraction and derivatization process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d3/d0 in standards and serum controls were highly correlated (r2(standard) = 0.99; r2(control) = 0.99) over a wide range of d3-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m2/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/m2/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotopic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production.

Adolescent↗
PubMed1999

Resistance to several steroids in two sisters.

A 14-yr-old native American girl from the Iroquois Nation was referred as a potential patient with the syndrome of apparent mineralocorticoid excess. Instead, her evaluation revealed resistance to glucocorticoids, mineralocorticoids, and androgens, but no resistance to vitamin D or thyroid hormones. She lacked Cushingoid features despite significantly high cortisol levels. Menstruation was regular, and there was no clinical evidence of masculinization despite high serum androgen levels in the male range. The patient's sister had similar clinical features. Partial resistance to exogenous glucocorticoid and mineralocorticoid administration was well demonstrated in both patients. It is proposed that these patients represent the first cases of partial resistance to multiple steroids, possibly due to a coactivator defect.

Adolescent↗
PubMed1996

Rapid loss of oestrogen and progesterone receptors in human leiomyoma and myometrial explant cultures.

Oestrogen and progesterone are promoters of uterine leiomyoma growth: oestrogen receptors (ER) and progesterone receptors (PR) are over-expressed in these tumours. Paradoxically, there is a heterogeneity in responsiveness of leiomyoma growth to oestrogen and progesterone in culture. In this study, leiomyoma and adjacent myometrium were obtained at hysterectomy. The effect of oestrogen and progesterone on steroid receptor maintenance was examined using minced explants. Quantitative enzyme-linked immunoassay and Northern analysis were performed to assess ER and PR protein and mRNA content respectively. There was an approximately 75% decrease in ER and PR protein content within 8 h of incubation in both leiomyoma and myometrium. The presence or absence of oestrogen and/or progesterone had no effect on receptor protein loss. Northern analysis indicated a parallel loss of ER and PR mRNA transcripts. These findings suggest that the ER and PR expression in leiomyoma may require other extracellular factors. In-vitro studies designed to test the effects of sex steroids and their respective inhibitors on growth and function of leiomyoma and myometrial cells should consider this phenomenon.

Estradiol↗
PubMed1995

Inhibition of dexamethasone binding to human glucocorticoid receptor by New World primate cell extracts.

To determine if New World primates express an inhibitor that influences glucocorticoid receptor (GR) binding characteristics, we examined [3H]dexamethasone binding in cytosol prepared from B95-8 lymphoid cells, derived from the cotton top tamarin (Saguinus oedipus), in combination with cytosol prepared from human or rat tissues. B95-8 cytosol inhibited specific binding of [3H]dexamethasone (P < 0.01) when mixed with cytosol prepared from either a human lymphoid cell line (HL) or rat thymus. The inhibitory activity was heat labile and trypsin sensitive. Peak inhibitory activity was found in the 150-200 kd fractions after Sephacryl G-200 ultrafiltration. Scatchard analysis of [3H]dexamethasone binding using mixed cytosol showed a diminished GR apparent binding affinity when compared to HL cytosol. Kinetic studies using mixed cytosol indicated that B95-8 cytosol did not affect the apparent dissociation rate of [3H]dexamethasone. These data demonstrate that B95-8 cells contain a competitive inhibitor that prevents binding of dexamethasone to its cognate receptor.

Animals↗
PubMed1995

Estrogen receptor gene expression in human uterine leiomyomata.

Estrogen and progestin are believed to be important physiological regulators of uterine leiomyoma growth. We recently showed that progesterone receptor messenger ribonucleic acid (mRNA) and protein levels are increased in human uterine leiomyomas compared with those in myometrial biopsy tissue obtained from the same patient. To further characterize the molecular mechanisms underlying abnormal growth of uterine leiomyomas, we analyzed biopsy samples of tumor and adjacent normal myometrium for estrogen receptor (ER) gene expression. Northern analysis indicated that ER mRNA levels were increased 1.4-to 12.6-fold in leiomyoma compared with myometrium in all patients examined (n = 11), whereas beta-actin mRNA was not different between the two groups. The size of the primary ER mRNA transcript was 6.2 kilobases in both leiomyoma and myometrium, indicating no gross mutation of the ER gene. An ER protein of 66 kilodaltons was detected by Western blot analysis, and quantitative immunoassay of ER revealed 9448 +/- 1955 fmol/mg DNA in leiomyoma compared to 2827 +/- 979 fmol/mg DNA in myometrial tissue. Scatchard analysis of 17 beta-estradiol binding to cell-free extracts revealed enhanced binding capacity (per mg DNA) in leiomyoma tissue (n = 6) of about 6-fold, whereas ER binding affinity was not substantially different between the leiomyoma and adjacent myometrial tissues. We propose that increased expression of progesterone receptor in leiomyoma is most likely a consequence of overexpression of functional ER that results in increased end-organ sensitivity to estradiol.

Adult↗
PubMed1993

Progesterone receptor messenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas.

OBJECTIVE: Our purpose was to identify molecular mechanisms underlying abnormal growth of uterine leiomyomas. STUDY DESIGN: Biopsy samples of tumor and adjacent "normal" myometrium from nine patients were analyzed for progesterone receptor gene expression and for proliferation-associated antigen Ki-67. RESULTS: Northern analysis indicated that progesterone receptor messenger ribonucleic acid levels were increased twofold to 15-fold in leiomyoma compared with adjacent myometrial biopsy tissue from all patients (n = 9), whereas beta-actin messenger ribonucleic acid was at similar levels in these samples. Quantitative immunoassay, immunohistochemistry studies, and Western blot analyses revealed increased amounts of progesterone receptor protein in the tumor tissue. Both the progesterone receptor A and B forms were expressed in the leiomyoma and adjacent myometrium. Corresponding to increased progesterone receptor gene expression, the proliferation-associated antigen Ki-67 was also significantly elevated in the leiomyoma tissue. CONCLUSION: These data provide the first evidence that progesterone receptor messenger ribonucleic acid is overexpressed in uterine leiomyomas, suggesting that amplified progesterone-mediated signaling is instrumental in the abnormal growth of these tumors.

Adult↗
PubMed1991

Genetic variation of the glucocorticoid receptor from a steroid-resistant primate.

The neotropical cotton-top marmoset (Saguinus oedipus) is a New World primate known to have markedly increased total and free plasma cortisol concentrations when compared with Old World primates including man. The relative end-organ 'resistance' to glucocorticoids found in various New World primates has been attributed to a glucocorticoid receptor (GR) with diminished affinity for glucocorticoids. It has been demonstrated that the marmoset GR has approximately tenfold lower binding affinity for dexamethasone when compared with the human GR. We have examined the primary structure of the marmoset GR by molecular cloning and sequencing of GR functional domains. A library of cDNA clones was constructed in the phage vector gamma gt10 using poly(A)+ RNA from a marmoset-derived lymphoid cell line, and screened using the human GR cDNA. DNA sequencing determined 76 individual nucleotide substitutions in the coding region of the marmoset GR. Comparison of the marmoset GR nucleotide sequence with the human GR cDNA coding region indicated an overall sequence homology of about 97%. Thirty of the nucleotide substitutions lead to alterations in the predicted amino acid sequence (28 amino acid substitutions) of the marmoset GR. The size of the marmoset GR predicted from the 778 amino acids is approximately 90,000 which is in agreement with previous size estimates of the human and marmoset GRs. Alterations of amino acid sequence in the marmoset GR were greatest towards the amino terminus, including the tau 1 domain putatively involved in transcriptional activation. The DNA-binding domain contained an additional codon (arginine).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the uterine estradiol receptor.

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.

Animals↗
PubMed1989

Glucocorticoid resistance in humans and nonhuman primates.

In humans, the syndrome of cortisol resistance is characterized by the absence of signs and symptoms of Cushing's syndrome, elevated total and unbound plasma cortisol concentrations, and increases in urinary free cortisol excretion and plasma adrenocorticotropic hormone. In one family, a severely affected member had hypertension and hypokalemic alkalosis associated with increased plasma concentrations of corticosterone and deoxycorticosterone. These patients are resistant to suppression of the pituitary-adrenal axis by dexamethasone. Dexamethasone therapy, however, effectively corrected hypertension and hypokalemic alkalosis in the severely affected patient, without causing signs of glucocorticoid excess. The glucocorticoid receptor from these patients has a low affinity for glucocorticoids and is unstable during thermal activation. Both the molecular weight of the glucocorticoid receptor and the size of the corresponding mRNA are similar to those of normal controls. Transformation of B-lymphocytes with Epstein-Barr virus leads to induction of glucocorticoid receptors. Receptor induction, however, is lower in patient cells than those obtained from normal controls. This decreased induction parallels decreased expression of glucocorticoid receptor mRNA. Thus, in this form of glucocorticoid resistance the glucocorticoid receptor is abnormal and leads to diminished target organ responsiveness. Many New World primates exhibit glucocorticoid "resistance," without apparent pathology. These species have markedly elevated plasma cortisol, both total and unbound concentrations, increased urinary free cortisol excretion, and marked increases in plasma adrenocorticotropic hormone and beta-endorphin. The glucocorticoid receptors of these primates have decreased affinity for glucocorticoids, are thermolabile, and are not induced by Epstein-Barr virus transformation as indicated by specific binding and mRNA expression. Both the molecular weight of the glucocorticoid receptor and the size of the corresponding mRNA are similar to those of normal controls. Despite the high plasma cortisol concentrations in these primates, there is no sodium retention and aldosterone levels are actually increased. The kidney aldosterone receptor cross-reacts poorly with cortisol, explaining the absence of sodium retention. New World primates also have progesterone, estrogen, aldosterone, and vitamin D insensitivity, suggesting a common factor linking steroid hormone receptors.

Animals↗
PubMed1989

Interaction of RU 486, a glucocorticoid and progestin antagonist with human circulating mononuclear leukocytes.

The glucocorticoid and progestin antagonist, RU 486 (17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-[1-propynyl]-estra-4, 9-dien-3-one), antagonizes the suppressive effect of dexamethasone on [14C]2-deoxyglucose uptake by intact human mononuclear leukocytes in a concentration-dependent fashion. The authors found at least two types of specific-binding sites for this compound in the mononuclear leukocytes. The first type of sites (receptor content [Ro], 10.8 +/- 1.6 fmoles/10(6) cells [mean +/- SD of 4 experiments]; equilibrium dissociation constant (Kd), 0.3 +/- 0.1 nM) have a capacity similar to that of the dexamethasone binding site (Ro, 11.2 fmoles/10(6) cells; Kd, 1.2 nM). The second type of sites (Ro, 56 +/- 27 fmoles/10(6) cells: Kd, 19 +/- 5 nM) have a higher capacity and lower affinity for RU 486 than the first type of sites and do not interact with dexamethasone. The authors were unable to demonstrate the presence of the second type of binding sites in subcellular fractions. This finding suggests that this site is unstable and lost in the fractionation process. The role of the second type of low-affinity, high-capacity RU 486 specific-binding site in intact human mononuclear leukocytes, as well as its possible occurrence in other tissue, requires further investigation.

Binding Sites↗
PubMed1988

Glucocorticoid receptor-mediated effects on rat fibrosarcoma growth.

Glucocorticoid receptors are present in most normal and malignant mammalian cells. To examine the hypothesis that the growth of methylcholanthrene-induced malignant sarcoma is glucocorticoid dependent, we evaluated the behavior of malignant fibrosarcoma (MCA) in adrenalectomized rats treated with either normal saline or deoxycorticosterone acetate and in intact rats treated with placebo or with the glucocorticoid receptor antagonist RU 486. Survival, tumor weight, and loss of body weight (an index of cachexia) were measured. In MCA-bearing rats, neither survival nor loss of body weight was affected by bilateral adrenalectomy or by treatment with RU 486. Tumor weight and time-integrated tumor volume, however, were significantly less in bilaterally adrenalectomized rats without deoxycorticosterone acetate replacement than in animals treated with deoxycorticosterone acetate. Similarly, tumor weight and time-integrated tumor volume were less in intact animals treated with RU 486 than in intact animals treated with placebo. The glucocorticoid receptors in the tumor cells had similar binding capacity (Ro) and equilibrium dissociation constant (Kd) as in control rat fibroblasts. These results suggest that the growth of MCA sarcoma cells is partially dependent upon glucocorticoids. This effect of glucocorticoids, however, was not of sufficient magnitude to improve survival and prevent cachexia. We conclude that glucocorticoids appear to influence MCA sarcoma growth in the rat, and that glucocorticoid receptor blockade, perhaps in combination with other antitumor agents, merits future study in the treatment of malignant tumors.

Adrenalectomy↗
PubMed1988

Receptor-mediated effects of glucocorticoids on inflammation: enhancement of the inflammatory response with a glucocorticoid antagonist.

Glucocorticoids suppress the inflammatory response by altering leukocyte traffic and function, cytokine secretion and action, and phospholipid metabolism. We employed the glucocorticoid receptor antagonist RU 486, to examine whether glucocorticoids suppress the inflammatory response through a receptor-mediated mechanism and whether basal glucocorticoid secretion exerts antiinflammatory effects in the resting (non-stress) state. To test these hypotheses we evaluated the effects of increasing doses of dexamethasone, RU 486, or dexamethasone plus RU 486 on the exudate volume and concentrations of leukocytes, prostaglandin E2, (PGE2) and leukotriene B4 (LTB4) in intact rats that received subcutaneous carrageenin. Exudate volume, leukocyte concentration and LTB4 and PGE2 levels were all suppressed by dexamethasone in a dose-dependent fashion (P less than 0.005). RU 486 was able to antagonize fully the suppressive effects of dexamethasone on the inflammatory response (P less than 0.001) and to cause increases of exudate volume and leukocyte, PGE2 and LTB4 concentrations when given alone (P less than 0.05). These increases ranged between 30 and 100% above the basal inflammatory response. We conclude that glucocorticoids most likely suppress the inflammatory response by a glucocorticoid receptor-mediated mechanism and under basal conditions exert tonic antiinflammatory effects.

Alprostadil↗
PubMed1987

Adrenal 11-hydroxylase activity in a hypercortisolemic New World primate: adaptive intra-adrenal changes.

The squirrel monkey, a representative New World primate, has high plasma cortisol and aldosterone concentrations when compared to Old World primates. We measured adrenal mitochondrial 11-hydroxylase (11-OHase) activity in squirrel monkeys and in two representative Old World species (cynomolgus and rhesus macaques) in an effort to explain these elevated plasma glucocorticoid and mineralocorticoid levels. The activity of 11-OHase was 5-fold higher in the squirrel monkey than in the Old World species tested. Calculated 11-OHase Vmax was different in the squirrel monkey and the cynomolgus. However, the Km values were similar in the New World primate when compared to cynomolgus. The ability of metyrapone to block 11-OHase was less in the former than in the latter. The data are consistent with the hypothesis that the squirrel monkey adrenal cortex possesses an increased number of 11-hydroxylase enzyme units compared to that of Old World primate species, and is therefore more efficient in producing cortisol. This difference in 11-OHase activity in the squirrel monkey, in addition to other previously reported adrenal steroidogenic enzyme alterations, may be adaptive in nature, favoring increased cortisol and aldosterone production in this and possibly other New World primate species.

Adaptation, Physiological↗
PubMed1987

The relationship of saline-induced changes in vasopressin secretion to basal and corticotropin-releasing hormone-stimulated adrenocorticotropin and cortisol secretion in man.

To investigate the effect of endogenous arginine vasopressin (AVP) on ACTH secretion, normal subjects were given infusions of either hypertonic saline (HS) or isotonic saline (NS) combined with human corticotropin-releasing hormone (CRH) or placebo. Basal plasma AVP was 2.3 +/- 0.3 (+/- SE) pg/ml, did not change with NS treatment, and rose to 5.4 +/- 0.6 pg/ml during HS infusion (P less than 0.01). Both basal and CRH-stimulated plasma ACTH and cortisol concentrations increased during HS infusion. Peak plasma ACTH and cortisol levels were 11.4 +/- 1.5 pg/ml and 8.6 +/- 0.8 micrograms/dl, respectively, during the HS (plus placebo) infusion. During the NS (plus placebo) infusion, plasma ACTH and cortisol gradually declined to 6.8 +/- 0.5 pg/ml and 2.6 +/- 0.4 micrograms/dl. The timing of the rise in ACTH during the HS infusion paralleled the rise in AVP. When an iv dose of 1 microgram/kg CRH was administered during the saline infusions, peak plasma ACTH and cortisol levels were 27.7 +/- 6.3 pg/ml and 17.5 +/- 1.0 micrograms/dl, respectively, during the HS infusion and 15.6 +/- 1.7 pg/ml and 13.4 +/- 1.2 micrograms/dl during the NS infusion. When the areas under the hormone response curves were compared, CRH stimulated ACTH and cortisol secretion to a greater extent than did HS (P less than 0.05). The hormonal stimulation due to combined CRH and hypertonic saline was greater than that attributable to either factor alone (P less than 0.025), but was not different than the sum of the effects of the individual factors. These results indicate that increases in endogenous AVP produced by HS are associated with increases in both basal and CRH-stimulated ACTH and cortisol release. The effect of HS appears to be additive to but not consistently synergistic with the effect of CRH.

Adrenocorticotropic Hormone↗
PubMed1987

Pharmacokinetic properties of the antiglucocorticoid and antiprogesterone steroid RU 486 in man.

RU 486 (17 beta-hydroxy-11 beta-[4-dimethylamino phenyl]-17 alpha-[1-propynyl]estra-4,9-dien-3-one) is a clinically useful glucocorticoid and progesterone antagonist. The authors studied the pharmacokinetic properties of this drug in normal volunteers and patients with Cushing's syndrome using a rat progesterone radioreceptor assay. This assay gave values similar to those obtained with a rat glucocorticoid radioreceptor assay. After a single oral dose of 25 mg/kg (n = 11) or 10 mg/kg (n = 11) to normal volunteers, plasma concentrations of progesterone receptor-reactive material reached maximal levels of 754 +/- 288 (mean +/- S.D.) and 517 +/- 183 micrograms/dl. This occurred at 3.1 +/- 1.9 and 2.5 +/- 1.0 h, respectively. The respective apparent plasma half-lives were 19.2 +/- 7.0 and 20.6 +/- 7.7 h. Four patients with Cushing's syndrome treated chronically (10-20 mg/kg/day) had relatively constant plasma levels of receptor reactivity ranging from 506 to 1184 micrograms/dl. Chromatographic characterization of circulating receptor reactivity showed that the active fraction corresponded to RU 486 and its hydrophilic N-mono- and N-didemethylated metabolites. Less than 0.5% of the daily dose was excreted in the urine of two of these patients as receptor reactivity. The drug bound extensively to circulating albumin, which competed with the glucocorticoid receptor of intact human mononuclear leukocytes for [3H]RU 486 in a concentration-dependent manner.

Adult↗