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Context The low-dose short synacthen test (LDSST) is recommended for patients with suspected central adrenal insufficiency (AI) if their basal serum cortisol (F) levels are not indicative of an intact hypothalamic–pituitary–adrenal (HPA) axis. Objective To evaluate diagnostic threshold for salivary F before and 30 min after administering 1 μg of synacthen, performed before 09:30 h. Design A cross-sectional study from 2014 to 2020. Setting A tertiary referral university hospital. Patients In this study, 174 patients with suspected AI, 37 with central AI and 137 adrenal sufficient (AS), were included. Main outcome measure The diagnostic accuracy (sensitivity (SE), specificity (SP)) of serum and salivary F levels measured, respectively, by chemiluminescence immunoassay and liquid chromatography-tandem mass spectrometry. Results Low basal serum or salivary F levels could predict AI. For the LDSST, the best ROC-calculated threshold for serum F to differentiate AI from AS was 427 nmol/L (SE 79%, SP 89%), serum F > 500 nmol/L reached SP 100%. A salivary F peak > 12.1 nmol/L after administering synacthen reached SE 95% and SP 84% for diagnosing central AI, indicating a conclusive reduction in the likelihood of AI. This ROC-calculated threshold for salivary F was similar to the 2.5th percentile of patients with a normal HPA axis, so it was considered sufficient to exclude AI. Considering AS those patients with salivary F > 12.1 nmol/L after LDSST, we could avoid unnecessary glucocorticoid treatment: 99/150 subjects (66%) had an inadequate serum F peak after synacthen, but salivary F was >12.1 nmol/L in 79 cases, who could, therefore, be considered AS. Conclusions Salivary F levels > 12.1 nmol/L after synacthen administration can indicate an intact HPA axis in patients with an incomplete serum F response, avoiding the need to start glucocorticoid replacement treatment.

Curing salts composed of mixtures of nitrates and nitrites are preservatives widely used in processed meats. Despite many desirable technological effects, their use in meat products has been linked to methemoglobinemia and the formation of nitrosamines. Therefore, an increasing “anti-nitrite feeling” has grown among meat consumers, who search for clean label products. In this view, the use of natural compounds as alternatives represents a challenge for the meat industry. Processing (including formulation and fermentation) induces chemical or physical changes of food matrix that can modify the bioaccessibility of nutrients and the formation of peptides, impacting on the real nutritional value of food. In this study we investigated the effect of nitrate/nitrite replacement with a combination of polyphenols, ascorbate, and nitrate-reducing microbial starter cultures on the bioaccessibility of fatty acids, the hydrolysis of proteins and the release of bioactive peptides after in vitro digestion. Moreover, digested salami formulations were investigated for their impacts on cell proliferation and genotoxicity in the human intestinal cellular model (HT-29 cell line). The results indicated that a replacement of synthetic nitrates/nitrites with natural additives can represent a promising strategy to develop innovative “clean label” salamis without negatively affecting their nutritional value.

Reducing the salt content in food is an important nutritional strategy for decreasing the risk of diet-related diseases. This strategy is particularly effective when applied to highly appreciated food having good nutritional characteristics, if it does not impact either upon sensory or nutritional properties of the final product. This work aimed at evaluating if the reduction of salt content by decreasing the brine soaking time modifies fatty acid and protein bioaccessibility and bioactive peptide formation in a 30-month-ripened Parmigiano Reggiano cheese (PRC). Hence, conventional and hyposodic PRC underwent in vitro static gastrointestinal digestion, and fatty acid and protein bioaccessibility were assessed. The release of peptide sequences during digestion was followed by LC–HRMS, and bioactive peptides were identified using a bioinformatic approach. At the end of digestion, fatty acid and protein bioaccessibility were similar in conventional and hyposodic PRC, but most of the bioactive peptides, mainly the ACE-inhibitors, were present in higher concentrations in the low-salt cheese. Considering that the sensory profiles were already evaluated as remarkably similar in conventional and hyposodic PRC, our results confirmed that shortening brine soaking time represents a promising strategy to reduce salt content in PRC.

Although epidemiological studies indicate a strong correlation between high sugar intake and metabolic diseases, the biological mechanisms underlying this link are still controversial. To further examine the modification and crosstalk occurring in enterocyte metabolism during sugar absorption, in this study we evaluate the diffusion and intestinal metabolism of glucose, fructose and sucrose, which were supplemented in equimolar concentration to Caco-2 cells grown on polyester membrane inserts. At different time points after supplementation, changes in metabolite concentration were evaluated in the apical and basolateral chambers by nuclear magnetic resonance (NMR) and gas-chromatography (GC). Sucrose was only minimally hydrolyzed by Caco-2 cells. Upon supplementation, we observed a faster uptake of fructose than glucose, the pentose sugar being also faster catabolized. Monosaccharide absorption was concomitant to the synthesis/transport of other metabolites, which occurred differently in glucose and fructose supplemented cells. Our results confirm the prominent role of intestinal cells in fructose metabolism and clearance after absorption, representing a further step forward in the understanding of the role of dietary sugars. Future research, including targeted analysis on specific transporters/enzymes and the use of labeled substrates, will be helpful to confirm the present results and their interpretation.

Success of adrenal vein sampling (AVS) is verified by the selectivity index (SI), i.e., by a step-up of cortisol levels between the adrenal vein and the infrarenal inferior vena cava samples, beyond a given cut-off. We tested the hypothesis that androstenedione, metanephrine, and normetanephrine, which have higher gradients than cortisol, could increase the rate of AVS studies judged to be bilaterally successful and usable for the clinical decision making. We prospectively compared within-patient, head-to-head, the selectivity index of androstenedione (SIA), metanephrine (SIM), and normetanephrine (SINM), and cortisol (SIC) in consecutive hypertensive patients with primary aldosteronism submitted to AVS. Main outcome measures were rate of bilateral success, SI values, and identification of unilateral PA. We recruited 136 patients (55 + 10 years, 35% women). Compared to the SIC, the SIA values were 3.5-fold higher bilaterally, and the SIM values were 7-fold and 4.4-fold higher on the right and the left side, respectively. With the SIA and the SIM the rate of bilaterally successful AVS increased by 14% and 15%, respectively without impairing the identification of unilateral PA. We concluded that androstenedione and metanephrine outperformed cortisol for ascertaining AVS success, thus increasing the AVS studies useable for the clinical decision making.

Introduction Lipemia in samples can cause analytical errors in coagulation tests using photometric assays. To define the level of this interference, some studies assessed lipemic interferences by in vitro 'spiking' of different types of lipids obtaining interesting information, but spiked samples do not represent a real-world situation as natively lipemic samples do. Methods A total of 101 samples flagged as 'lipemic' by a Sysmex CS-5100 coagulometer were analyzed for PT, aPTT, fibrinogen Clauss assay, antithrombin activity, D-dimer concentration, before and after a double high-speed centrifugation procedure to reduce lipemic interference. We evaluated using Bland-Altman test if high-speed centrifugation and retesting are justified, considering that's a resource-consuming procedure; when a statistically significant difference was found, quality specification for imprecision was considered and compared to the observed delta. Results Statistically significant differences were found for PT, antithrombin activity and fibrinogen. Considering the Bland-Altman plot, fibrinogen results were split into two groups, and statistically significant difference was confirmed only for samples >2 g/L. Conclusions For PT and antithrombin activity a mean percentual difference between the two determinations lower than within-subject biologic variation and one of the Fraser's quality specifications can be considered as a confounding 'noise' factor that is neither analytically nor clinically relevant. If the instrument determines a result on the first run, for PT, aPTT, D-dimer concentration and antithrombin activity tests, the double plasma high-speed centrifugation is unnecessary. It is instead necessary if fibrinogen >2 g/L or if the instrument cannot determine a result on the first run.

Aims A blood pressure (BP)-independent metabolic shift towards a catabolic state upon high sodium (Na+) diet, ultimately favouring body fluid preservation, has recently been described in pre-clinical controlled settings. We sought to investigate the real-life impact of high Na+ intake on measures of renal Na+/water handling and metabolic signatures, as surrogates for cardiovascular risk, in hypertensive patients. Methods and results We analysed clinical and biochemical data from 766 consecutive patients with essential hypertension, collected at the time of screening for secondary causes. The systematic screening protocol included 24 h urine (24 h-u-) collection on usual diet and avoidance of renin–angiotensin–aldosterone system-confounding medications. Urinary 24 h-Na+ excretion, used to define classes of Na+ intake (low ≤2.3 g/day; medium 2.3–5 g/day; high >5 g/day), was an independent predictor of glomerular filtration rate after correction for age, sex, BP, BMI, aldosterone, and potassium excretion [P = 0.001; low: 94.1 (69.9–118.8) vs. high: 127.5 (108.3–147.8) mL/min/1.73 m2]. Renal Na+ and water handling diverged, with higher fractional excretion of Na+ and lower fractional excretion of water in those with evidence of high Na+ intake [FENa: low 0.39% (0.30–0.47) vs. high 0.81% (0.73–0.98), P, Graphical Abstract

Coenzyme Q10 (CoQ10) is a lipid-soluble molecule with a dual role: it transfers electrons in the mitochondrial transport chain by promoting the transmembrane potential exploited by the ATPase to synthesize ATP and, in its reduced form, is a membrane antioxidant. Since the high CoQ10 hydrophobicity hinders its bioavailability, several formulations have been developed to facilitate its cellular uptake. In this work, we studied the bioenergetic and antioxidant effects in I407 and H9c2 cells of a CoQ10 phytosome formulation (UBIQSOME®, UBQ). We investigated the cellular and mitochondrial content of CoQ10 and its redox state after incubation with UBQ. We studied different bioenergetic parameters, such as oxygen consumption, ATP content and mitochondrial potential. Moreover, we evaluated the effects of CoQ10 incubation on oxidative stress, membrane lipid peroxidation and ferroptosis and highlighted the connection between the intracellular concentration of CoQ10 and its antioxidant potency. Finally, we focused on the cellular mechanism that regulates UBQ internalization. We showed that the cell lines used in this work share the same uptake mechanism for UBQ, although the intestinal cell line was less efficient. Given the limitations of an in vitro model, the latter result supports that intestinal absorption is a critical step for the oral administration of Coenzyme Q10 formulations.

Impaired cortisol rhythm is a characteristic feature of Cushing's Syndrome, nevertheless late night salivary cortisol (LNSC) is not suitable to detect subclinical hypercortisolism in patients with adrenal incidentaloma (AI). We studied daily salivary cortisol (F) and cortisone (E) rhythm in patients with AI.Six saliva samples were collected from awakening to night in 106 patients with AI and 40 controls. F and E were measured with LC-MS/MS and daily F exposure was calculated with the area under the curve (AUC).Patients with serum cortisol after dexamethasone suppression test (DST) 50 nmol/L showed higher morning F (15.5 ± 14.5 vs. 8.6 ± 5.5 nmol/L, p = 0.001), suppressed corticotropin levels (76 vs. 35%, p 0.001) and increased daily F exposure (3795 ± 1716 vs. 2898 ± 1478, p = 0.012), especially in the morning (2035 ± 1267 vs. 1365 ± 777, p = 0.003), otherwise LNSC levels were similar. Salivary E and AUC levels were higher in patients with DST 50 nmol/L. AUC was not correlated with urinary cortisol levels or adenoma size. F and E levels were similar among patients with unilateral or bilateral adenoma, or considering the presence of hypertension, dyslipidemia, diabetes, or cardiovascular events.Daily cortisol exposure, evaluated with AUC from multiple saliva collections, is increased in AI patients with serum cortisol 50 nmol/L after DST, especially in the morning, leading to reduced corticotropin levels. Cortisol rhythm is preserved in patients with AI, remarking that LNSC is not a screening test for subclinical hypercortisolism.

Background & Aims Relative adrenal insufficiency (RAI) is defined by insufficient production of cortisol relative to organ demand. RAI is observed frequently in hospitalized patients with cirrhosis, but there is disagreement over the clinical effects of RAI in these patients. We evaluated the prevalence and the clinical effects of RAI in hospitalized patients with cirrhosis. Methods We performed a prospective study of 160 patients admitted to a hospital in Italy for acute decompensation of cirrhosis from May 2011 through September 2016. Patients were followed up until death, liver transplantation, or a maximum of 90 days. Serum and salivary levels of cortisol were measured before and after a 1-hour Short Synacthen Test. A diagnosis of RAI was given to patients with an increase in serum cortisol of less than 9 μg/dL, after Synacthen administration, in patients with baseline serum levels of cortisol less than 35 μg/dL. We collected blood samples before the Synacthen test and analyzed them for blood cell counts, liver and renal function, levels of C-reactive protein, and lipid profiles (total cholesterol, high-density lipoprotein cholesterol, apolipoprotein-A1). Results A diagnosis of RAI was made for 78 patients (49%). Age (odds ratio [OR], 0.95; P = .030), number of leukocytes (OR, 3.10; P = .006), and levels of high-density lipoprotein cholesterol (OR, 0.30; P = .039) were associated independently with RAI. Patients with RAI had a significantly higher risk of developing bacterial infections (hazard ratio [HR], 1.60; P = .038), sepsis (HR, 2.95; P = .001), septic shock (HR, 4.94; P = .038), new organ failures (HR, 2.45; P = .014), and acute-on-chronic liver failure (HR, 2.27; P = .037) than patients without RAI. RAI was associated independently with death within 90 days of diagnosis (subdistribution HR, 4.83; P = .001). Patients with RAI and mild renal dysfunction or hepatic encephalopathy had no significant difference in cumulative incidence of 28-day mortality vs patients with acute-on-chronic liver failure grade 1 (25% vs 22%). Conclusions We found RAI to occur in almost half of patients admitted to a hospital for acute decompensation of cirrhosis. RAI was associated with a deficit of substrates for steroidogenesis and an increase in markers of inflammation. Patients with RAI have a high risk of developing sepsis, septic shock, organ failure, and death within 90 days. RAI has similar prognostic value to nonrenal organ failures.

Background: A metabolic shift in energy expenditure toward a catabolic state upon high sodium (Na + ) diet, ultimately favouring endogenous water accrual and body fluid preservation, has recently been described in a rodent model. Objective: To investigate the impact of high Na + intake on renal Na + /water handling and metabolic signatures in a large real-life cohort of patients. Methods and Results: We analysed biochemical data from 767 hypertensive patients (429 males, 55.9%; age 47 ± 13 years; BMI 25.6 [23.0-29.0] kg/m 2 ) in washout from drugs affecting the renin-angiotensin-aldosterone system at the time of screening for secondary causes and with a conclusive diagnosis of essential hypertension (2012-2017). Classes of Na + intake ( L ow ≤2.3g/d; M edium 2.3-5g/d; H igh >5g/d) were defined based on urinary 24h Na + excretion (uNaV). The fractional excretion (FE) of Na + increased with increasing Na + intake (L: 0.39% [0.30-0.47] vs H: 0.81% [0.73-0.98], p < 0.001), while FE of free water decreased (L: 1.13% [0.73-1.72] vs H: 0.89% [0.69-1.12], p = 0.015). uNaV was an independent predictor of glomerular filtration rate (GFR, estimated by creatinine clearance; H: 130 ± 33 vs M: 111 ± 52 and L: 96 ± 39 ml/min/1.73m 2 ; p < 0.001 for comparisons) after correction for age, sex, BMI, renin, aldosterone and uKV (p = 0.001). This resulted in a marked increase in the 24h tubular Na + reabsorption and, accordingly, in the estimated energy expenditure (Δ H vs L= 18 [12-24] kcal/d, p < 0.001). At non-targeted LC/MS metabolomics (n = 67), metabolites increased (p < 0.05) in H vs L Na + intake mostly entailed intermediates or end products of the urea cycle and products of protein catabolism. Urinary 24h cortisol excretion, as a potential determinant of catabolism, increased with Na + intake (n = 137; L: 63 [36-72] nmol, M: 60 [47-86] nmol, H: 86 [75-139] nmol; p < 0.001), but its strongest predictor at multivariate regression analysis was GFR (p = 0.001). Conclusions: Kidneys can effectively dissociate Na + and water handling upon high Na + diet, at the cost of glomerular hyperfiltration, enhanced glomerulotubular balance, increased tubular energy expenditure and, ultimately, protein catabolism from endogenous or excess exogenous sources. This has broad implications on global cardiovascular risk.

Dexamethasone Suppression Test (DST), recommended for Cushing’s Syndrome (CS) diagnosis, explores the pituitary feedback to glucocorticoids. Its diagnostic accuracy could be affected by dexamethasone bioavailability, and therefore, we have developed and validated a dexamethasone threshold after 1-mg DST. We studied 200 subjects: 125 patients were considered retrospectively and 75 were enrolled prospectively as the validation cohort. Serum dexamethasone, Late Night Salivary Cortisol (LNSC), and Urinary Free Cortisol (UFC) were measured with LC–MS/MS. Normal LNSC and UFC levels were used to exclude CS. The lower 2.5th percentile of dexamethasone distribution in non-CS patients with cortisol ≤ 50 nmol/L after 1-mg DST was used as threshold. 16 patients were CS and 184 non-CS (108 adrenal incidentaloma and 76 excluded CS); 4.5 nmol/L resulted the calculated threshold. Cortisol after 1-mg DST confirmed high sensitivity (100% at 50 nmol/L cut-off) and moderate–low specificity (63%, increased to 91% at 138 nmol/L) to diagnose CS in the whole cohort of patients. We could reduce the number of false-positive results (from 10 to 6 and from 7 to 4 in AI and excluded CS) considering adequate dexamethasone levels. Dexamethasone levels were not affected by hypercortisolism, age, gender, smoke, weight, and creatinine. 6% of non-CS patients did not achieve adequate dexamethasone levels (40% of tests with serum cortisol > 138 nmol/L after 1-mg DST). We developed and validated the routine dexamethasone measurement during 1-mg DST: it is independent from patient’s clinical presentation, and it should be used to increase the specificity of serum cortisol levels.

Introduction and Aim The purpose of replacement therapy in adrenal insufficiency (AI) is mimicking endogenous cortisol levels as closely as possible: dual release hydrocortisone (DR-HC) has been introduced to replicate the circadian cortisol rhythm. Multiple daily saliva collections could be used to assess the cortisol rhythm during real life: our aim was to study the salivary cortisol profile in AI. Materials and Methods We prospectively evaluated, in an observational study, 18 adult outpatients with AI (11 primary and 7 secondary AI), switched from conventional treatment (conv-HC, 25 mg/day) to the same dose of DR-HC. We collected six samples of saliva in a day, measuring cortisol (F) and cortisone (E) with LC-MS/MS. Forty-three matched healthy subjects served as controls. Results F levels were similar in the morning (and higher than controls) in patients treated with conv-HC or DR-HC; otherwise F levels and exposure were lower in the afternoon and evening in patients with DR-HC, achieving a cortisol profile closer to healthy controls. Daily cortisol exposure, measured with area under the curve, was lower with DR-HC. Morning F and E presented sensitivity and specificity >90% to diagnose AI (respectively threshold of 3 and 9.45 nmol/L). Total cholesterol and HbA1c levels reduced with DR-HC. Conclusions Salivary cortisol daily curve could be used as a new tool to assess the cortisol profiles in patients treated with conv-HC and DR-HC. A lower daily cortisol exposure was achieved with DR-HC (despite the same HC dose), especially in the afternoon-evening.

Medical treatment is increasingly used in patients with Cushing’s syndrome (CS). Metyrapone (MET) is an inhibitor of 11β-hydroxylase: retrospective studies reported a decrease of cortisol secretion in 50% of cases. We evaluated the effectiveness of MET in an observational study, considering the normalization of urinary-free cortisol (UFC) and late-night salivary cortisol (LNSC) levels. We enrolled 31 patients with CS, treated with MET for at least 1 month (16 for primary treatment and 15 after surgical failure). A planned dose-titration regimen considering baseline UFC levels was adopted; MET dose was uptitrated until UFC normalization, surgery, or side effect occurrence. UFC and LNSC levels were routinely measured by liquid chromatography–tandem mass spectrometry. Patients were treated with a median dose of 1000 mg for 9 months. UFC and LNSC decreased quickly after the first month of treatment (−67 and −57% from baseline), with sustained UFC normalization up to 12 and 24 months (in 13 and 6 patients, respectively). UFC and LNSC normalized later (after 3–6 months) in patients with severe hypercortisolism (>5-fold baseline UFC). Regarding the last visit, 70 and 37% of patients normalized UFC and LNSC, respectively. Body weight reduction (−4 kg) was observed after UFC normalization. Severe side effects were not reported, half of the female patients complained of hirsutism, and blood pressure was not increased. MET therapy is a rapid-onset, long-term effective, and safe medical treatment in CS patients, achieving UFC normalization (in 70% of patients) more than cortisol rhythm recovery (in 37% of subjects).

Background Measurement uncertainty (MU) estimation has been introduced by ISO 15189 for the accreditation of clinical laboratories. Although MU reporting is not required, its inclusion in medical reports is of potential assistance to physicians in results interpretation. Methods MU reporting was evaluated with respect to different test purposes, namely comparison with reference intervals (RI), patient monitoring or comparison with clinical decision limits. Clinical Biochemistry, Hematology, Coagulation and Clinical Immunology measurands were used as examples. Assuming Gaussian RI distribution, the probability of retesting due to MU was determined by simulations. Significant MU variations were compared against the reference change value (RCV) and clinical decision limits. Results Three potential scenarios emerged for RI. For 12 measurands, depending on the MU interval, a potential change in results interpretation was found only for Sodium and S-Protein. On considering only the results within RI, simulations confirmed that up to 8.6% of MU intervals encompassed the RI limits, thus potentially leading to retesting. For tests used in patient monitoring, significant MU variations were comparable to those calculated by RCV, with the exception of CEA. For tests results evaluated with respect to clinical decision limits, on including MU, the clinical interpretation may be improved (e.g. for tPSA). Conclusion The findings made in the present study, which considers real MU data and hypothetical results obtained for a series of measurands, support the concept that MU may aid the physician's interpretation thus ensuring reliable clinical decision making.

Background The ISO 15189:2012 describes the requirements for quality and competence, which are specific for all sectors of a medical laboratory. Laboratory-developed methods, standard methods used outside their intended scope and validated methods subsequently modified shall be validated. The main objective of validation of an examination procedure (EP) is to demonstrate its fitness-for-purpose. The aim of the present study is to illustrate a model to validate laboratory-developed methods in agreement with the ISO 15189:2012 requirement, through the example of salivary cortisol (sF) measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods The proposed validation model for a laboratory developed method includes 6 steps: 1) analysis of available scientific documentation pertinent to specific EP; 2) evaluation of EP intended use; 3) identification of performance characteristics to evaluate; 4) definition of experimental procedure; 5) identification of acceptability criterion for results evaluation; 6) preparation of a validation plan; 7) production of a validation certificate. Results sF is an EP sampled during the accreditation audit and resulted in compliance with the Standard. A structured procedure to certify that the achieved performance specifications are appropriate for the purpose of the test is therefore necessary. Conclusions A high competence is required for laboratory professionals in order to guarantee that the validated quality specifications have a positive impact on patients' management. The ISO 15189 accreditation is an important tool that allows demonstrating the value of a medical laboratory in the clinical context.

For identification of potentially surgically curable primary aldosteronism, guidelines recommend use of adrenal vein sampling (AVS) that requires selective catheterization of both adrenal veins as verified by using the cortisol-derived selectivity index. Unfortunately, bilaterally selective studies are not obtained under unstimulated conditions in a proportion of the cases ranging between 15% and 50% depending on the cutoff used. We therefore investigated whether 17-α-hydroxyprogesterone and androstenedione, which showed a higher step-up between adrenal vein and inferior vena cava blood than cortisol, can ascertain selectivity when cortisol failed to do so. We prospectively recruited 32 hypertensive patients with confirmed primary aldosteronism, who underwent bilaterally simultaneous sampling without cosyntropin stimulation and with the same predefined AVS protocol. All were consecutively selected because of a cortisol-based selectivity index P

Background: One of the most frequent reasons to determine total bile acids (TBA) in a routine laboratory is for the diagnosis and management of intrahepatic cholestasis of pregnancy (ICP). The analytical methods that can be carried out for TBA determination are enzymatic and immunometric methods. However, the enzymatic methods suffer of significant cross-reactivity with ursodeoxycholic acid (UDCA), the principal drug administered in the ICP management. The aim of this paper is to evaluate the clinical utility of a new fully-automated chemiluminescence immunoassay (CLIA) to measure cholylglycine (CG) in the ICP. Methods: Two cohorts of patients with a singleton pregnancy, in the third trimester, were retrospectively recruited. The first cohort (controls, n=56, 21–48 years) was represented by patient hospitalized, for a different reason respect to ICP. The second cohort (ICP women, n=32, 22–45 years) was composed of women with a final diagnosis of ICP, before the treatment with UDCA. Results: Intra-assay and intra-laboratory imprecisions were less than 6%. The 95% CI of CG in the control cohort were 0.9–9.1 µmol/L. The levels of CG in the ICP women cohort ranged from 2.3 to 57.4 µmol/L. The optimal cut-off of CG for ICP was 5.5 µmol/L (sensitivity of 84% and specificity of 92%). Conclusions: CG determination by CLIA method has demonstrated a reliability from the analytical point of view. The obtained diagnostic accuracy for ICP was similar to the traditional assays carried out for TBA determination. Moreover, the turnaround time of CG analysis allows a rapidly answer to clinical suspects of cholestasis in pregnancies ensuring the patient-foetus safety.

Objectives Although, due to its high specificity and sensitivity, LC-MS/MS is an efficient technique for the routine determination of immunosuppressants in whole blood, it involves time-consuming manual sample preparation. The aim of the present study was therefore to develop an automated sample-preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by LC-MS/MS using a liquid handling platform. Methods Six-level commercially available blood calibrators were used for assay development, while four quality control materials and three blood samples from patients under immunosuppressant treatment were employed for the evaluation of imprecision. Barcode reading, sample re-suspension, transfer of whole blood samples into 96-well plates, addition of internal standard solution, mixing, and protein precipitation were performed with a liquid handling platform. After plate filtration, the deproteinised supernatants were submitted for SPE on-line. The only manual steps in the entire process were de-capping of the tubes, and transfer of the well plates to the HPLC autosampler. Results Calibration curves were linear throughout the selected ranges. The imprecision and accuracy data for all analytes were highly satisfactory. The agreement between the results obtained with manual and those obtained with automated sample preparation was optimal ( n = 390, r = 0.96). In daily routine (100 patient samples) the typical overall total turnaround time was less than 6 h. Conclusions Our findings indicate that the proposed analytical system is suitable for routine analysis, since it is straightforward and precise. Furthermore, it incurs less manual workload and less risk of error in the quantification of whole blood immunosuppressant concentrations than conventional methods.

Background:The present study was prompted by the ISO 15189 requirements that medical laboratories should estimate measurement uncertainty (MU).Methods:The method used to estimate MU included the: a) identification of quantitative tests, b) classification of tests in relation to their clinical purpose, and c) identification of criteria to estimate the different MU components. Imprecision was estimated using long-term internal quality control (IQC) results of the year 2016, while external quality assessment schemes (EQAs) results obtained in the period 2015–2016 were used to estimate bias and bias uncertainty.Results:A total of 263 measurement procedures (MPs) were analyzed. On the basis of test purpose, in 51 MPs imprecision only was used to estimate MU; in the remaining MPs, the bias component was not estimable for 22 MPs because EQAs results did not provide reliable statistics. For a total of 28 MPs, two or more MU values were calculated on the basis of analyte concentration levels. Overall, results showed that uncertainty of bias is a minor factor contributing to MU, the bias component being the most relevant contributor to all the studied sample matrices.Conclusions:The model chosen for MU estimation allowed us to derive a standardized approach for bias calculation, with respect to the fitness-for-purpose of test results. Measurement uncertainty estimation could readily be implemented in medical laboratories as a useful tool in monitoring the analytical quality of test results since they are calculated using a combination of both the long-term imprecision IQC results and bias, on the basis of EQAs results.

Patients with adrenal incidentaloma present a wide range of cortisol secretion, which is not always properly defined by first-line screening tests recommended to rule out Cushing's syndrome (CS), such as 1-mg dexamethasone suppression test (1-mg DST), late night salivary cortisol (LNSC), or 24-h urinary free cortisol (UFC). Therefore, we examined the diagnostic performance of each screening test in patients with adrenal incidentaloma.In a series of 164 consecutive patients with adrenal incidentaloma, we measured serum cortisol after 1-mg DST, LNSC, and UFC (with LC-MS/MS). Medical history was investigated for cardiovascular events (CVE) in a subgroup of 93 patients with at least 2 years of follow-up.Serum cortisol50 nmol/L after 1-mg DST presented the highest sensitivity (100%) to rule out CS, despite a low specificity (62%). UFC 170 nmol/24 h achieved the highest diagnostic accuracy (sensitivity 98%, specificity 91%, and negative/positive likelihood ratios of 0.02/10.83, respectively). The prevalence of CVE was higher in patients with non-suppressed cortisol after 1-mg DST and high UFC levels (p = 0.018). Traditional cardiovascular risk factors (hypertension, diabetes mellitus, dyslipidemia, BMI 30 kg/mThe 1-mg DST at its lowest threshold presented high sensitivity in identifying CS, but its low specificity encourages us to consider UFC levels, measured with LC-MS/MS, to reduce false-positive test results. High UFC levels could also be considered as markers to stratify cardiovascular risk in patients with adrenal incidentaloma.

Background The International Standard ISO 15189 is recognized as a valuable guide in ensuring high quality clinical laboratory services and promoting the harmonization of accreditation programmes in laboratory medicine. Examination procedures must be verified in order to guarantee that their performance characteristics are congruent with the intended scope of the test. The aim of the present study was to propose a practice model for implementing procedures employed for the verification of validated examination procedures already used for at least 2 years in our laboratory, in agreement with the ISO 15189 requirement at the Section 5.5.1.2. Methods In order to identify the operative procedure to be used, approved documents were identified, together with the definition of performance characteristics to be evaluated for the different methods; the examination procedures used in laboratory were analyzed and checked for performance specifications reported by manufacturers. Then, operative flow charts were identified to compare the laboratory performance characteristics with those declared by manufacturers. Results The choice of performance characteristics for verification was based on approved documents used as guidance, and the specific purpose tests undertaken, a consideration being made of: imprecision and trueness for quantitative methods; diagnostic accuracy for qualitative methods; imprecision together with diagnostic accuracy for semi-quantitative methods. Conclusions The described approach, balancing technological possibilities, risks and costs and assuring the compliance of the fundamental component of result accuracy, appears promising as an easily applicable and flexible procedure helping laboratories to comply with the ISO 15189 requirements.

The communication of laboratory results to physicians and the quality of reports represent fundamental requirements of the post-analytical phase in order to assure the right interpretation and utilization of laboratory information. Accordingly, the International Standard for clinical laboratories accreditation (ISO 15189) requires that "laboratory reports shall include the information necessary for the interpretation of the examination results". Measurement uncertainty (MU) is an inherent property of any quantitative measurement result which express the lack of knowledge of the true value and quantify the uncertainty of a result, incorporating the factors known to influence it. Even if the MU is not included in the report attributes of ISO 15189 and cannot be considered a post-analytical requirement, it is suggested as an information which should facilitate an appropriate interpretation of quantitative results (quantity values). Therefore, MU has two intended uses: for laboratory professionals, it gives information about the quality of measurements, providing evidence of the compliance with analytical performance characteristics; for physicians (and patients) it may help in interpretation of measurement results, especially when values are compared with reference intervals or clinical decision limits, providing objective information. Here we describe the way that MU should be added to laboratory reports in order to facilitate the interpretation of laboratory results and connecting efforts performed within laboratory to provide more accurate and reliable results with a more objective tool for their interpretation by physicians.

A relationship between natriuretic peptides, reproduction, and the elderly has been known for some time. To investigate the relation between Nt-pro C-type natriuretic peptide (CNP) and reproduction, a group of 71 elderly men and women were studied. In particular, the relation between prolactin (PRL) and Nt-proCNP was examined. Nt-proBNP (N-terminal Brain NP), pro Atrial (ANP)(1-98) (Atrial NP), Nt-proCNP, luteinizing and follicle-stimulating hormones, PRL, progesterone, and 17-estradiol were measured in 51 (23 males/28 females) hypertensive patients. A total of 20 normotensive elderly subjects formed the control group. The hypertensive patients (TH) were divided into subgroups, accordingly to their main other clinical diagnosis, namely Diabetes, Hypertension, Peripheral Arterial Oc- clusive Disease. A direct correlation was observed between Nt-proCNP and PRL in all subjects, patients, and healthy volunteers (r 0.56; P 0.001). An inverse correlation between Nt-proCNP and follicle-stimulating hor- mones was observed in TH patients (r 0.55; P 0.05). proANP(1- 98) and Nt-proBNP correlated with PRL respectively in peripheral arterial occlusive disease (r 0.57; P 0.05) and in TH (r 0.52; P 0.05). A direct correlation was found between the latter and luteinizing hormone in Diabetes (r 0.64; P 0.05). We demonstrate, for the first time, a correlation between Nt-proCNP and PRL in sick and healthy elderly subjects. Also proANP(1-98) and Nt-proBNP appeared to coregu- lated with PRL.

Background: Quality indicators (QIs) used as performance measurements are an effective tool in accurately estimating quality, identifying problems that may need to be addressed, and monitoring the processes over time. In Laboratory Medicine, QIs should cover all steps of the testing process, as error studies have confirmed that most errors occur in the pre- and post-analytical phase of testing. Aim of the present study is to provide preliminary results on QIs and related performance criteria in the post-analytical phase. Methods: This work was conducted according to a previously described study design based on the voluntary participation of clinical laboratories in the project on QIs of the Working Group “Laboratory Errors and Patient Safety” (WG-LEPS) of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Results: Overall, data collected highlighted an improvement or stability in performances over time for all reported indicators thus demonstrating that the use of QIs is effective in the quality improvement strategy. Moreover, QIs data are an important source for defining the state-of-the-art concerning the error rate in the total testing process. The definition of performance specifications based on the state-of-the-art, as suggested by consensus documents, is a valuable benchmark point in evaluating the performance of each laboratory. Conclusions: Laboratory tests play a relevant role in the monitoring and evaluation of the efficacy of patient outcome thus assisting clinicians in decision-making. Laboratory performance evaluation is therefore crucial to providing patients with safe, effective and efficient care.

Objectives: To examine an immunoassay for measuring free IGF-I in a saliva specimen (free sIGF-I) and to study the levels in relation to the training conditions comparing young athletes and sedentary females. Design and methods: The analysis was carried out by modifying a commercial kit for plasma matrix to measure the free sIGF-I. The plasma free and total IGF-I fractions, hGH and salivary total proteins were also measured. Saliva and blood specimens were collected from 15 well-trained young female volleyball athletes and from a control group of 14 young sedentary females. Results: The calibration curve to assay free sIGF-I covered the range 0.05–5.00 μg/L. The detection limit was 0.07 μg/L. The within-run and between-run imprecision CVs were 10% and 13% respectively. The average recovery was 88%. Free sIGF-I, measured in 15 athletes and in 14 young sedentary females, was 0.10 ± 0.03 and 0.20 ± 0.05 μg/L respectively ( p Conclusions: There were decreased levels of free sIGF-I in well-trained athletes, compared with sedentary females. This decrease could be related to a greater tissue requirement by the active muscle subjected to intense exercise for several days.

The Endocrine Society recommends late-night salivary cortisol (LNS-F) as a first-line screening test for Cushing's syndrome (CS). In the parotid gland, 11β-hydroxysteroid dehydrogenase type 2 inactivates cortisol (F) to cortisone (E), a known source of interference in the more frequently used immunoassays. A highly specific method is mandatory in determining salivary F and E: it is widely accepted that liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the best available technique for this purpose.A LC-MS/MS method with SPE of saliva samples was developed and validated. Appropriate awakening and bedtime reference ranges were established. The diagnostic performance for F, E and the ratio at bedtime was evaluated in 25 cases of CS.The method was linear, with up to 55.4 nmol/L and 51.0 nmol/L, LLOQ of 0.51 nmol/L and 0.55 nmol/L, for F and E, respectively. Within-run and between-run imprecisions were10% for both analytes. No ion suppression was observed. A cut-off of 2.4 nmol/L for LNS-F yielded a sensitivity of 100% and a specificity of 98% in the diagnosis of CS.The analytical performance of this method justifies its introduction into clinical practice, thus allowing clinicians the opportunity to further investigate CS and other endocrine diseases.

A functional and basic method for the quantitative analysis of urine cortisol ( F ) and cortisone ( E ) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists ( F : 136 ± 93 nmol/l versus 67 ± 50 nmol/l ( p E : 797 ± 400 nmol/l versus 408 ± 252 nmol/l ( p F / E ratio was 0.18 ± 0.11 versus 0.16 ± 0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11 ± 0.07) and cyclist individuals at rest ( p

As initial screening for Cushing's syndrome (CS), The Endocrine Society guidelines recommend one of the following: the 1-mg dexamethasone suppression test (DST) or late-night salivary cortisol (LNSC) or urinary free cortisol (UFC) measurement. We examined the diagnostic performance of the above-mentioned tests in a series of patients.We retrospectively analyzed 137 patients with clinical conditions suggestive of hypercortisolism: 38 with confirmed CS diagnosis and 99 without (termed non-CS). UFC was measured by liquid chromatography tandem-mass spectrometry, whereas LNSC by the radioimmunometric method and serum cortisol were measured by a chemiluminescence immunoassay.Comparing CS vs non-CS, a cutoff of 138 nmol/L after 1-mg DST revealed the best specificity (SP; 97%), whereas the 50-nmol/L cutoff confirmed the best sensitivity (SE; 100%); the SE and SP for LNSC greater than 14.46 nmol/L were, respectively, 84% and 89%, whereas the SE and SP for UFC greater than 170 nmol per 24 hours, they were 97% and 91%. Overall, UFC revealed both a combined higher positive and a lower negative likelihood ratio among first-line tests (respectively 10.7 and 0.03). Computing a receiver-operating curve -contrast analysis to compare the power of each single test with that of the others, alone or combined (DST+LNSC, DST+UFC, and LNSC+UFC) or with that of all the tests together (DST+LNSC+UFC), the UFC assay was at least as good as all the other possible combinations.Measuring UFC by liquid chromatography tandem-mass spectrometry achieves the best accuracy in diagnosing CS among patients presenting with suspected hypercortisolism.

Patient safety, defined by the Institute of Medicine (IOM) as “the prevention of harm to patients”, is the ultimate goal of medical laboratory services. Although it has been demonstrated that the error rate in medical laboratories is very low compared to the billions of tests daily performed, and that most of these errors rarely become unfavourable events, adverse outcome may occur and may also become the object of daily news. Risk management principles should therefore be considered as integral parts of laboratory in assuring quality and safety, so that they have become actual requirements (4.14.6) of International Organization for Standardization (ISO) 15189:2012, the international standard for accreditation of medical laboratories. Risk is no longer thought in negative sense, but becomes a tool to identify improvement opportunities and preventing negative outcomes. ISO/TS 22367 and some Clinical and Laboratory Standards Institute (CLSI) guidelines (i.e., EP18-A2 and EP23-A) introduce risk management principles and they can be used for driving application of ISO 15189 as a system for reducing laboratory error and improving patient safety. Laboratory goals, organization within which the laboratory operates along with available resources, are all key elements for selecting the technique to be used for risk assessment. However, although different approaches have been suggested, failure modes and effects analysis (FMEA) is the most commonly applied. There are currently few reports on active use of risk management tools in medical laboratories. Some of these are limited to risk identification and estimation steps, whilst others report corrective measures without verifying their effectiveness. Risk management, however, is a process consisting of three main phases (i.e., risk identification, estimation and control), and should hence embrace all its phases to achieve its goal, that is ensuring patient safety. This paper summarizes the principles of risk management process applied to clinical laboratory and discusses some practical applications available in the literature.

The International Standard Organization (ISO) 15189 accreditation has raised new enthusiasm among laboratory professionals. On the one hand, it satisfies the desire to undertake a pathway attesting the high degree of quality of laboratory performance measured according to the important role of Laboratory Medicine throughout patient care. On the other hand, it allows demonstrating how the competence of each staff member produces a great impact on the level of achieved performance. However, laboratory staff must recognize that application of ISO 15189 needs great commitment in terms of both time and efforts but, before all, in terms of ability to express competence. The application of ISO 15189 is needed for collecting experiences from different laboratories and identifying the most suitable approaches to comply with requirements and oversee challenges. A close cooperation among national Scientific Societies, as expression of medical laboratories, manufacturers of in vitro diagnostic medical devices (IVD) and national Accreditation Body is needed to guarantee success of ISO 15189 Accreditation.

The latest revision of ISO 15189 standard in 2012 is now universally considered as the most important document for accreditation of medical laboratories. This standard is aimed to verify and validate examination procedures (EPs). Verification is “provision of objective evidence that a given item fulfils specified requirements”, while validation is “verification, where the specified requirements are adequate for the intended use”. When used without modification, validated EPs shall be verified, whilst non-standard methods, home brew methods, validated methods which have been modified or are being used outside their intended scope shall be validated before being introduced into clinical and laboratory practice. In order to fulfil the above requirements, three different approaches were used for: (I) verification of EPs already in use in laboratory; (II) verification of newly introduced EPs; (III) validation of EPs. For verification, at least two performance characteristics should be verified according to the intended use of specific EPs. Different operative flows are described for quantitative, qualitative and semi-quantitative methods. For validation procedure, a validation plan should be planned, including performance characteristics for specific EP, the experimental procedure and acceptability criterion. The final step of the validation process is production of a validation certificate. This is aimed to summarizing the validation process and defining the approval for being used in clinical practice. The proposed verification and validation procedures were actually fulfilling the ISO 15189 requirements during the accreditation audit. They can hence represent a model to follow for other medical laboratories.

ObjectiveThe Endocrine Society Clinical Guidelines recommend measuring 24-h urinary free cortisol (UFF) levels using a highly accurate method as one of the first-line screening tests for the diagnosis of Cushing's Syndrome (CS). We evaluated the performance of UFF, urinary free cortisone (UFE), and the UFF:UFE ratio, measured using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method.Subjects and methodsThe LC–MS/MS was used to analyze UFF and UFE levels in 43 surgically confirmed CS patients: 26 with Cushing's disease (CD, 16de novoand ten recurrences), 11 with adrenal CS and six with ectopic CS; 22 CD patients in remission; 14 eu-cortisolemic CD patients receiving medical therapy; 60 non-CS patients; and 70 healthy controls. Sensitivity and specificity were determined in the combined groups of non-CS patients, healthy controls, and CD in remission.ResultsUFF>170 nmol/24 h showed 98.7% specificity and 100% sensitivity forde novoCS, while sensitivity was 80% for recurrent CD patients, who were characterized by lower UFF levels. The UFF:UFE and UFF+UFE showed lower sensitivity and specificity than UFF. Ectopic CS patients had the highest UFF and UFF:UFE levels, which were normal in the CD remission patients and in those receiving medical therapy.ConclusionsOur data suggest high diagnostic performance of UFF excretion measured using LC–MS/MS, in detectingde novoCS. UFF:UFE and UFF+UFE assessments are not useful in the first step of CS diagnosis, although high levels were found to be indicative of ectopic CS.

The determination of urinary cortisol/cortisone ratio is of clinical utility in cases of Cushing’s syndrome, apparent mineralocorticoid excess, and also provides information on 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2 activity. It is therefore of utmost importance to ensure accurate cortisol and cortisone measurement and establish appropriate reference ranges.After the isotopic dilution of urine, sample cleanups were obtained with on-line solid-phase extraction and cortisol and cortisone, separated using a Zorbax Eclipse XDB-C18 HPLC analytical column, were analyzed by tandem mass spectrometry with an electrospray ionization source in positive ion mode operation.The method was linear, with concentrations of up to 625 and 1125 nmol/L and lower limit of quantitation (LLOQ) of 5 and 6 nmol/L, for cortisol and cortisone, respectively. Within-run and between-run coefficients of variation were A simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the measurement of cortisol and cortisone in urine. Our findings indicate that the proposed analytical method is suitable for routine purposes and useful in many pathological conditions.

The ISO 15189 has endorsed the use of measurement uncertainty (MU) to estimate the quantity of measurement error of a given method, although the total error (TE) approach, originally postulated by Westgard et al. in 1974, is still widely used and appreciated in laboratory medicine. Both MU and TE present some struggles, especially regarding the estimation of both bias and imprecision. In particular, bias may be seen as a complicating factor because it needs specification of a reference quantity, which may be obtained by different sources. Different formulations of MU have been proposed by different guidelines so far, and each approach presents advantages or limitations. These methods are not comparable and there are considerable differences not only in the theoretic approach but also in MU formulation. Moreover, only few practical results have been reported in the literature, thus limiting comprehension and applicability of these methods, ultimately supporting the hypothesis that implementation of MU in medical laboratories remains in embryo. Additional research and developments in this area are hence advisable for establishing consensus on practical methods for medical laboratories.

SUMMARY Background: A colorimetric method based on acid violet pigment, namely AV17, to analyse salivary total protein content, was assessed. Methods: Human saliva sample or standard (50 µL) was added to 1.5 mL of AV17 working solution (1 mg/mL in 75 mmol/L sodium chloride and 1.7 mol/L phosphoric acid). Total protein concentration was measured at 546 nm. Salivary total protein of healthy subjects was analyzed. Results: The standard protein was Human Serum Albumin and the detection range was 38 mg/L - 900 mg/L with a LOD and LOQ of 26 mg/L and 64 mg/L, respectively. Intraday CVs were 3% - 5% and interday CVs were 3% 6%. The dilution test demonstrated a correlation coefficient of 0.999 and the recovery tests ranged from 108% to 111%. Saliva sample stability was also demonstrated. No intra-individual salivary total protein variation was found during the morning. Conclusions: The method suitability for laboratory diagnostic purposes to analyse human saliva protein content and stability was demonstrated. (Clin. Lab. 2012;58:835-839. DOI: 10.7754/Clin.Lab.2012.111006)

Aims To study the relationship between C-type natriuretic peptide (NT-proCNP) and other natriuretic peptides, such as pro-atrial natriuretic peptide [proANP(1-98)] and N-terminal pro-brain natriuretic peptide (NT-proBNP), in the elderly, investigating also their correlation with other traditional clinical markers of the hypertensive condition. Methods NT-proCNP, NT-proBNP and proANP(1-98) were measured in 57 elderly patients. They were hypertensive patients (nU 36) and normotensive controls (nU 21). Their anthropometric parameters, including Winsor’s index and total and high-density lipoprotein cholesterol, were determined. Results A diagnostic role of NT-proBNP in hypertensive patients was detected by a model of logistic regression, which gave a significant result [odds ratio (OR) 1.0115, PU 0.0184]. By this model the area (AUC) under the receiver-operating characteristic (ROC) curve was 0.69W 0.071 (PU 0.0075). On the basis of the ROC curve, the calculated serum NT-proBNP cut-off for the prediction of hypertension was greater than 164 pmol/l – the value being provided with a sensitivity of 89% coupled with a specificity of 55%. NT-proCNP and proANP(1-98) did not predict the hypertensive condition, although significant correlations were detected with serum lipid profile and creatinine levels. Conclusions By using the logistic regression analysis, NT-proBNP was identified as a significant predictor of hypertension, whereas NT-proCNP and proANP circulating levels were not shown to reliably predict the hypertensive condition. Further validation by means of larger cohort studies is undoubtedly needed to assess the use of all three peptides to increase the performance of a possible test for the prediction of the hypertensive condition in humans.

Background/aims: The offer of human saliva IGF-I (sIGF-I) measurement in athletes investigation is a new proposal. The aim was to investigate the physical exercise effect on sIGF-I and explore plasma free IGF-I relation. Materials and methods: Saliva and blood were collected from well-trained athletes, investigated immediately before and at the end of a physical exercise test. Results: sIGF-I was significantly increased at the end of the physical exercise. The plasma free IGF-I concentrations did not demonstrate any difference. The saliva total protein level (sTP) was also significantly increased. A positive correlation between sTP and sIGF-I, was observed, both before and after physical exercise, and between salivary and plasma free IGF-I only after physical exercise. The salivary free IGF-I level significantly increased after physical exercise, moreover a correlation with the plasma levels exists in post-exercise condition. Conclusion: The physical exercise affects sIGF-I as well as the sTP. The correlation between plasma and salivary free IGF-I levels only in post-exercise condition suggests further studies to investigate the effects of different type and duration of physical exercise. The comparison with other salivary biochemical parameter investigation would also further increase comprehension on the role of salivary IGF-I.

The overview of cortisol physiology, action and pathology is achieved in relation to the hypothalamic-pituitary-adrenal axis alteration by laboratory investigation. The measurements of cortisol and related compound levels in blood, urine and saliva used to study the physiological and pathological cortisol involvement, are critically reviewed. The immunoassay and chromatographic methods for cortisol measurement in the various biological fluids are examined in relation to their analytical performances, reference ranges and diagnostic specificity and sensitivity. Moreover, blood, urine and saliva cortisol level measurements are described taking into account the diagnostic implications. The deduction is that each method requires the definition of its own reference range and its related diagnostic cut-off levels. Thus, this review, stressing the analysis procedures, could help to understand and compare the results of the different assays.

The assay of saliva is an increasing area of research with implications for basic and clinical purposes. Although this biological fluid is easy to manipulate and collect, careful attention must be directed to limit variation in specimen integrity. Recently, the use of saliva has provided a substantial addition to the diagnostic armamentarium as an investigative tool for disease processes and disorders. In addition to its oral indications, the analysis of saliva provides important information about the functioning of various organs within the body. In this respect, endocrine research certainly occupies a central role. The present review considers the laboratory aspects of salivary assays with respect to the different analytes including ions, drugs and various non-protein/protein compounds such as hormones and immunoglobulins. This review also examines the consequences of preanalytical variation with respect to collection strategy and subsequent storage conditions. It is likely that the use of saliva in assays will continue to expand thus providing a new instrument of investigation for physiologic as well as pathophysiologic states.

Background Human growth hormone (hGH) responds to bouts of exercise by increasing, while the insulin-like growth factor-I (IGF-I) responses are conflicting. Methods Twenty well-trained male cyclists completed a brief duration exercise (A: warm up + increasing workload until exhaustion, lasting 25 min) and a medium duration exercise (B: warm up + 70–80%VO2 max + increasing workload until exhaustion, lasting 40 min). The immunoreactivity of plasma hGH, the IGF-I in its total and free fraction were measured before and at the end of the exercise, and the free/total IGF-I ratio response to the two cycling exercise bouts was examined. Results Both A and B demonstrated increased hGH (from 77 ± 122 to 544 ± 327 and 28 ± 68 to 369 ± 276 pmol/l respectively) and total IGF-I (from 67 ± 10 to 70 ± 10 and 55 ± 14 to 61 ± 15 nmol/l respectively). The free IGF-I was decreased only in A (from 0.38 ± 0.16 to 0.32 ± 0.14 nmol/l). Both A and B demonstrated a decreased free/total IGF-I ratio (from 0.57 ± 0.30 to 0.46 ± 0.22 and 0.61 ± 0.37 to 0.52 ± 0.29). Conclusion Brief and medium duration physical exercise influences the hGH, the total and free IGF-I concentrations. The free/total IGF-I ratio was also influenced and it might be related to the GH/IGF system. Its investigation might be a way of studying the training condition.