Relation between the level of self-mutilation and theconcentration of fecal metabolites of glucocorticoids incaptive chimpanzees (Pan troglodytes)

The influence of stress in an environment, according with the behavioral and endocrine variables of primates, are increasingly being studied by a diversity of authors, and have shown that abnormal behaviors associated with increased glucocorticoids may be directly related with the impairment of their well-being. In this work were used 22 adult chimpanzees (Pan troglodytes), 11 males and 11 females, kept in captivity in three different institutions. All animals had their behavior registered by focal session using a 30 seconds sample interval, during six months, totaling 4,800 registries per each animal. During this period, fecal samples were collected 3 times a week for the extraction and measurement of the concentration of fecal metabolites of glucocorticoid by radioimmunoassay. Of the total observed, stereotypical behaviors represented 13,45±2.76%, and among them, self-mutilation represented 38.28±3.98 %. The animals were classified into three different scores, according with the percentage of body surface with alopecia due to self-mutilation. It was found a positive correlation of high intensity between the scores of alopecia due to the observed mutilation and the average concentrations of fecal metabolites of glucocorticoids. This result strongly suggests that this measurement of self-mutilation in a chimpanzee can be used as an important auxiliary tool to evaluate de conditions of adaptation of an animal in captivity, functioning as a direct indicator of the presence of chronic stress.

Comparative allelopathic potential of metabolites of two Alternanthera species against germination and seedling growth of rice

A laboratory study was conducted to investigate the allelopathic effect of aqueous extracts of plant parts of Alternanthera philoxeroides and A. sessilis and soil incorporated residues on germination and seedling growth of rice (Oryza sativa). Aqueous extracts prepared from different plant parts of Alternanthera species delayed rice germination. Alternanthera philoxeroides and A. sessilis inhibited rice germination by 9-100% and 4-49%, respectively. Germination of rice seeds was reduced with increasing concentration of aqueous leaf extracts of both weed species. Early seedling growth (root and shoot lengths) and seedling vigor index were significantly reduced by 5% aqueous leaf extract compared with distilled water treated control. Germination, root and shoot lengths, root and shoot dry weights and seedling vigor index of rice were drastically reduced by 3 and 4% in residue infested soil compared with residue free soil. The inhibitory effect of A. philoxeroides in terms of germination and seedling growth of rice was greater than that of A. sessilis. Five percent aqueous leaf extract and 4% residue infested soil of A. philoxeroides caused complete failure of rice seed germination. Alternanthera philoxeroides contained water soluble phenolics, namely 4 hydroxy-3-methoxy benzoic acid (16.19 mg L-1) and m-coumaric acid (1.48 mg L-1), whereas Alternanthera sessilis was rich in chlorogenic acid (17.85 mg L-1), gallic acid (11.03 mg L-1) and vanillic acid (9.88 mg L-1). The study indicates that the allelopathic potential of Alternanthera species may play an important role in enhancing the invasiveness of these species and may suppress rice plants in the vicinity.

Plasma cortisol levels in captive wild felines after chemical restraint

Eight Panthera onca (Po), 13 Felis concolor (Fc), 7 Felis yagouaroundi (Fy), 7 Felis tigrina (Ft) and 5 Felis pardalis (Fp) specimens from São Paulo State zoos were used. All animals were restrained with darts containing 10 mg/kg ketamine and 1 mg/kg xylazine. Venous blood samples were collected as soon as possible (within 15-20 min) and serum was frozen until the time for cortisol quantification. Cortisol was determined using a solid phase radioimmunoassay with an intra-assay coefficient of 8.51%. Data were analyzed statistically by the Kruskal-Wallis test, followed by Dunn's multiple comparisons test, and the one-sample t-test, with the level of significance set at P<0.05. Data are reported as means ± SEM. Cortisol levels differed among the captive felines: Po = 166 ± 33a, Fc = 670 ± 118b, Fy = 480 ± 83b, Ft = 237 ± 42ab, Fp = 97 ± 12a nmol/l (values followed by different superscript letters were significantly different (P<0.001)). Since most of the veterinary procedures on these species involve chemical restraint, these results show the necessity of preventive measures in order to minimize the effect of restraint stress on more susceptible species

Salivary cortisol as a tool for physiological studies and diagnostic strategies

Salivary cortisol is an index of plasma free cortisol and is obtained by a noninvasive procedure. We have been using salivary cortisol as a tool for physiological and diagnostic studies, among them the emergence of circadian rhythm in preterm and term infants. The salivary cortisol circadian rhythm in term and premature infants was established between 8 and 12 postnatal weeks. In the preterm infants the emergence of circadian rhythm was parallel to the onset of sleep rhythm. We also studied the use of salivary cortisol for screening for Cushing's syndrome (CS) in control and obese outpatients based on circadian rhythm and the overnight 1 mg dexamethasone (DEX) suppression test. Salivary cortisol was suppressed to less than 100 ng/dl after 1 mg DEX in control and obese patients. A single salivary cortisol measurement at 23:00 h and again after 1 mg DEX above the 90th percentile of the obese group values had sensitivity and specificity of 93 and 93% (23:00 h), and 91 and 94% (after DEX), respectively. The sensitivity improved to 100% when we combined both parameters. We also studied 11 CS children and 21 age-matched primary obese children for whom salivary cortisol sensitivity and specificity were 100/95% (23:00 h), and 100/95% (1 mg DEX), respectively. Similar to adults, sensitivity and specificity of 100% were obtained by combining 23:00 h and 1 mg DEX. The measurement of salivary cortisol is a useful tool for physiological studies and for the diagnosis of CS in children and adults on an outpatient basis.

Glutamine and glutamate as vital metabolites

Glucose is widely accepted as the primary nutrient for the maintenance and promotion of cell function. This metabolite leads to production of ATP, NADPH and precursors for the synthesis of macromolecules such as nucleic acids and phospholipids. We propose that, in addition to glucose, the 5-carbon amino acids glutamine and glutamate should be considered to be equally important for maintenance and promotion of cell function. The functions of glutamine/glutamate are many, i.e., they are substrates for protein synthesis, anabolic precursors for muscle growth, they regulate acid-base balance in the kidney, they are substrates for ureagenesis in the liver and for hepatic and renal gluconeogenesis, they act as an oxidative fuel for the intestine and cells of the immune system, provide inter-organ nitrogen transport, and act as precursors of neurotransmitter synthesis, of nucleotide and nucleic acid synthesis and of glutathione production. Many of these functions are interrelated with glucose metabolism. The specialized aspects of glutamine/glutamate metabolism of different glutamine-utilizing cells are discussed in the context of glucose requirements and cell function.

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37ºC) of intact human red blood cells to doxorubicinol (40 µM) and to aglycone derivatives of doxorubicin (40 µM) induced, compared with untreated red cells: i) a ~2-fold stimulation of the pentose phosphate pathway (PPP) and ii) a marked inhibition of the red cell antioxidant enzymes, glutathione peroxidase (~20%) and superoxide dismutase (~60%). In contrast to doxorubicin-derived metabolites, doxorubicin itself induced a slighter PPP stimulation (~35%) and this metabolic event was not associated with any alteration in glutathione reductase, glutathione peroxidase, catalase or superoxide dismutase activity. Furthermore, the interaction of hemoglobin with doxorubicin and its metabolites induced a significant increase (~22%) in oxygen affinity compared with hemoglobin incubated without drugs. On the basis of the results obtained in the present study, a new hypothesis, involving doxorubicinol and aglycone metabolites, has been proposed to clarify the mechanisms responsible for the doxorubicin-induced red blood cell toxicity.

Effect of the establishment of dominance relationships on cortisol and other metabolic parameters in Nile tilapia (Oreochromis niloticus)

The objective of the present study was to investigate the influence of the establishment of dominance relationships and social stress on plasma cortisol and metabolite levels in Nile tilapia (Oreochromis niloticus). During the 30-day experiment, the fish weighing 236 ± 29 g were kept in individual aquaria, except for two pairings lasting 6 h each. Blood samples were taken from the animals before and after pairing. Display, approach, attack, rebuff, chase flight, and coloration were carried out on days 16 and 30. Activities and behaviors characteristic of the establishment of dominance relationships were described. It was possible to classify all experimental fish (N = 30) as dominant or subordinate. No differences were detected between dominant (N = 15) and subordinate (N = 15) fish during isolation or after pairing in cortisol (isolated: 5.76 ± 0.98 vs 5.42 ± 0.63; paired: 10.94 ± 1.62 vs 11.21 ± 2.45 µg/dl), glucose (isolated: 60.02 ± 4.9 vs 67.85 ± 16.16; paired: 110.44 ± 15.72 vs 136.26 ± 22.46 mg/dl), triglyceride (isolated: 167.87 ± 5.06 vs 185.68 ± 7.24; paired: 210.85 ± 13.40 vs 221.82 ± 12.70 mg/dl) or total protein levels (isolated: 7.01 ± 0.42 vs 6.69 ± 0.59; paired: 9.21 ± 0.62 vs 9.51 ± 0.66 g/dl). However, when isolated (N = 30) and paired (N = 30) tilapia were compared, there were significant differences in cortisol and metabolite levels. The similar response presented by dominant and subordinate tilapia indicates that establishment of dominance relationships was a stressor for both groups.

An improved HPLC method for the quantitation of 6-mercaptopurine and its metabolites in red blood cells

A procedure is described for the rapid determination of the intra-erythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10(8) cells) in 350 µl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 µl 70% perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100°C for 45 min. After cooling, 100 µl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (r² > 0.998), and the analytical recoveries were 73.2% for 6-TG, 119.1% for 6-MP and 97.4% for 6-MMP. The intra- and inter-assay variations were highest for 6-MP (9.6 and 14.3%, respectively). The lowest detectable concentrations were 3, 3 and 25 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The quantification limits (coefficients of variation <15%) were 8, 10 and 70 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The method was applied to the analysis of 183 samples from 36 children under chemotherapy for acute lymphoblastic leukemia. The concentrations of the metabolites in the red cells of the patients ranged from 0 to 1934 pmol/8 x 10(8) erythrocytes for 6-TGN, and from 0 to 105.8 and 0 to 45.9 nmol/8 x 10(8) erythrocytes for 6-MP and 6-MMP, respectively. The procedure gave results that were in agreement with those obtained with other methods designed to detect cases of non-compliance with treatment, including patient interviews and medical evaluation, among others, demonstrating its applicability to monitoring the treatment of leukemic children.

The relationship between salivary cortisol concentrations and anxiety in adolescent and non-adolescent pregnant women

The main purpose of the present study was to determine the relationship between salivary cortisol concentrations and self-report anxiety in 50 adolescent and 178 non-adolescent women during the last month of pregnancy. The subjects were randomly selected from a previous study involving women who attended antenatal care from September 1997 to August 2000 at 17 health services in Southeast Brazil. Salivary cortisol was measured with an enzyme immunoassay kit, and anxiety was assessed by the State-Trait Anxiety Inventories (STAI) of Spielberger. After saliva collection, the participants completed the STAI. Mean concentrations of cortisol for both pregnant adolescents (14.17 ± 6.78 nmol/l) and non-adolescents (13.81 ± 8.51 nmol/l) were similar (P = 0.89). Forty-three percent of the pregnant adolescents and 30.5% of the non-adolescents felt anxious at the time of being questioned (State Anxiety Inventory (SAI) scores >40; P = 0.06). Cortisol concentrations in adolescents were negatively related to the SAI scores (r = -0.39; P = 0.01) which assess a temporary condition of anxiety. There was a statistically significant difference in mean cortisol concentrations between adolescents with low (<=40) and high (>40) SAI scores (P = 0.03, t-test), but no differences for non-adolescents. The negative relationship between salivary cortisol concentrations and anxiety scores in adolescents may be due to puberty-related hormone differences during this period of life. Pregnant adolescents may possess unique biological or psychological characteristics compared to adults and non-pregnant adolescents. Thus, we need to know more about the hypothalamic-pituitary-adrenocortical axis of pregnant adolescents.

Quantification of fecal estradiol and progesterone metabolites in Syrian hamsters (Mesocricetus auratus)

Alternative methods to the utilization of laboratory animal blood and its by-products are particularly attractive, especially regarding hamsters due to their small size and difficulties in obtaining serial blood samples. Steroid hormone metabolite quantification in feces, widely used in studies of free-ranging or intractable animals, is a non-invasive, non-stressor, economical, and animal saving technique which allows longitudinal studies by permitting frequent sampling of the same individual. The present study was undertaken to determine the suitability of this method for laboratory animals. Estradiol and progesterone metabolites were quantified by radioimmunoassay in feces of intact, sexually mature female Syrian hamsters during the estrous cycle (control) and in feces of superovulated females. Metabolites were extracted by fecal dilution in ethanol and quantified by solid phase radioimmunoassay. Median estrogen and progesterone concentrations were 9.703 and 180.74 ng/g feces in the control group, respectively. Peaks of estrogen (22.44 ± 4.54 ng/g feces) and progesterone (655.95 ± 129.93 ng/g feces) mean fecal concentrations respectively occurred 12 h before and immediately after ovulation, which is easily detected in this species by observation of a characteristic vaginal postovulatory discharge. Median estrogen and progesterone concentrations (28.159 and 586.57 ng/g feces, respectively) were significantly higher in superovulated animal feces (P < 0.0001). The present study demonstrated that it is possible to monitor ovarian activity in Syrian hamsters non-invasively by measuring fecal estradiol and progesterone metabolites. This technique appears to be a quite encouraging method for the development of new endocrinologic studies on laboratory animals.

Prolactin and cortisol levels in women with endometriosis

Endometriosis is a progressive estrogen-dependent disease affecting women during their reproductive years. The objective of the present study was to investigate whether endometriosis is associated with stress parameters. We determined cortisol and prolactin levels in serum, peritoneal and follicular fluid from infertile women with endometriosis and fertile women without the disease. The extent of the disease was staged according to the revised American Fertility Society classification (1997). Serum and peritoneal fluid were collected from 49 women aged 19 to 39 years undergoing laparoscopy. Eighteen women had stage I-II endometriosis and 10 had stage III-IV. Controls were 21 women undergoing laparoscopy for tubal sterilization. Follicular fluid was obtained from 39 women aged 25-39 years undergoing in vitro fertilization (21 infertile women with endometriosis and 18 infertile women without endometriosis). Serum prolactin levels were significantly higher in infertile women with stage III-IV endometriosis (28.9 ± 2.1 ng/mL) than in healthy controls (13.2 ± 2.1 ng/mL). Serum cortisol levels were significantly higher in infertile women with stage III-IV endometriosis (20.1 ± 1.3 ng/mL) than in controls (10.5 ± 1.4 ng/mL). Cortisol and prolactin levels in follicular fluid and peritoneal fluid did not differ significantly between groups. The high levels of cortisol and prolactin in the serum from women with endometriosis might contribute to the subfertility frequently associated with the disease. Moreover, since higher levels of cortisol and prolactin are often associated with stress, it is probable that stress might contribute to the development of endometriosis and its progression to advanced stages of the disease.

Cortisol and 17-a-hydroxy-progesterone levels in infants with refractory hypotension born at 30 weeks of gestation or less

Refractory hypotension is frequent in very low-birth weight infants, whose hypothalamic-pituitary-adrenal axis has been suggested to be immature. The objective of the present study was to evaluate basal cortisol and 17-a-OH-progesterone in the first 36 h of life in preterm infants with and without refractory hypotension (mean arterial blood pressure below the lower limit for gestational age throughout the study despite aggressive volume expansion and use of vasopressors). Thirty-five infants with £30 weeks of gestation and a birth weight £1250 g, with no postnatal use of corticosteroid or death in the first 48 h were studied. Mean arterial pressure was measured every 4 h during the first 48 h. Cortisol and 17-a-OH-progesterone were determined at 12 and 36 h and patients were divided into refractory hypotensive (N = 15) and control (N = 20) groups. The groups were not different regarding type of delivery, use of prenatal corticosteroid, requirement of mechanical ventilation, use of vasopressor drugs, morphine, fentanyl, prophylactic indomethacin, and mean sample timing. Although refractory hypotensive newborns were more immature, were smaller, suffered more deaths after 48 h of life and had a higher SNAPPE-2 score, their cortisol and 17-a-OH-progesterone levels were not different from controls at 12 h and at 36 h. The increase of cortisol in newborns with refractory hypotension 36 h after birth was significantly higher than in controls. Despite the fact that refractory hypotensive very low-birth weight neonates were submitted to a very stressful condition, their cortisol and 17-a-OH-progesterone levels were similar to controls.

Do whole-body vibration exercise and resistance exercise modify concentrations of salivary cortisol and immunoglobulin A?

A single bout of resistance exercise (RE) induces hormonal and immune responses, playing an important role in a long-term adaptive process. Whole-body vibration (WBV) has also been shown to affect hormonal responses. Evidence suggests that combining WBV with RE may amplify hormonal and immune responses due to the increased neuromuscular load. Therefore, the aim of this study was to evaluate salivary cortisol (Scortisol) and salivary IgA (SIgA) concentrations following a RE session combined or not with WBV. Nine university students (22.9 ± 5.1 years, 175.8 ± 5.2 cm, and 69.2 ± 7.3 kg) performed five sets of squat exercise (70% one-repetition-maximum) combined (R+V30) or not (R) with WBV at 30 Hz. Saliva samples were obtained before and after exercise. Subjects also rated their effort according to the Borg CR-10 scale (RPE). Data were analyzed by a mixed model. RPE was higher after R+V30 (8.3 ± 0.7) compared to R (6.2 ± 0.7). However, Scortisol (pre: 10.6 ± 7.6 and 11.7 ± 7.6, post: 8.3 ± 6.3 and 10.2 ± 7.2 ng/mL for R and R+V30, respectively) and SIgA concentrations (pre: 98.3 ± 22.6 and 116.1 ± 51.2, post: 116.6 ± 64.7 and 143.6 ± 80.5 µg/mL for R and R+V30, respectively) were unaffected. No significant correlations were observed between Scortisol and RPE (r = 0.45, P = 0.22; r = 0.30, P = 0.42, for R and R+V30, respectively). On the basis of these data, neither protocol modified salivary cortisol or IgA, although RPE was higher after R+V30 than R.

Measurement of serum estrogen and estrogen metabolites in pre- and postmenopausal women with osteoarthritis using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

Although 17β-estradiol (E2) deficiency has been linked to the development of osteoarthritis (OA) in middle-aged women, there are few studies relating other estrogens and estrogen metabolites (EMs) to this condition. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to measure the levels of six EMs (i.e., estrone, E2, estriol, 2-hydroxyestrone, 2-hydroxyestradiol, and 16a-hydroxyestrone) in healthy pre- and postmenopausal women and women with OA. This method had a precision ranging from 1.1 to 3.1% and a detection limit ranging from 10 to 15 pg. Compared to healthy women, serum-free E2 was lower in the luteal and postmenopausal phases in women with OA, and total serum E2 was lower in postmenopausal women with OA. Moreover, compared to healthy women, total serum 2-hydroxyestradiol was higher in postmenopausal women with OA and total serum 2-hydroxyestrone was lower in both the luteal and follicular phases in women with OA. In conclusion, our HPLC-ESI-MS/MS method allowed the measurement of multiple biochemical targets in a single assay, and, given its increased cost-effectiveness, simplicity, and speed relative to previous methods, this method is suitable for clinical studies.

Adrenal function in 23 children with paracoccidioidomycosis

Adrenal involvement by Paracoccidioides brasiliensis was described at necropsies and in many clinical studies, but only in adults. Therefore, the aim of this study was to evaluate adrenal function in children with paracoccidioidomycosis. Twenty-three children with the systemic form of paracoccidioidomycosis were evaluated and divided in two Groups: Group A (n = 8) included children before treatment and Group B (n = 15) children after the end of treatment. Plasma cortisol (basal and after ACTH test), ACTH, renin activity, aldosterone, sodium and potassium were measured. They were within normal range in all cases, except for renin activity and aldosterone, which were elevated in some cases. Group A patients showed basal and post-ACTH cortisol levels significantly greater than Group B patients. The results showed that adrenal function was not compromised in these children with paracoccidioidomycosis.