Salivary cortisone as potential predictor of occupational exposure to noise and related stress.

Salivary cortisone strongly correlates with serum cortisol, and since it is less invasive to measure salivary cortisone than serum cortisol and easier than to measure cortisol in saliva, as its concentrations are much lower, we wanted to compare salivary cortisone and cortisol levels as markers of noise-induced stress reaction. The study included 104 participants aged 19-30 years, 50 of whom were exposed to occupational noise ≥85 dB(A) and 54 non-exposed, control students. All participants took samples of their saliva with Salivette® Cortisol synthetic swabs on three consecutive working days first thing in the morning. Salivary cortisone and cortisol levels were determined with high-performance liquid chromatography. In addition, they completed a 10-item Perceived Stress Scale (PSS-10) questionnaire, and occupationally noise-exposed participants also completed the Health and Safety Executive (HSE) questionnaire on occupational psychosocial risks. The exposed participants had significantly higher cortisone (P<0.001) and cortisol (P<0.001) levels than controls, and the correlation between cortisone and cortisol levels in the exposed participants was strong (ϱ =0.692, P<0.001), which suggests that salivary cortisone can replace cortisol measurements in saliva as a more reliable method than salivary cortisol and less invasive than serum cortisol. However, the level of perceived stress scored on PSS-10 in the exposed participants did not differ significantly from stress reported by controls, but correlated negatively with cortisone levels, which is contrary to our expectations and raises questions as to why.

Beta-Cyclodextrin-Decorated Magnetic Activated Carbon as a Sorbent for Extraction and Enrichment of Steroid Hormones (Estrone, ß-Estradiol, Hydrocortisone and Progesterone) for Liquid Chromatographic Analysis.

A ß-cyclodextrin-decorated magnetic activated carbon adsorbent was prepared and characterized using various analytical techniques (X-ray diffraction (XRD), scanning electron microscopy-electron diffraction spectroscopy (SEM-EDS) and transmission electron microscopy (TEM)), and the adsorbent was used in the development of a magnetic solid-phase microextraction (MSPE) method for the preconcentration of estrone, ß-estradiol, hydrocortisone and progesterone in wastewater and river water samples. This method was optimized using the central composite design in order to determine the experimental parameters affecting the extraction procedure. The quantification of hormones was achieved using high-performance liquid chromatography equipped with a photodiode array detector (HPLC-DAD). Under optimum conditions, the linearity ranged from 0.04 to 300 µg L-1 with a correlation of determinations of 0.9969-0.9991. The limits of detection and quantification were between 0.01-0.03 and 0.033-0.1 µg L-1, with intraday and interday precisions at 1.1-3.4 and 3.2-4.2. The equilibrium data were best described by the Langmuir isotherm model, and high adsorption capacities (217-294 mg g-1) were obtained. The developed procedure demonstrated high potential as an effective technique for use in wastewater samples without significant interferences, and the adsorbent could be reused up to eight times.

Stress-Related Trajectories of Diurnal Cortisol in Older Adulthood Over 12 Years.

OBJECTIVE: Although evidence shows that stress experiences can predict both hyper- and hypo-cortisol regulation, there is a lack of research examining these associations longitudinally. Our study assessed whether levels and increases in psychological stress experiences predicted 12-year changes in circadian cortisol levels (area under the curve; AUC) and cortisol slopes in a sample of community-dwelling older adults. METHODS: In 2004, 190 community dwelling older adults (57 to 94 years) started providing three days of diurnal cortisol and stress experience data every two years for a total of seven waves of data. All analyses controlled for relevant covariates including: SES, BMI, age, sex, cortisol-related medication, chronic illness, and smoking status. RESULTS: Growth-curve modeling documented that compared to participants who reported generally lower stress experiences (T-ratio = -5.57, p < .01), their counterparts with higher stress experiences showed significantly steeper declines in cortisol AUC over time (T-ratio = -9.23, p < .01). Higher stress experience was associated with generally flatter cortisol slopes. In addition, among participants with high and increasing stress experience over 12 years, cortisol slopes became increasingly flatter over time (T-ratio = 2.78, p < .01). CONCLUSIONS: Among individuals with high, as compared to low, levels of chronic stress experience, cortisol levels displayed steeper declines across the study period. Moreover, cortisol slopes became increasingly flatter as a function of high and increasing stress experience. Implications for theory and research on the associations between stress experience and cortisol in the context of longitudinal observations are discussed.

Childhood trauma and dysregulation of multiple biological stress systems in adulthood: Results from the Netherlands Study of Depression and Anxiety (NESDA).

BACKGROUND: Childhood trauma (CT) is a risk factor for depressive and anxiety disorders. Although dysregulated biological stress systems may underlie the enduring effect of CT, the relation between CT and separate and cumulative activity of the major stress systems, namely, the hypothalamic-pituitary-adrenal (HPA)-axis, the immune-inflammatory system, and the autonomic nervous system (ANS), remains inconclusive. METHODS: In the Netherlands Study of Depression and Anxiety (NESDA, n = 2778), we determined whether self-reported CT (as assessed by the Childhood Trauma Interview) was associated with separate and cumulative markers of the HPA-axis (cortisol awakening response, evening cortisol, dexamethasone suppression test cortisol), the immune-inflammatory system (C-reactive protein, interleukin-6, tumor necrosis factor-α), and the ANS (heart rate, respiratory sinus arrhythmia, pre-ejection period) in adulthood. RESULTS: Almost all individuals with CT (n = 1330) had either current or remitted depressive and/or anxiety disorder (88.6%). Total-sample analyses showed little evidence for CT being significantly associated with the separate or cumulative stress systems' activity in adulthood. These findings were true for individuals with and without depressive and/or anxiety disorders. To maximize contrast, individuals with severe CT were compared to healthy controls without CT. This yielded slight, but significantly higher levels of cortisol awakening response (AUCg, ß = .088, p = .007; AUCi, ß = .084, p = .010), cumulative HPA-axis markers (ß = .115, p = .001), C-reactive protein (ß = .055, p = .032), interleukin-6 (ß = .053, p = .038), cumulative inflammation (ß = .060, p = .020), and cumulative markers across all systems (ß = .125, p = .0003) for those with severe CT, partially explained by higher rates of smoking, body mass index, and chronic diseases. CONCLUSION: While our findings do not provide conclusive evidence on CT directly dysregulating stress systems, individuals with severe CT showed slight indications of dysregulations, partially explained by an unhealthy lifestyle and poorer health.

Designing an anti-inflammatory and tissue-adhesive colloidal dressing for wound treatment.

Wound dressing materials are widely used to protect wounds from the external environment and to promote wound healing. However, conventional wound dressings lack tissue adhesive properties and anti-inflammatory functions, which lead to fibrosis and stricture, in cases such as gastrointestinal wounds after endoscopic surgery. In the current study, we report tissue-adhesive and anti-inflammatory properties of a wound dressing composed of corticosteroid-modified gelatin particles. Hydrocortisone (HC), which is a class of anti-inflammatory corticosteroid, was used to modify Alaska-pollock gelatin (ApGltn) to synthesize HC-modified ApGltn (HC-ApGltn). Microparticles (MPs) of HC-ApGltn were fabricated by adding ethanol in HC-ApGltn aqueous solution and performing thermal crosslinking (TC) without the use of toxic surfactants and crosslinking reagents. Modification of ApGltn with hydrophobic HC containing cholesterol backbone structure improved its adhesion strength to gastric submucosal tissues under wet conditions owing to hydrophobic interactions. This retention of adhesive property under wet conditions allows for stable protection of wounds from the external environment. We found that HC-ApGltn MPs were taken up by macrophages and they effectively suppressed morphological changes of LPS-activated macrophages and the expression level of the inflammatory cytokine. Robust tissue adhesive and anti-inflammatory MPs may serve as an advanced wound dressing that can protect wounds and suppress inflammatory responses for promoting wound healing.

Influence of season and pituitary pars intermedia dysfunction on hair cortisol concentration in horses.

Hair cortisol has been demonstrated to reflect hypothalamic-pituitary-adrenal axis activity (including Cushing's disease and stress) in several species. We hypothesized that hair cortisol concentrations are increased in horses with pituitary pars intermedia dysfunction (PPID) compared with healthy adult horses and that this difference is magnified in the fall, when circulating adrenocorticotropin (ACTH) is generally the highest. Cortisol from hair collected using clippers with a #40 blade from the neck was compared between PPID horses and control horses over several months in the fall (August-December) and 1 mo in spring (May). Cortisol from hair at several sampling sites (neck, jugular furrow, sternum, and submandibular) were compared between PPID (n = 6) and control (n = 8) horses in May. Relationships between hair cortisol and ACTH were assessed in the fall. Hair cortisol when measured by weight was higher in PPID vs control horses in October and November (P ≤ 0.01) but not December (P = 0.15), May (P > 0.7), or August-September (P = 0.18). When normalized for hair length, hair cortisol was higher in PPID vs control horses in November (P = 0.0006), but not October or December (P ≥ 0.06). Hair cortisol concentrations did not differ between PPID and control horses from any collection site in May (P > 0.7). There were no consistent relationships between ACTH and hair cortisol concentrations in October, November, or December (P ≥ 0.05). These findings suggest that PPID horses have increased hair cortisol accumulation in the fall compared with control horses. Additional work is needed to clarify whether assessing cortisol per weight or per hair length is most relevant in the horse.

Esters of terpene alcohols as highly potent, reversible, and low toxic skin penetration enhancers.

Skin penetration/permeation enhancers are compounds that improve (trans)dermal drug delivery. We designed hybrid terpene-amino acid enhancers by conjugating natural terpenes (citronellol, geraniol, nerol, farnesol, linalool, perillyl alcohol, menthol, borneol, carveol) or cinnamyl alcohol with 6-(dimethylamino)hexanoic acid through a biodegradable ester linker. The compounds were screened for their ability to increase the delivery of theophylline and hydrocortisone through and into human skin ex vivo. The citronellyl, bornyl and cinnamyl esters showed exceptional permeation-enhancing properties (enhancement ratios up to 82) while having low cellular toxicities. The barrier function of enhancer-treated skin (assessed by transepidermal water loss and electrical impedance) recovered within 24 h. Infrared spectroscopy suggested that these esters fluidized the stratum corneum lipids. Furthermore, the citronellyl ester increased the epidermal concentration of topically applied cidofovir, which is a potent antiviral and anticancer drug, by 15-fold. In conclusion, citronellyl 6-(dimethylamino)hexanoate is an outstanding enhancer with an advantageous combination of properties, which may improve the delivery of drugs that have a limited ability to cross biological barriers.

Comparison of Steroid Hormone Hydroxylations by and Docking to Human Cytochromes P450 3A4 and 3A5.

PURPOSE: Hydroxylation activity at the 6ß-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (P450 or CYP) 3A4 and CYP3A5 and their molecular docking energy values were compared to understand the catalytic properties of the major forms of human CYP3A, namely, CYP3A4 and CYP3A5. METHODS: Testosterone, progesterone, and cortisol 6ß-hydroxylation activities of recombinant CYP3A4 and CYP3A5 were determined by liquid chromatography. Docking simulations of these substrates to the heme moiety of reported crystal structures of CYP3A4 (Protein Data Bank code ITQN) and CYP3A5 (6MJM) were conducted. RESULTS: Michaelis constants (Km) for CYP3A5- mediated 6ß-hydroxylation of testosterone and progesterone were approximately twice those for CYP3A4, whereas the value for cortisol 6ß-hydroxylation mediated by CYP3A5 was similar to the value for that by CYP3A4. Maximal velocities (Vmax) of the three steroid hormones 6ß-hydroxylation catalyzed by CYP3A5 were 30%-63% of those by CYP3A4. Thus, Vmax/ Km values of these hormones for CYP3A5 resulted in 22%- 31% of those for CYP3A4. The differences in the docking energies between CYP3A4 and CYP3A5 for steroid hormones were slightly correlated to the logarithm of CYP3A5/CYP3A4 ratios for Km values (substrate affinity). CONCLUSIONS: The Vmax, rather than Km values, for CYP3A5-mediated 6ß-hydroxylation of three steroid hormones were different from those for CYP3A4. Molecular docking simulations could partially explain the differences in the accessibility of substrates to the heme moiety of human CYP3A molecules, resulting in the enzymatic affinity of CYP3A4 and CYP3A5.

Spectroscopic evaluation of synthesized 5ß-dihydrocortisol and 5ß-dihydrocortisol acetate binding mechanism with human serum albumin and their role in anticancer activity.

Our study focus on the biological importance of synthesized 5ß-dihydrocortisol (Dhc) and 5ß-dihydrocortisol acetate (DhcA) molecules, the cytotoxic study was performed on breast cancer cell line (MCF-7) normal human embryonic kidney cell line (HEK293), the IC50 values for MCF-7 cells were 28 and 25 µM, respectively, whereas no toxicity in terms of cell viability was observed with HEK293 cell line. Further experiment proved that Dhc and DhcA induced 35.6 and 37.7% early apoptotic cells and 2.5, 2.9% late apoptotic cells, respectively, morphological observation of cell death through TUNEL assay revealed that Dhc and DhcA induced apoptosis in MCF-7 cells. The complexes of HSA-Dhc and HSA-DhcA were observed as static quenching, and the binding constants (K) was 4.7 ± .03 × 104 M-1 and 3.9 ± .05 × 104 M-1, and their binding free energies were found to be -6.4 and -6.16 kcal/mol, respectively. The displacement studies confirmed that lidocaine 1.4 ± .05 × 104 M-1 replaced Dhc, and phenylbutazone 1.5 ± .05 × 104 M-1 replaced by DhcA, which explains domain I and domain II are the binding sites for Dhc and DhcA. Further, FT-IR, synchronous spectroscopy, and CD results revealed that the secondary structure of HSA was altered in the presence of Dhc and DhcA. Furthermore, the atomic force microscopy and transmission electron microscopy showed that the dimensions like height and molecular size of the HSA-Dhc and HSA-DhcA complex were larger compared to HSA alone. Detailed analysis through molecular dynamics simulations also supported greater stability of HSA-Dhc and HSA-DhcA complexes, and root-mean-square-fluctuation interpreted the binding site of Dhc as domain IB and domain IIA for DhcA. This information is valuable for further development of steroid derivative with improved pharmacological significance as novel anti-cancer drugs.

Hair cortisol and progesterone detection in dairy cattle: interrelation with physiological status and milk production.

Hair cortisol concentrations (HCCs) and hair progesterone concentrations (HPCs) allow monitoring long-term retrospective steroid levels. However, there are still gaps in the knowledge of the mechanisms of steroid deposition in hair and its potential application in dairy cattle research. This study aimed to evaluate the potential uses of hair steroid determinations by studying the interrelations between HCC, HPC, physiological data from cows, and their milk production and quality. Cortisol and progesterone concentrations were analyzed in hair from 101 milking Holstein Friesian cows in a commercial farm. Physiological data were obtained from the 60 d prior to hair collection. Moreover, productive data from the month when hair was collected and the previous one were also obtained as well as at 124 d after hair sampling. Significant but weak correlations were found between HCC and HPC (r = 0.25, P < 0.0001) and between HPC and age (r = 0.06, P = 0.0133). High HCC were associated with low milk yields from the 2 previous months to hair sampling (P = 0.0396) and during the whole lactation (P < 0.0001). High HCC were also related to high somatic cell count (P = 0.0241). No effect of HCC on fat or protein content was detected. No significant correlations were detected between hair steroid concentrations and pregnancy status, days of gestation, parturition category (primiparous vs multiparous), number of lactations or days in milk. The relationship between physiological variables and HCC or HPC could depend on the duration of the time period over which hair accumulates hormones. Steroid concentrations in hair present high variability between individuals but are a potential tool for dairy cattle welfare and production research by providing a useful and practical tool for long-term steroid monitoring.

Potentiation of Otoprotective Effect of Hydrocortisone Immobilized on Povidone Nanoparticles under Conditions of Intravenous Injection.

The otoprotective effect of immobilized hydrocortisone was studied on the model of acute acoustic injury to the auditory analyzer in male Wistar rats. The effects of true solution and suspension where polyvinylpyrrolidone particles (100-500 nm) served as dispersed phase (hydrocortisone concentration 5 mg/kg). The agents were administered immediately after continuous acoustic stimulation: 5 kHz tone, 110 dB for 2 h. The hearing status was evaluated by the amplitude of otoacoustic emission at the distortion product frequency (4-6.4 kHz) 1 and 24 h and 7 days after acoustic stimulation. Single injection of hydrocortisone suspension caused a more pronounced therapeutic effect within 1 day after acoustic stimulation.

Conscientiousness, hair cortisol concentration, and health behaviour in older men and women.

Conscientious is associated with greater longevity and other favourable health outcomes, but the processes underlying these links are poorly understood. Health behaviours such as physical activity and avoidance of smoking and excessive alcohol consumption may contribute, but direct associations with neuroendocrine and inflammatory processes may also be relevant. We tested the associations between conscientiousness and hair cortisol concentration in 2318 older men and women (mean age 66.2 years) from the English Longitudinal Study of Ageing. Conscientiousness was positively associated with physical activity and fruit and vegetable consumption, and negatively related to alcohol intake, sedentary behaviour, body mass index and depressive symptoms (all p<0.001). We found an inverse association between conscientiousness and hair cortisol concentration that was independent of age, sex, education and wealth (ß=-0.053, p=0.012), and the relationship remained significant with additional adjustment for health behaviour and depressive symptoms (ß=-0.048, p=0.025). The observation that greater conscientiousness was correlated with lower hair cortisol indicates that this trait might impact central nervous regulation of hypothalamic-pituitary-adrenocortical function, with effects that are possibly advantageous for health.

The effects of intrauterine infusion of peanut oil on endometrial health, salivary cortisol and interovulatory period in mares.

Intrauterine infusion of peanut oil at Day 10 post-ovulation has been reported to prolong dioestrus in mares. However, the effects of peanut oil treatment on the endometrium and whether the technique is painful have not been assessed. The objectives of this study were, (i) to determine the effect of intrauterine infusion of peanut oil on endometrial health, (ii) to determine whether use of intrauterine peanut oil is painful and (iii) to confirm that peanut oil causes prolonged dioestrus. Six mares aged 3-12 years old were used in a cross-over design with each mare administered both 1 ml of intrauterine peanut oil and a sham treatment on different oestrous cycles. The effect of intrauterine infusion of 1 ml peanut oil or sham treatment were measured using interovulatory period, uterine fluid accumulation as determined by transrectal ultrasonography, serum progesterone levels, endometrial Kenney biopsy scores and histological features, endometrial eosinophil numbers and salivary cortisol measurements. The individual mare response to intrauterine infusion of peanut oil was variable. Peanut oil infusion did not statistically prolong the luteal phase, nor elevate salivary cortisol levels but did cause superficial erosion of the endometrial surface epithelium in all mares and significantly increased eosinophil numbers in the endometrium (P = 0.0068). The Kenney grade for biopsies from 2/6 mares worsened transiently following infusion. In conclusion, intra-uterine peanut oil does not statistically increase the duration of the luteal phase but results in an inflammatory response and increase in endometrial eosinophil numbers suggesting treatment may be associated with a hypersensitivity-type reaction. Those contemplating using peanut oil to suppress oestrus should also be aware of the legislative and regulatory implications.

Investigation of techniques to measure cortisol and testosterone concentrations in coyote hair.

Long-term noninvasive sampling for endangered or elusive species is particularly difficult due to the challenge of collecting fecal samples before hormone metabolite desiccation, as well as the difficulty in collecting a large enough sample size from all individuals. Hair samples may provide an environmentally stable alternative that provides a long-term assessment of stress and reproductive hormone profiles for captive, zoo, and wild mammals. Here, we extracted and analyzed both cortisol and testosterone in coyote (Canis latrans) hair for the first time. We collected samples from 5-week old coyote pups (six female, six male) housed at the USDA-NWRC Predator Research Facility in Millville, UT. Each individual pup was shaved in six different locations to assess variation in concentrations by body region. We found that pup hair cortisol (F5,57.1 = 0.47, p = 0.80) and testosterone concentrations (F5,60 = 1.03, p = 0.41) did not differ as a function of body region. Male pups generally had higher cortisol concentrations than females (males = 17.71 ± 0.85 ng/g, females = 15.48 ± 0.24 ng/g; F1,57.0 = 5.06, p = 0.028). Comparatively, we did not find any differences between male and female testosterone concentrations (males = 2.86 ± 0.17 ng/g, females = 3.12 ± 0.21 ng/g; F1,60 = 1.42, p = 0.24). These techniques represent an attractive method in describing long-term stress and reproductive profiles of captive, zoo-housed, and wild mammal populations.

The effects of ovarian biopsy and blood sampling methods on salivary cortisol and behaviour in sows.

In reproductive physiology research, experimental animals are often subjected to stressful procedures, including blood sampling and biopsy. In this present study, presence of pain or distress induced by four different procedures was examined using a measurement of salivary cortisol levels and activity observations in sows. The treatments were: 1) PAL: The ovary was palpated through the rectum without snaring, 2) TUB: transvaginal ultrasound-guided biopsy of the ovary was conducted without snaring, 3) SNA: a soft rope snare was placed around the maxilla, 4) CAT: A soft rope snare was placed around the maxilla, and an intravenous catheter was inserted through the ear vein of the sows. Activities, social cohesion and other pain-related behaviour, and salivary cortisol concentrations were recorded. Salivary cortisol concentrations in CAT sows increased in response to the procedure (P<0.05), whereas the other treatments did not trigger a significant response. The CAT sows had higher cortisol concentrations than the other groups for 10min after initiation of the procedures (P<0.01), and they maintained higher cortisol levels than the PAL and TUB groups 15min post-treatment (P<0.05). Furthermore, the CAT sows showed the highest frequency of head shaking (P<0.001) and trembling behaviour (P<0.05) during the 1h post-treatment. Summarizing, the catheterization procedure might induce a short-term pain or stress response during and after the procedure in terms of pain-related behaviour and salivary cortisol status. We suggest that TUB might not cause appreciable pain or distress.

Variation of Human Milk Glucocorticoids over 24 hour Period.

Human milk (HM) contains a complex array of hormones, including members of the glucocorticoid family. The predominant glucocorticoids, cortisol and cortisone may influence the growth and behaviour of the breastfed infant. However, little is understood of the factors regulating the levels of these hormones within HM. The aim of the study was to examine HM cortisol and cortisone concentration, measured in samples collected at each feed during a 24 hour period. Twenty three exclusively breastfeeding mothers collected milk, prior to and after each breastfeeding session over 24 hour period at 3.2(1.60) months. HM cortisol and cortisone levels were measured using high pressure liquid chromatography mass spectroscopy. Cortisone was the predominant glucocorticoid (3.40 ng/ml), and cortisol was detected in all samples (1.62 ng/ml). A positive correlation was found between cortisone and cortisol (r = 0.61, y = 1.93 ± 0.24, p < 0.0001). Cortisol and cortisone concentrations were significantly higher in feeds in the morning (2.97 ng/ml and 4.88 ng/ml), compared to afternoon (1.20 ng/ml and 3.54 ng/ml), evening (0.69 ng/ml and 2.13 ng/ml) and night (1.59 and 3.27 ng/ml). No difference was found between glucocorticoids level of the milk expressed for collection either before or immediately after the breastfeed, or between milk collected from the left or right breast. This study shows that HM glucocorticoid concentrations exhibit a 24 hour pattern, with highest peak levels in the early morning, reflecting the circadian pattern as previously reported in plasma. Thus, HM glucocorticoid concentrations are likely to reflect those in the maternal circulation.

Catalytic photodegradation of pharmaceuticals - homogeneous and heterogeneous photocatalysis.

Photocatalytic degradation of pharmaceuticals (hydrocortisone, estradiol, and verapamil) and personal care product additives (parabens-methyl, ethyl, and propyl derivatives) was investigated in the homogeneous phase (with ferric ions as the catalyst) and on TiO2. Ferric ions in concentrations corresponding to concentrations in natural water bodies were shown to be a significant accelerator of the degradation in homogeneous reaction mixtures. In heterogeneous photocatalytic reactions on TiO2, lower reaction rates, but mineralisation to higher extents, were observed.

Non-invasive monitoring of reproductive and stress hormones in the endangered red panda (Ailurus fulgens fulgens).

The red panda (Ailurus fulgens fulgens) is classified as endangered due to its declining population, habitat fragmentation and poaching. Efforts are being made to breed them in captivity as part of nationwide conservation breeding program. This study aimed to standardize Enzyme immunoassays (EIAs) to monitor reproductive (Progesterone metabolite, Testosterone) and stress hormone (Cortisol) in red panda. For this purpose, we collected 1471 faecal samples from four females and one male over a period of one year from Padmaja Naidu Himalayan Zoological Park, Darjeeling, India. HPLC confirmed the presence of immunoreactive 5α-pregnan-3α-ol-20-one, testosterone and cortisol metabolites in faecal samples. Using 5α-pregnan-3α-ol-20-one EIA, we were able to monitor reproduction and detect pregnancy in one of the females, which successfully conceived and delivered during the study period. We were also able to monitor testosterone and cortisol in faecal samples of the red panda. Faecal testosterone levels were found in higher concentration in breeding season than in non-breeding season. Faecal cortisol concentrations showed a negative relationship with ambient temperature and peaked during winter months in all animals. Standardization of EIAs and faecal hormone monitoring would facilitate red panda conservation breeding programs in India and elsewhere.

Stability of four standardized preparations of methotrexate, cytarabine, and hydrocortisone for intrathecal use.

INTRODUCTION: Intrathecal administration of methotrexate, cytarabine, and hydrocortisone is commonly used to treat and prevent central nervous system involvement in leukemias and lymphomas. The use of intrathecal solutions with pH and osmolarity values close to physiologic range of CSF (pH 7.31-7.37, osmolarity 281-306 mOsm/kg) and standardization of the methotrexate, cytarabine, and hydrocortisone doses in children and adults based on age is highly recommended. Stability studies of standardized intrathecal mixtures under these conditions have not yet been published. OBJECTIVE: The purpose of this study was to evaluate the physical and chemical stabilities of four standardized mixtures of methotrexate, cytarabine, and hydrocortisone stored at 2-8℃ and 25℃ up to 7 days after preparation. METHODS: Four different standardized intrathecal mixtures were prepared and stored at 2-8℃ and 25℃ and protected from light. Triplicate samples were taken at different times and precipitation, appearance, color, pH, and osmolarity were analyzed. Methotrexate, cytarabine, and hydrocortisone concentrations were measured using a modified high-performance liquid chromatography method. RESULTS: No variation greater than 10% of the initial concentration of methotrexate, cytarabine, and hydrocortisone was observed in any of the four standardized mixtures for the 7 days of study when stored at 2-8℃ and 25℃ and protected from light. The osmolarity of the four preparations was within the physiologic range of CSF for 7 days at both 2-8℃ and 25℃. The pH values close to the physiologic range of CSF were stable for 48 h at 25℃ and for 120 h at 2-8℃. CONCLUSIONS: Triple intrathecal standardized preparations of methotrexate, cytarabine, and hydrocortisone sodium phosphate are physically and chemically stable at 25℃ for 48 h and at 2-8℃ for 5 days.