Limited Role of Hair Cortisol and Cortisone Measurement for Detecting Cortisol Autonomy in Patients With Adrenal Incidentalomas.

Several studies demonstrated the diagnostic accuracy of hair glucocorticoid measurement in patients with overt Cushing syndrome, but few data are available for patients with adrenal incidentaloma (AI) and cortisol autonomy. The aim of our study was to assess whether measurement of 5 corticosteroid hormones with the ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method in the keratin matrix is useful to stratify patients with AI by the presence of autonomous cortisol secretion [ACS] (defined as serum cortisol after 1 mg dexamethasone suppression test (DST) > 138 nmol/l) or possible ACS [PACS] (defined as serum cortisol after 1 mg DST > 50 nmol/l but ≤138 nmol/l). We analysed data of 67 AI patients (32 with cortisol autonomy) and 81 healthy subjects. We did not find any significant statistical difference comparing hair cortisol, cortisone, and 20ß-dihydrocortisol concentrations between healthy controls and AI patients, while 6ß-hydroxycortisol and 11-deoxycortisol were undetectable. Moreover, no significant difference was found in hair cortisol, cortisone, and 20ß-dihydrocortisol levels of AI patients with or without cortisol autonomy. Finally, we did not find any correlation in patients with AI between hormonal concentrations in the keratin matrix and serum, salivary, and urinary cortisol levels, or with body mass index. In conclusion, our findings suggest that hair glucocorticoid measurement is not suitable as a diagnostic test for cortisol autonomy (ACS and PACS).

Multitarget Antioxidant NO-Donor Organic Nitrates: A Novel Approach to Overcome Nitrates Tolerance, an Ex Vivo Study.

Chronic use of glyceryl trinitrate (GTN) is limited by serious side effects, such as tolerance and endothelial dysfunction of coronary and resistance arteries. Although GTN is used as a drug since more than 130 years, the mechanisms of the vasodilatory effects and of tolerance development to organic nitrates are still incompletely elucidated. New synthesized organic nitrates with and without antioxidant properties were characterized for their ex vivo tolerance profile, in order to investigate the oxidative stress hypothesis of nitrate tolerance. The organic nitrates studied showed different vasodilation and tolerance profiles, probably due to the ability or inability of the compounds to interact with the aldehyde dehydrogenase-2 enzyme (ALDH-2) involved in bioactivation. Furthermore, nitrooxy derivatives endowed with antioxidant properties did not determine the onset of tolerance, even if bioactivated by ALDH-2. The results of this study could be further evidence of the involvement of ALDH-2 in the development of nitrate tolerance. Moreover, the behavior of organic nitrates with antioxidant properties supports the hypothesis of the involvement of ROS in inactivating ALDH-2.

Effects of truck transportation and slaughtering on the occurrence of prednisolone and its metabolites in cow urine, liver, and adrenal glands.

BACKGROUND: The recognition of illegal administration of synthetic corticosteroids in animal husbandry has been recently challenged by the case of prednisolone, whose occasional presence in the urine of bovines under strong stressful conditions was attributed to endogenous biosynthesis, not to exogenous administration. The study of the natural stress sources possibly inducing endogenous prednisolone production represents a stimulating investigation subject. The biochemical effects of transportation and slaughtering were verified in untreated cows by studying the possible occurrence of prednisolone and its metabolites in urine, liver and adrenal glands, and the cortisol/cortisone quantification. RESULTS: Cortisol, cortisone, prednisolone and its metabolites were measured in urine, collected at farm under natural micturition and then at the slaughterhouse. The study was performed on 15 untreated cows reared in different farms at the end of their productive cycle. 2-3 days after the first urine collection, the animals were transported by trucks to the abattoir, slaughtered, and subjected to a second urine sampling from the bladder. Specimens of liver and adrenal gland were also collected and analysed by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated method. The stressful conditions of transportation and slaughtering proved to increase considerably the urinary levels of cortisol and cortisone as compared to those collected at farm. Prednisolone was detected in the urine collected at the slaughterhouse of two cows only, at a concentration level (≈0.6 µg L- 1) largely below the official cut off (5.0 µg L- 1) established to avoid false non-compliances. These two animals exhibited the highest urinary cortisol levels of the series. Prednisolone and prednisone were also detected in the adrenal glands of a different cow. Prednisolone metabolites were not detected in any urine, liver, and adrenal gland sample. CONCLUSION: Within the constraints of the condition adopted, this study confirms the sporadic presence of prednisolone traces (2 samples out of 15) and the consistently increased concentration of cortisone and cortisol in the urines collected from cows subjected to truck transportation and subsequent slaughtering. No prednisolone metabolites were detected in any liver and adrenal gland samples, nor in urine specimens, unlike what was previously reported for cows artificially stressed by pharmacological treatment.

Urine volume as a predicting factor for furosemide clearance during continuous infusion in AKI septic shock patients on hemodiafiltration.

BACKGROUND: This study assessed the contribution of intracorporeal (IC) and extracorporeal clearance (EC) of furosemide in patients with septic acute kidney injury (AKI), and the relationship between plasma concentrations and urine volume. METHODS: Prospective cohort observational study of 15 patients with septic AKI undergoing continuous veno-venous hemodiafiltration (CVVHDF) divided according to urine volume (< 500 ml/12 h, Oliguria group, n = 5; > 500 ml/12 h, Diuresis group, n = 10) during continuous infusion of furosemide (120 mg/12 h) at steady-state condition. Plasma and effluent furosemide concentrations were determined by high-performance liquid chromatography (HPLC)-mass spectrometry every 12 h for 48 h. RESULTS: Furosemide plasma concentrations and total body clearance (TBC) were 6.14 mg/l and 22.1 ml/min for the Oliguria group, and 2.63 mg/l and 54.4 ml/min for the Diuresis group, respectively (p < 0.05). When urine volume was < 500 ml/24 h, the furosemide plasma concentrations peaked at the potentially toxic value of 13.0 mg/l. Furosemide EC was not relevant for the Diuresis group, but it represented 18% of TBC for the Oliguria group. Furosemide plasma concentrations correlated positively with dose infusion for both groups (r = 0.728 and 0.685, p < 0.05), and negatively with urine volume only for the Diuresis (r = - 0.578, p < 0.01) but not for the Oliguria group (r = - 0.089, p = 0.715). CONCLUSIONS: For patients with urine volume > 500 ml/12 h continuous infusion of furosemide up to 480 mg/24 h leads to increasing urine volume, which can predict furosemide plasma levels within its safety range. When the urine volume is lower, the furosemide plasma levels are increased beyond any further diuretic efficacy.

Development and validation of an UHPLC-MS/MS method for ß2-agonists quantification in human urine and application to clinical samples.

A fast analytical method for the simultaneous detection of 24 ß2-agonists in human urine was developed and validated. The method covers the therapeutic drugs most commonly administered, but also potentially abused ß2-agonists. The procedure is based on enzymatic deconjugation with ß-glucuronidase followed by SPE clean up using mixed-phase cartridges with both ion-exchange and lipophilic properties. Instrumental analysis conducted by UHPLC-MS/MS allowed high peak resolution and rapid chromatographic separation, with reduced time and costs. The method was fully validated according ISO 17025:2005 principles. The following parameters were determined for each analyte: specificity, selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, matrix effect, recovery and carry-over. The method was tested on real samples obtained from patients subjected to clinical treatment under chronic or acute therapy with either formoterol, indacaterol, salbutamol, or salmeterol. The drugs were administered using pressurized metered dose inhalers. All ß2-agonists administered to the patients were detected in the real samples. The method proved adequate to accurately measure the concentration of these analytes in the real samples. The observed analytical data are discussed with reference to the administered dose and the duration of the therapy.

Determination of endogenous and exogenous corticosteroids in bovine urine and effect of fighting stress during the "Batailles des Reines" on their biosynthesis.

Natural corticosteroids include two families of substances: mineralocorticoids and glucocorticoids. Several drugs of similar structure and biological activity have been synthesized and are currently used in the clinical practice. Beside legal pharmacological treatments, these drugs have been consistently misused in animal breeding. One of the most abused corticosteroids is prednisolone. For many years, prednisolone has been considered of exclusive synthetic origin, but nowadays a debate about its possible endogenous production is under way. Several studies have been addressed to ascertain the potential relationship between stressful conditions, such as transportation and slaughtering, and endogenous production of prednisolone. In order to verify further the effect of stressful conditions, our laboratory analysed urine samples collected from the cows participating to the "Batailles des Reines" (a traditional contest based on ritual and spontaneous fights of pregnant cows), to verify if an endogenous prednisolone production may occur in these animals. We developed and validated a LC-MS/MS method for the simultaneous determination of cortisol, cortisone, prednisolone and five of its metabolites. The method was applied to the analysis of urine samples collected from "Batailles des Reines" competitions in 2012 and 2013. All these samples had been previously analysed within an anti-doping control program and tested compliant to all screenings.

Determination of Anticoagulant Rodenticides and α-Chloralose in Human Hair. Application to a Real Case.

Anticoagulant rodenticides are the largest group of poisons used to kill harmful rodents. Their fundamental mode of action consists in the inhibition of the vitamin K epoxide reductase, which causes blood-clotting alteration, ultimately leading to hemorrhagic events as the cause of death. In this study, we developed an UHPLC-MS-MS for the simultaneous determination of 10 anticoagulant hydroxycoumarine rodenticides, plus α-chloralose in human hair, with the scope of detecting potential trace of chronological poison exposure in clinical and forensic cases. The method was fully validated and applied to a case of intentional poisoning perpetrated by administration of difenacoum and α-chloralose to a 97-year-old woman, who was hospitalized because of severe symptoms, including drowsiness, convulsions, pallor and hematoma. Hair sample from the victim was segmentally analyzed. Difenacoum was detected in the proximal 3-cm hair segment at the concentration of 2.9 pg/mg. To our knowledge, this is the first study to report that exposure to difenacoum is detectable in real hair samples. The other target analyte found in the hair sample was α-chloralose, which was detected in the 0-3 cm segment at the concentration of 85 pg/mg. The two subsequent and consecutive segments (3-6 cm and 6-9 cm) showed only traces of difenacoum (below LOQ) and low but quantifiable concentrations of α-chloralose (29 and 6 pg/mg, respectively). Therefore, hair segmental analysis allowed us to conclude that the victim was repeatedly exposed to two poisons in the period corresponding to the first segment of hair.

Driving under the influence of alcohol. A 5-year overview in Piedmont, Italy.

Alcohol consumption represents a major health issue worldwide and a crucial factor in road accidences. This study provides information on the prevalence of alcohol in blood testing performed on 2752 subjects involved in vehicle accidents, which occurred in Piedmont (northern Italy) between 2008 and 2013. Blood alcohol concentration (BAC) was determined by an ISO 17025 accredited GC/MS procedure. Fifty-one % of positive samples showed BAC concentrations above 1.5 g/L, with a legal cut-off fixed at 0.5 g/L (and 0 g/L for specified categories such as novice and professional drivers). BAC values proved statistically different regarding the day of sampling (week or weekend days), age and gender, with a prevalence of positive results that reflects different drinking habits of a multifaceted population of alcohol consumers.

Efficient removal of colistin A and B in critically ill patients undergoing CVVHDF and sorbent technologies.

BACKGROUND: Colistin pharmacokinetics data are scarce regarding patients undergoing renal replacement therapy (RRT), or even absent as in patients treated with sorbent technologies potentially capable of removing colistin by extensive absorption on many polymeric materials. METHODS: Twelve septic shock patients with acute kidney injury (AKI) undergoing RRT [continuous venovenous hemodiafiltration (CVVHDF) n = 7, coupled-plasma filtration adsorption-HF (CPFA-HF) n = 4, hemoperfusion n = 1] treated with colistin methanesulfonate at a dose of 4.5 × 10(6) U bid were studied. Colistin A (Col-A) and colistin B (Col-B) concentrations on plasma and effluent at time 0, 0.2, 1, 3, 6, 12, 24 and 48 h were determined by the liquid chromatography-tandem mass spectrometry method. RESULTS: With CVVHDF the sieving coefficient was lower for Col-A, peaked early (0.40 for Col-A at 10 min, and 0.59 for Col-B at 3 h) and declined after 48 h (0.22 and 0.30 for Col-A and Col-B, respectively). Colistin's filter clearance showed a similar pattern, with the highest clearance value of 18.7 ml/min for Col-B at 1 h. With CPFA-HF after the cartridge the Col-A and Col-B levels were negligible (<0.2 mg/l) or not detectable. The sum of the effluent and cartridge clearances reached values of 30 and 40 ml/min for Col-A and Col-B, respectively. With hemoperfusion the postcartridge concentrations for Col-A and Col-B were about 30 % lower than those determined precartridge. CONCLUSIONS: During CPFA-HF and CVVHDF, the extent of colistin removal is high, and patients should receive an unreduced dosage. However, due to risk of accumulation in long-term administration colistin plasma levels determination is recommended.

Profile of the urinary excretion of prednisolone and its metabolites in finishing bulls and cows treated with a therapeutic schedule.

BACKGROUND: Prednisolone was one of the first glucocorticoids to be synthesised, but it is still widely applied to cattle. Illegal uses of prednisolone include its uses for masking a number of diseases before animal sale and, at lower dosages for extended periods of time, for the improvement of feed efficiency and carcass characteristics. Since occasional presence of prednisolone has been detected at trace level in urine samples from untreated cattle, the Italian Ministry of Health introduced a provisional limit of 5 ng/mL to avoid false non-compliances. However, this limit proved ineffective in disclosing prednisolone misuse as a growth-promoter. In the present study, prednisolone acetate was administered to finishing bulls and cows according to a therapeutic protocol (2 × 0.4-0.5 mg/kg bw i.m. at 48 h interval) to further verify the practical impact of this cut-off limit and develop sound strategies to distinguish between exogenous administration and endogenous production. Urinary prednisolone, prednisone, 20ß-dihydroprednisolone, 20α-dihydroprednisolone, 20ß-dihydroprednisone, 6ß-hydroxyprednisolone, cortisol, and cortisone were determined using a validated LC/MS-MS method. RESULTS: The urinary excretion profile showed the simultaneous presence of prednisolone, 20ß-dihydroprednisolone, and prednisone, the latter at lower concentrations, up to 33 days after the first dosing. Higher analyte levels were detected in bulls even after correction for dilution in the urine. Prednisolone concentrations below 5 ng/ml were determined in half of the samples collected at 19 days, and in all the samples obtained 26 and 33 days after the first administration. No measurable concentrations of prednisolone or its metabolites were found in the samples collected before the treatment, while cortisol and cortisone levels lower than the respective LOQs were observed upon treatment. CONCLUSIONS: The present study confirms the criticism of the coarse quantitative approach currently adopted to ascertain illegal prednisolone administration in cattle. As previously shown for growth-promoting treatments of meat cattle, the simultaneous determination of urinary prednisolone, prednisone, 20ß-dihydroprednisolone, along with cortisol and cortisone, may represent a more reliable approach to confirm the exogenous origin of prednisolone. Such a strategy would facilitate unequivocal detection of animals treated with prednisolone acetate using a therapeutical protocol, even 3 to 4 weeks after the treatment.

Determination by LC-MS/MS of colistins A and B in plasma and ultrafiltrate from critically ill patients undergoing continuous venovenous hemodiafiltration.

BACKGROUND: Colistin is a 50-year-old antibiotic, the use of which was ceased in the 70s and recently resumed as a "salvage therapy" against multidrug-resistant gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii. The narrow therapeutic range of colistin makes the choice of its correct dosage crucial, and monitoring of blood concentration is occasionally necessary for critically ill patients, including intensive care patients subjected to continuous renal replacement therapy. METHODS: Two LC-MS/MS methods were developed and fully validated for the quantitative determination of colistins A and B in plasma and dialysis ultrafiltrate (UF) samples, ultimately arising from 4 patients undergoing continuous venovenous hemodiafiltration (CVVHDF). RESULTS: The developed methods proved to be both specific and selective. They showed good fit and linearity over the entire range of interest. Trueness and accuracy proved satisfactory. Both methods have excellent intraassay precision (percent coefficient of variations were lower than 10%) and limit of detection values in the range 20-100 ng/mL, about 1-2 orders of magnitude below the concentrations commonly detected in real samples. The mean sieving coefficient (SC) values, measured after 10 minutes of CVVHDF, were 0.42 for colistin A and 0.48 for colistin B. SC values proved to be quite stable for 24 hours, but then declined to 0.24 for colistin A and 0.32 for colistin B, respectively, after 48 hours. At the median blood flow and effluent flow rate of 120 and 28 mL/min, clearance values for colistin B were higher than 15 mL/min. During the entire duration of CVVHDF sessions, the SC and clearance values for colistin A were significantly lower than colistin B. CONCLUSIONS: Two simple methods for the simultaneous determination of colistins A and B have been developed and validated. Their application in the clinical setting demonstrates that CVVHDF treatment lasting 48 hours produces a relatively constant and efficient removal of the drug.

Fit-for-purpose in veterinary drug residue analysis: development and validation of an LC-MS/MS method for the screening of thirty illicit drugs in bovine urine.

A selective and sensitive method for screening 31 analytes (nine corticosteroids, eight ß-agonists, seven anabolic steroids, six promazines and zeranol) in bovine urine was validated according to 2002/657/EC guidelines. Upon optimization of sample treatment conditions, the extraction was performed by diethylether at pH 9, after deconjugation. Extraction yields (R%) proved higher than 70% for 19 analytes, 50