An LC-MS/MS method for the determination of drugs of abuse included THC-COOH, EtG, and NPS, using a single hair extraction sample.

Hair analysis plays an important role in the determination of drugs of abuse in both forensic and clinical toxicology investigations. The analysis of different substances often requires the use of different sample preparation methods, thereby increasing the amount of hair sample and time required. In the present study, a fast method involving a combination of a single 25 mg hair extraction procedure and four liquid chromatography-tandem mass spectrometry methods using the same chromatographic phases and column was developed and validated. The target was the identification and quantification of various commonly abused drugs and their metabolites, including amphetamines, cocaine, opioids, cannabinoids, THC-COOH and EtG, and more than 140 new psychoactive substances, including synthetic cannabinoids, phenethylamines, synthetic opioids, methylphenidate, cathinone, piperidine, and tryptamines.

Comparison of two methods for the extraction of ethylglucuronide from hair.

The aim was the comparison between the Society of Hair Testing (SoHT) consensus for the use of alcohol markers which powdering hair for the extraction of ethylglucuronide (EtG) in water and extraction using the patented M3 Reagent Test kit on cut hair. Hair samples were cut into small segments and washed twice with methanol and diethyl ether. The SoHT-Consensus entails the extraction of pulverised hair in water. This is obtained by incubation of 25 mg of hair at room temperature overnight and 2 h sonication, even if the overnight incubation is not mandatory. The M3 method entails incubation of 25 mg of cut hair with the M3-Reagent at 100°C for 60 min. After centrifugation, the supernatant is injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples (191) were collected in the APSS laboratory in Trento, Italy, between 2021 and 2022. The limit of quantification (LOQ) was set at 5 pg/mg for the pulverised and M3-Reagent methods. Assays showed good linearity above the range of LOQ-300 pg/mg. Precision (within 20%) values were also obtained using both methods. In the Passing-Bablock linear regression, the final regression curve between M3 (y) and the pulverising method (x) showed good agreement; the Bland-Altman analysis did not show any significant bias between the two methods. The M3-Reagent method, due to cut hair use, is easy to perform, saves time and allows for a smaller sample quantity loss with use of nondisposable grinding jars for the ball mill to obtain the extraction of EtG.

Activation of pituitary axis according to underlying critical illness and its effect on outcome.

PURPOSE: Critical illness is a life threatening condition inducing a severe acute physical stress. The aim of the study was to investigate the activation of pituitary axis early after ICU admission in patients with critical illnesses of different etiology and its association with outcome. MATERIALS AND METHODS: Patients admitted for acute respiratory distress syndrome (ARDS), severe traumatic brain injury (TBI), subarachnoid hemorrhage (SAH) and neurocritically ill patients at the moment of brain death (BD) diagnosis were included in the present post-hoc analysis. On day 1, 2-3 and 4-5 after admission the following pituitary axes were assessed: hypothalamic-pituitary-adrenal, hypothalamic-pituitary-thyroid, somatotroph, prolactin and copeptin. ICU mortality was used as outcome measure. RESULTS: One hundred-thirteen critical ill patients were studied. Thyroid axis suppression and activation of copeptin axis were the most frequent pituitary hormone alterations, present in almost 60% of patients. Activation of the hypothalamic pituitary adrenal axis was a predictor of ICU mortality independently from the underlying critical illness [OR 3.952 (C.I.95% 1.129-13.838)]. CONCLUSIONS: Pituitary axis function is frequently altered early after ICU admission, the magnitude of hormonal response being different according to the underlying critical illness. The activation of the hypothalamic pituitary adrenal axis was a strong predictor of ICU mortality.

LC-MS/MS based 25(OH)D status in a large Southern European outpatient cohort: gender- and age-specific differences.

BACKGROUND: Developed countries have a high prevalence of vitamin D deficiency. In previous studies, 25(OH)D was predominantly measured by immunoassays. The present study assessed serum 25(OH)D in a very large Southern European outpatient cohort by liquid chromatography tandem mass spectrometry (LC-MS/MS). MATERIALS AND METHODS: 74,235 serum 25(OH)D results generated under routine conditions between 2015 and 2016 were extracted from the laboratory information system of the Department of Clinical Pathology at Bolzano Hospital (Italy). In 3801 cases, parathyroid hormone (PTH) was requested in parallel. Serum 25(OH)D was measured by a NIST-972 aligned commercial LC-MS/MS method. The distribution of serum 25(OH)D concentrations in males and females of different age groups, the prevalence of 25(OH)D2 and seasonal variability were studied. RESULTS: The average 25(OH)D concentration in the entire cohort was 68.6 nmol/L (7.5-1880 nmol/L). Females had a 7 nmol/L higher average 25(OH)D concentration than males, which increased significantly with age. 37.9 and 28.3% of males and females, respectively, had a deficient 25(OH)D concentration of < 50 nmol/L. 620 samples (0.84%) had measureable amounts of 25(OH)D2. In samples with a normal PTH, 25(OH)D was 11 nmol/L higher than in the entire cohort. Seasonal variation ranged between 20 and 30% and was most pronounced in young individuals. 25(OH)D2 remained constant throughout the year. CONCLUSION: Average serum 25(OH)D in South Tyrol is higher than in other parts of Europe. 25(OH)D and PTH show a continuous inverse relationship. Seasonal variation of serum 25(OH)D is an important aspect in young and middle-aged adults, but becomes less relevant in elderly subjects. 25(OH)D2 is of minor practical importance in South Tyrol.

NTproBNP in insulin-resistance mediated conditions: overweight/obesity, metabolic syndrome and diabetes. The population-based Casale Monferrato Study.

BACKGROUND AND AIMS: NTproBNP and BNP levels are reduced in obese subjects, but population-based data comparing the pattern of this relationship in the full spectrum of insulin-resistance mediated conditions, overweight/obesity, metabolic syndrome and diabetes, are limited. METHODS: The study-base were 3244 individuals aged 45-74 years, none of whom had heart failure, 1880 without diabetes and 1364 with diabetes, identified as part of two surveys of the population-based Casale Monferrato Study. All measurements were centralized. We examined with multiple linear regression and cubic regression splines the relationship between NTproBNP and BMI, independently of known risk factors and confounders. A logistic regression analysis was also performed to assess the effect of overweight/obesity (BMI ≥ 25 kg/m2), diabetes and metabolic syndrome on NTproBNP values. RESULTS: Out of the overall cohort of 3244 people, overweight/obesity was observed in 1118 (59.4%) non-diabetic and 917 (67.2%) diabetic subjects, respectively. In logistic regression, compared to normal weight individuals, those with a BMI ≥ 25 kg/m2 had a OR of 0.70 (95% CI 0.56-0.87) of having high NTproBNP values, independently of diabetes. As interaction between diabetes and NTproBNP was evident (p < 0.001), stratified analyses were performed. Diabetes either alone or combined with overweight/obesity or metabolic syndrome enhanced fourfold and over the OR of having high NTproBNP levels, while the presence of metabolic syndrome alone had a more modest effect (OR 1.54, 1.18-2.01) even after having excluded individuals with CVD. In the non-diabetic cohort, obesity/overweight and HOMA-IR ≥ 2.0 decreased to a similar extent the ORs of high NTproBNP [0.76 (0.60-0.95) and 0.74 (0.59-0.93)], but the association between overweight/obesity and NTproBNP was no longer significant after the inclusion into the model of HOMA-IR, whereas CRP > 3 mg/dl conferred a fully adjusted OR of 0.65 (0.49-0.86). CONCLUSIONS: NT-proBNP levels are lower in overweight/obesity, even in those with diabetes. Both insulin-resistance and chronic low-grade inflammation are involved in this relationship. Further intervention studies are required to clarify the potential role of drugs affecting the natriuretic peptides system on body weight and risk of diabetes.

Thrombopoietin as Early Biomarker of Disease Severity in Patients With Acute Pancreatitis.

OBJECTIVES: To study the concentrations of thrombopoietin (TPO), a growth factor recently involved in the pathogenesis of experimental acute pancreatitis (AP), and its potential role as an early diagnostic and prognostic biomarker in patients with AP. METHODS: Thrombopoietin was measured in 44 AP patients, 18 patients with nonpancreatic acute abdominal pain, and 18 healthy volunteers. Acute pancreatitis severity was classified on the basis of the 2012 International Atlanta Symposium on Acute Pancreatitis criteria. RESULTS: Thrombopoietin levels did not differ between AP patients and control subjects, whereas these were higher in patients with moderately severe or severe AP compared with those with mild AP. Receiver operating characteristic curve analysis of TPO for severe AP diagnosis showed an area under the curve of 0.80. A cutoff value of 31.48 pg/mL showed the highest sensitivity, allowing to rule out severe AP when TPO was lower, whereas TPO higher than 98.23 pg/mL was associated with severe AP with high specificity (93.5%). Furthermore, TPO levels were greater in AP patients developing organ dysfunction or sepsis and in nonsurvivors compared with survivors. CONCLUSIONS: Our data provide the first evidence for TPO as potential early prognostic biomarker in AP patients. High TPO levels at hospital admission may predict organ dysfunction, sepsis, and fatal outcome in AP patients.

Assessment of Diagnostic and Prognostic Role of Copeptin in the Clinical Setting of Sepsis.

The diagnostic and prognostic usefulness of copeptin were evaluated in septic patients, as compared to procalcitonin assessment. In this single centre and observational study 105 patients were enrolled: 24 with sepsis, 25 with severe sepsis, 15 with septic shock, and 41 controls, divided in two subgroups (15 patients with gastrointestinal bleeding and 26 with suspected SIRS secondary to trauma, acute coronary syndrome, and pulmonary embolism). Biomarkers were determined at the first medical evaluation and thereafter 24, 48, and 72 hours after admission. Definitive diagnosis and in-hospital survival rates at 30 days were obtained through analysis of medical records. At entry, copeptin proved to be able to distinguish cases from controls and also sepsis group from septic shock group, while procalcitonin could distinguish also severe sepsis from septic shock group. Areas under the ROC curve for copeptin and procalcitonin were 0.845 and 0.861, respectively. Noteworthy, patients with copeptin concentrations higher than the threshold value (23.2 pmol/L), calculated from the ROC curve, at admission presented higher 30-day mortality. No significant differences were found in copeptin temporal profile among different subgroups. Copeptin showed promising diagnostic and prognostic role in the management of sepsis, together with its possible role in monitoring the response to treatment.

Diagnostic accuracy of aldosterone and renin measurement by chemiluminescent immunoassay and radioimmunoassay in primary aldosteronism.

OBJECTIVE: Up to 50% of hypertensive patients should be screened for primary aldosteronism, using the aldosterone to renin (or plasma renin activity) ratio [aldosterone to active renin ratio (AARR) and aldosterone to plasma renin activity ratio (ARR), respectively]. Aim of the study was to prospectively compare the diagnostic accuracy of AARR (measured by chemiluminescent immunoassay) and ARR (measured by radioimmunoassay) as screening tests for primary aldosteronism and aldosterone assays (measured by chemiluminescence and radioimmunoassay) during confirmatory testing. METHODS: One hundred patients were screened for primary aldosteronism and 34 underwent confirmatory testing. The cut-offs for ARR and AARR were 30 ng/dl/ng/ml/h and 3.7 ng/dl/mU/l, respectively. Patients with positive confirmatory test underwent subtype diagnosis. RESULTS: Seventy-five patients were essential hypertensive patients, 15 had idiopathic hyperaldosteronism, five aldosterone-producing adenoma (APA) and five with undefined diagnosis. The AARR displayed a sensitivity of 90% and a specificity of 99%, the ARR had a sensitivity of 100% and a specificity of 73%. Of the two of 20 primary aldosteronism patients missed by AARR, none resulted affected by APA. All primary aldosteronism patients were correctly diagnosed by chemiluminescence at confirmatory testing. In the total sample of 168 measurements both the correlation for plasma renin activity with renin and for aldosterone in chemiluminescence and radioimmunoassay were highly significant (ρ = 0.70, P < 0.001 and ρ = 0.78, P < 0.001, respectively). On receiver operator characteristics curves, the area under the curve for AARR was 0.989 [95% confidence interval (CI) 0.97-1] and 0.934 for ARR (95% CI 0.89-0.98), which were not significantly different. CONCLUSION: The automated aldosterone and renin chemiluminescent assay is a reliable alternative to the radioimmunometric method, especially for APA detection.

Tenofovir clearance is reduced in HIV-positive patients with subclinical tubular impairment.

OBJECTIVE: To assess if tenofovir (TFV) clearance is associated with urinary retinal-binding protein (RBP) in HIV-positive patients with normal estimated filtration rate. DESIGN: A cross-sectional diagnostic study. METHODS: HIV-positive patients with estimated creatinine clearance above 60 ml/min, on tenofovir disoproxil fumarate (TDF)-containing combination since at least 6 months, taking TDF at night, and without significant comorbidities (diabetes, untreated hypertension, known renal malformations, recurrent nephrolithiasis) and nephrotoxic drugs were included. TFV plasma and urinary concentrations were measured 12 h after drug intake (C12). RBP was measured through enzyme immunoassay kit on spot urines and corrected per urinary creatinine (uRBP/uCr); normality ranges were below 130 µg/g (in patients aged <50 years) and below 172 µg/g (in patients aged ≥50 years). RESULTS: Two hundred and eighty-nine patients were included (median age of 45.8 years, 71.6% male and 85.4% whites); patients were concomitantly treated with nonnucleoside reverse transcriptase inhibitors (155, 53.6%), protease inhibitors (118, 40.8%), or integrase inhibitors (16, 5.5%)-containing regimens. Estimated creatinine clearance was 89.4 ml/min (78.6-105.9). Urinary RBP (uRBP) and uRBP/uCr were 204.6 ng/ml (92-380) and 169.7 µg/g (85.8-318.3), respectively; abnormally high uRBP/uCr was observed in 157 patients (54.3%). A multivariate binary logistic regression confirmed that both ethnicity (P = 0.004, ß 2.93, 95% confidence interval 1.41-6.10) and TFV urinary C12 less than 21 mg/ml (P = 0.006, ß 2.04, 95% confidence interval 1.12-3.41) were significantly associated with abnormal uRBP/uCr. CONCLUSION: HIV-positive TDF-treated patients showed a high prevalence of proximal tubular impairment: ethnicity (whites) and low urinary TFV concentrations were significantly associated with elevated uRBP. SDC VIDEO:: http://links.lww.com/QAD/A852.

Improvement of anthropometric and metabolic parameters, and quality of life following treatment with dual-release hydrocortisone in patients with Addison's disease.

In patients with Addison's disease (AD), a dual-release preparation of hydrocortisone (Plenadren, PLEN) has been demonstrated to maintain cortisol levels in a more physiological range than conventional glucocorticoid therapy, and to exert positive effects. This study aimed to assess variations of anthropometric, metabolic, and hormonal parameters in patients with AD after switching from conventional hydrocortisone (HC) treatment to PLEN. In nineteen AD patients (15 F and 4 M, age 27-65 years) treated with HC 20 mg/day thrice daily, body weight, BMI, waist circumference, fasting glucose, HbA1c, serum lipids, plasma renin activity, electrolytes, and blood pressure were evaluated at baseline, and 1, 3, 6, and 12 months after switching from HC to PLEN. At baseline, and after 1 and 12 months of PLEN, blood ACTH and cortisol (at 0800 h at fasting, and 30, 60, 90, 120, and 240 min after drug ingestion), and health-related quality of life (HRQoL), using 30-AddiQoL questionnaire, were evaluated. During PLEN, waist and serum lipid progressively decreased. After 12 months of PLEN, a significant difference was observed in waist circumference (P = 0.007), HbA1c (P = 0.002), total and LDL-cholesterol levels (P < 0.05). ACTH levels at 240 min and the area under the curve (AUC) were lower (P < 0.05) during PLEN than HC, while cortisol peaks and AUC were similar. 30-AddiQoL total score also improved (P = 0.04) during PLEN. In AD patients, PLEN reduces central adiposity, and improves glucose and metabolism parameters and HRQoL.

Role of presepsin for the evaluation of sepsis in the emergency department.

Sepsis, severe sepsis and septic shock are among the most common conditions handled in the emergency department (ED). According to new Sepsis Guidelines, early diagnosis and treatment are the keys to improve survival. Plasma C-reactive protein (CRP) and procalcitonin (PCT) levels, when associated with documented or suspected infection, are now part of the definitions of sepsis. Blood culture is the gold standard method for detecting microorganisms but it requires too much time for results to be known. Sensitive biomarkers are required for early diagnosis and as indexes of prognosis sepsis. CRP is one of the acute phase proteins synthesized by the liver: it has a great sensitivity but a very poor specificity for bacterial infections. Moreover, the evolution of sepsis does not correlate with CRP plasma changes. In recent years PCT has been widely used for sepsis differential diagnosis, because of its close correlation with infections, but it still retains some limitations and false positivity (such as in multiple trauma and burns). Soluble CD14 subtype (sCD14-ST), also known as presepsin, is a novel and promising biomarker that has been shown to increase significantly in patients with sepsis, in comparison to the healthy population. Studies pointed out the capability of this biomarker for diagnosing sepsis, assessing the severity of the disease and providing a prognostic evaluation of patient outcome. In this mini review we mainly focused on presepsin: we evaluate its diagnostic and prognostic roles in patients presenting to the ED with systemic inflammatory response syndrome (SIRS), suspected sepsis or septic shock.

Diagnostic and prognostic value of presepsin in the management of sepsis in the emergency department: a multicenter prospective study.

INTRODUCTION: Sepsis, severe sepsis and septic shock are common conditions with high mortality. Their early diagnosis in the Emergency Department (ED) is one of the keys to improving survival. Procalcitonin (PCT) has been used as a biomarker in septic patients but has limited specificity and can be elevated in other scenarios of systemic inflammatory response syndrome (SIRS). Soluble CD14 (sCD14) or presepsin is the free fragment of a glycoprotein expressed on monocytes and macrophages. Preliminary reports suggest that levels of presepsin are significantly higher in septic patients than in healthy individuals. The aim of this study is to investigate the diagnostic and prognostic value of presepsin compared to PCT in people presenting at the ED with SIRS and suspected sepsis or septic shock. METHODS: This study was conducted in two major hospitals in Turin, Italy. One hundred six patients presenting to the EDs with suspected sepsis or septic shock were included, and another eighty-three patients affected by SIRS, but with no clinical evidence of infection, were recruited as controls. Blood samples were collected at first medical evaluation and for some patients after 24 and 72 h. The samples were analyzed using the PATHFAST Presepsin assay for sCD14, and commercial kits were used for other determinations (for example, PCT). Definitive diagnosis and survival rates were obtained afterward by analysis of digital medical records. RESULTS: Elevated concentrations of presepsin at presentation were observed in septic patients compared to control patients. The same trend was observed for mean values of PCT. Higher values of presepsin were observed in septic patients at presentation (time 0). The diagnostic accuracy of PCT was generally higher, and areas under the curve (AUCs) were 0.875 for PCT and 0.701 for presepsin. Mean presepsin values were significantly higher in nonsurvivor septic patients (60-day mortality) than in survivors. No significant correlation was noted between PCT and survival. CONCLUSIONS: In our experience, presepsin was useful in the early diagnosis of infection in a complex population of patients with SIRS, sepsis, severe sepsis and septic shock who presented to the ED. Presepsin showed a significant prognostic value, and initial values were significantly correlated with in-hospital mortality of patients affected by sepsis, severe sepsis or septic shock.

Rule out of acute aortic dissection with plasma matrix metalloproteinase 8 in the emergency department.

INTRODUCTION: Matrix metalloproteinases (MMPs) are involved in aortic pathophysiology. Preliminary studies have detected increased plasma levels of MMP8 and MMP9 in patients with acute aortic dissection (AAD). However, the performance of plasma MMP8 and MMP9 for the diagnosis of AAD in the emergency department is at present unknown. METHODS: The levels of MMP8 and MMP9 were measured by ELISA on plasma samples obtained from 126 consecutive patients evaluated in the emergency department for suspected AAD. All patients were subjected to urgent computed tomography (CT) scan for final diagnosis. RESULTS: In the study cohort (N = 126), AAD was diagnosed in 52 patients and ruled out in 74 patients. Median plasma MMP8 levels were 36.4 (interquartile range 24.8 to 69.3) ng/ml in patients with AAD and 13.2 (8.1 to 31.8) ng/ml in patients receiving an alternative final diagnosis (P <0.0001). Median plasma MMP9 levels were 169.2 (93.0 to 261.8) ng/ml in patients with AAD and 80.5 (41.8 to 140.6) ng/ml in patients receiving an alternative final diagnosis (P = 0.001). The area under the curve (AUC) on receiver-operating characteristic (ROC) analysis of MMP8 and MMP9 for the diagnosis of AAD was respectively 0.75 and 0.70, as compared to 0.87 of D-dimer. At the cutoff of 3.6 ng/ml, plasma MMP8 had a sensitivity of 100.0% (95% CI, 93.2% to 100.0%) and a specificity of 9.5% (95% CI, 3.9% to 18.5%) and ruled out AAD in 5.6% of patients. Combination of plasma MMP8 with D-dimer increased the AUC on ROC analysis to 0.89. Presence of MMP8 <11.0 ng/ml and D-dimer <1.0 or <2.0 µg/ml provided a negative predictive value of 100% and ruled out AAD in 13.6% and 21.4% of patients respectively. CONCLUSIONS: Low levels of plasma MMP8 can rule out AAD in a minority of patients. Combination of plasma MMP8 and D-dimer at individually suboptimal cutoffs could safely rule out AAD in a substantial proportion of patients evaluated in the emergency department.