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The M4F project brings together the fusion and fission materials communities working on the prediction of radiation damage production and evolution and their effects on the mechanical behaviour of irradiated ferritic/martensitic (F/M) steels. It is a multidisciplinary project in which several different experimental and computational materials science tools are integrated to understand and model the complex phenomena associated with the formation and evolution of irradiation induced defects and their effects on the macroscopic behaviour of the target materials. In particular the project focuses on two specific aspects: (1) To develop physical understanding and predictive models of the origin and consequences of localised deformation under irradiation in F/M steels; (2) To develop good practices and possibly advance towards the definition of protocols for the use of ion irradiation as a tool to evaluate radiation effects on materials. Nineteen modelling codes across different scales are being used and developed and an experimental validation programme based on the examination of materials irradiated with neutrons and ions is being carried out. The project enters now its 4th year and is close to delivering high-quality results. This paper overviews the work performed so far within the project, highlighting its impact for fission and fusion materials science.

The study investigated the effects of periarticular knee pressure stimuli on quadriceps strength and neuromuscular activity in subjects with knee osteoarthritis. Twenty-five subjects with knee osteoarthritis and 25 age-matched healthy controls performed maximal voluntary knee extension tasks on an isometric dynamometer. Three different pressure stimuli (no-pressure, 60-mmHg, 120-mmHg) were applied using a sphygmomanometer via the cuff covering the knee joint. Peak torque and root-mean-square peak of rectus femoris (RF), vastus medialis (VM), and vastus lateralis (VL) were collected and normalized for the no-pressure condition (nTorque-peak and nRMS-peak). Normalized Torque-peak increased from no-pressure to 60-mmHg and 120-mmHg in patients, which revealed higher nTorque-peak during 60-mmHg (MD: 10.9%, IC 95 : 1.8%, 20.1%, p = 0.020) and 120-mmHg (MD: 16.0%, IC 95 : 4.4%, 27.6%, p = 0.008) conditions than healthy subjects. Moreover, nRMS-peak increased from no-pressure to 60-mmHg for RF, from no-pressure to 120-mmHg for RF, VM and VL, and from 60-mmHg to 120 mm-Hg for VL in patients. Patients revealed higher nRMS-peak of RF and VM during 60-mmHg and 120-mmHg conditions than healthy subjects. Periarticular knee pressure stimuli enhanced quadriceps strength and neuromuscular activity in subjects with knee osteoarthritis. This approach may represent a new strength training modality in patients with neuromuscular activation deficits for knee osteoarthritis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objective: To investigate neuromuscular activation of quadriceps bellies during different tasks in patients before and after total knee arthroplasty (TKA)., Methods: Twenty-six patients scheduled for TKA and 16 control subjects performed three isometric tasks: knee extension (KE), hip flexion (HF), hip flexion with contralateral hip extension (HFE). Surface electromyography signals of rectus femoris, vastus medialis and vastus lateralis were collected the day before (T0), at one (T1) and three (T2) days after surgery, whereas control subjects underwent a single evaluation. The Root Mean Square peak normalized for its highest value during the three tasks (nRMS-peak) was used as index of maximum neuromuscular activation for each belly. Sixteen patients performed the postoperative assessment, due to the placement of an elastomeric pump aimed at reducing pain in 10 patients., Results: Patients showed lower rectus femoris nRMS-peak during KE compared to HF and HFE before and after surgery (p < 0.001), as occurred in control subjects. Differently from control subjects, patients showed higher vastus medialis and vastus lateralis nRMS-peak during HF compared to KE at T1 (p = 0.008) and T2 (p = 0.039)., Conclusion: TKA modified quadriceps neuromuscular activation during different tasks performed the same biomechanical condition. These findings may be considered in planning physiotherapy interventions after TKA., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

An inverted U-shape relationship between cognitive performance and indoor temperature with best performance peaking at 21.6 °C was previously described. Little is known on classroom temperature reduction effects on cognitive performances and cardiac autonomic profile, during the cold season. Fifteen students underwent electrocardiogram recording during a lecture in two days in December when classroom temperatures were set as neutral (NEUTRAL, 20-22 °C) and cool (COOL, 16-18 °C). Cognitive performance (memory, verbal ability, reasoning, overall cognitive C-score) was assessed by Cambridge Brain Science cognitive evaluation tool. Cardiac autonomic control was evaluated via the analysis of spontaneous fluctuations of heart period, as the temporal distance between two successive R-wave peaks (RR). Spectral analysis provided the power in the high frequency (HF, 0.15-0.40 Hz) and low frequency (LF, 0.04-0.15 Hz) bands of RR variability. Sympatho-vagal interaction was assessed by LF to HF ratio (LF/HF). Symbolic analysis provided the fraction of RR patterns composed by three heart periods with no variation (0 V%) and two variations (2 V%), taken as markers of cardiac sympathetic and vagal modulations, respectively. The students' thermal comfort was assessed during NEUTRAL and COOL trials. Classroom temperatures were 21.5 ± 0.8 °C and 18.4 ± 0.4 °C during NEUTRAL and COOL. Memory, verbal ability, C-Score were greater during COOL (13.01 ± 3.43, 12.32 ± 2.58, 14.29 ± 2.90) compared to NEUTRAL (9.98 ± 2.26, p = 0.002; 8.57 ± 1.07, p = 0.001 and 10.35 ± 3.20, p = 0.001). LF/HF (2.4 ± 1.7) and 0 V% (23.2 ± 11.1%) were lower during COOL compared to NEUTRAL (3.7 ± 2.8, p = 0.042; 28.1 ± 12.2.1%, p = 0.031). During COOL, 2 V% was greater (30.5 ± 10.9%) compared to NEUTRAL (26.2 ± 11.3, p = 0.047). The students' thermal comfort was slightly reduced during COOL compared to NEUTRAL trial. During cold season, a better cognitive performance was obtained in a cooler indoor setting enabling therefore energy saving too., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

One-stage bilateral total hip arthroplasty (THA) implies similar complication rate and hospitalization time to unilateral THA, but no studies have evaluated the functional and postural recovery in these patients. The aim of this study was to assess short-term functional and postural recovery in patients after one-stage bilateral or unilateral THA. Forty patients undergoing bilateral (n = 20) or unilateral (n = 20) THA were assessed by Timed Up and Go (TUG), Numeric Rating Scale (NRS), Tampa Scale of Kinesiophobia (TSK) and Body Weight Distribution Symmetry Index (BWDSI) during stand-to-sit (STS). Centre of Pressure (CoP) parameters and BWDSI during standing with eyes open (EO) and closed (EC) were also assessed. Data were collected one day before surgery, at three and seven days. No between-group differences were found for TUG, NRS and TSK at any time-point, showing similar mobility, pain and fear of movement in both groups. BWDSI during STS (P = 0.001) and standing (OE P = 0.007; CE P = 0.012) revealed differences over time in favor of patients with bilateral THA, who showed better symmetry in weight distribution. Shorter CoP path length was observed during standing in patients with unilateral THA (OE P = 0.023; CE P = 0.018), who mainly used their non-affected limb to maintain balance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Question: In people after stroke, does virtual reality based rehabilitation (VRBR) improve walking speed, balance and mobility more than the same duration of standard rehabilitation? In people after stroke, does adding extra VRBR to standard rehabilitation improve the effects on gait, balance and mobility?, Design: Systematic review with meta-analysis of randomised trials., Participants: Adults with a clinical diagnosis of stroke., Intervention: Eligible trials had to include one these comparisons: VRBR replacing some or all of standard rehabilitation or VRBR used as extra rehabilitation time added to a standard rehabilitation regimen., Outcome Measures: Walking speed, balance, mobility and adverse events., Results: In total, 15 trials involving 341 participants were included. When VRBR replaced some or all of the standard rehabilitation, there were statistically significant benefits in walking speed (MD 0.15 m/s, 95% CI 0.10 to 0.19), balance (MD 2.1 points on the Berg Balance Scale, 95% CI 1.8 to 2.5) and mobility (MD 2.3 seconds on the Timed Up and Go test, 95% CI 1.2 to 3.4). When VRBR was added to standard rehabilitation, mobility showed a significant benefit (0.7 seconds on the Timed Up and Go test, 95% CI 0.4 to 1.1), but insufficient evidence was found to comment about walking speed (one trial) and balance (high heterogeneity)., Conclusion: Substituting some or all of a standard rehabilitation regimen with VRBR elicits greater benefits in walking speed, balance and mobility in people with stroke. Although the benefits are small, the extra cost of applying virtual reality to standard rehabilitation is also small, especially when spread over many patients in a clinic. Adding extra VRBR time to standard rehabilitation also has some benefits; further research is needed to determine if these benefits are clinically worthwhile., (Copyright © 2015 Australian Physiotherapy Association. Published by Elsevier B.V. All rights reserved.)

Background and Aims: We investigated the effect of berberine (BBR), an alkaloid showing antiatherogenic properties beyond the cholesterol lowering capacity, on macrophage cholesterol handling upon exposure to human serum and on macrophage responses to excess free cholesterol (FC) loading., Methods and Results: Mouse and human macrophages were utilized as cellular models. Cholesterol content was measured by a fluorimetric assay; cholesterol efflux, cytotoxicity and membrane FC distribution were evaluated by radioisotopic assays. Monocyte chemotactic protein-1 (MCP-1) secretion was measured by ELISA; membrane ruffling and macropinocytosis were visualized by confocal microscopy. Exposure of cholesterol-enriched MPM to serum in the presence of 1 μM BBR resulted in a reduction of intracellular cholesterol content twice greater than exposure to serum alone (-52%; p < 0.01 and -21%; p < 0.05), an effect not mediated by an increase of cholesterol efflux, but rather by the inhibition of cholesterol uptake from serum. Consistently, BBR inhibited in a dose-dependent manner cholesterol accumulation in human macrophages exposed to hypercholesterolemic serum. Confocal microscope analysis revealed that BBR inhibited macropinocytosis, an independent-receptor process involved in LDL internalization. Macrophage FC-enrichment increased MCP-1 release by 1.5 folds, increased cytotoxicity by 2 fold, and induced membrane ruffling; all these responses were markedly inhibited by BBR. FC-enrichment led to an increase in plasma membrane cholesterol by 4.5 folds, an effect counteracted by BBR., Conclusion: We showed novel potentially atheroprotective activities of BBR in macrophages, consisting in the inhibition of serum-induced cholesterol accumulation, occurring at least in part through an impairment of macropinocytosis, and of FC-induced deleterious effects., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A simple, sensitive and fast hydrophilic interaction liquid chromatography (HILIC) method using ultraviolet diode-array detector (UV-DAD)/electrospray ionization tandem mass spectrometry was developed for the automated high performance liquid chromatography (HPLC) determination of sodium risedronate (SR) and its degradation products in new pharmaceuticals. The chromatographic separations were performed on Ascentis Express HILIC 2.7μm (150mm×2.1mm, i.d.) stainless steel column (fused core). The mobile phase consisted of formate buffer solution (pH 3.4; 0.03M)/acetonitrile 42:58 and 45:55 (v/v) for granules for oral solution and effervescent tablet analysis, respectively, at a flow-rate of 0.2mL/min, setting the wavelength at 262nm. Stability characteristics of SR were evaluated by performing stress test studies. The main degradation product formed under oxidation conditions corresponding to sodium hydrogen (1-hydroxy-2-(1-oxidopyridin-3-yl)-1-phosphonoethyl)phosphonate was characterized by high performance liquid chromatography-electrospray ionization-mass tandem mass spectrometry (HPLC-ESI-MS/MS). The validation parameters such as linearity, sensitivity, accuracy, precision and selectivity were found to be highly satisfactory. Linear responses were observed in standard and in fortified placebo solutions. Intra-day precision (relative standard deviation, RSD) was ≤1.1% for peak area and ≤0.2% for retention times (tR) without significant differences between intra- and inter-day data. Recovery studies showed good results for all the examined compounds (from 98.7 to 101.0%) with RSD ranging from 0.6 to 0.7%. The limits of detection (LOD) and quantitation (LOQ) were 1 and 3ng/mL, respectively. The high stability of standard and sample solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of many samples and consecutive chromatographic analyses by using an autosampler. The developed stability indicating method is suitable for the quality control of SR in new and commercial pharmaceutical formulations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The use of menadione (MD) as a pre-column reagent for high performance liquid chromatography (HPLC) analysis of aliphatic thiols is proposed. The reaction was carried out for 5 min at room temperature and pH 8.5. The developed method was applied to the N-acetylcysteine (NAC) analysis of alimentary supplements and pharmaceutical formulations. The effect of the complex matrix was evaluated by the study of the thiol derivatization reaction both in standard and in placebo solutions. The yield of NAC-MD adduct was found to be quantitative at a reagent to thiol molar ratio of about 4 in comparison with an authentic specimen of synthesized NAC adduct, which was characterized by (1)H NMR, IR and UV. The routine chromatographic separations were performed on a Synergi MAX-RP column using a mobile phase consisting of methanol/triethylammonium (TEA) phosphate buffer (pH 3; 0.05 mol L(-1)) 70:30 (v/v) at a flow-rate of 0.4 mL min(-1). UV-diode array detection was used setting the wavelength at λ=260 nm. The validation parameters such as linearity, sensitivity, accuracy, precision, selectivity and ruggedness were found to be highly satisfactory. Similar linear responses were observed by standard and placebo solutions (determination coefficient: 0.9996). Limit of detection was about 0.019 μg g(-1). Intra-day precision (relative standard deviation, R.S.D.) was ≤0.81% for NAC to internal standard (IS) peak area ratio, ≤0.28% and ≤0.32%, respectively, for NAC and IS retention times (tR), without significant differences between intra- and inter-day data. NAC recovery studies gave good results (100.12%) with R.S.D.=1.05%., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The preference for in-phase association of coupled cyclic limbs movements is well described (mirror-symmetrical patterns) and this is demonstrated by the ease of performing in-phase movements compared to anti-phase ones. The hypothesis of this study is that the easiest movement patterns are those with minor postural activity. The aim of this study was to describe postural activity in standing subjects in the sagittal and frontal planes during the execution of three upper limbs tasks (single arm, in-phase, anti-phase) at four different frequencies (from 0.6 to 1.2Hz). We employed six infrared cameras for recording kinematics information, a force platform for measuring forces exerted on the ground, and a system for surface electromyography (SEMG). Outcome measures were: upper limb range of movement and relative-phase, centre of pressure displacement (COP), screw torque (Tz) exerted on the ground, and SEMG recordings of postural muscles (adductor longus, gluteus medius, rectus femoris, and biceps femoris). Our results show that in both the planes the in-phase task resulted in less COP displacement, torque production, and postural muscles involvement than the anti-phase and single arm tasks. This reduced need of postural control could explain the ease of performing in-phase coupled limb movements compared with anti-phase movements., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A RP-HPLC method with pre-column derivatization was developed and validated for the simultaneous quantification of carnosine (Carn), acetylcarnitine taurinate (AC-Tau), asparagine (Asn), potassium aspartate (Asp) and for the determination of phosphoserine (p-Ser) in new and commercial alimentary supplements. The effect of complex matrices was evaluated by the study of the amino acid derivatization reaction with 2,4-dinitrofluorobenzene (DNFB) both in standard and placebo solutions. The reaction was carried out for 20 min at 70 °C in alkaline medium (pH10) for p-Ser analysis, whereas for 60 min in the case of Carn, AC-Tau, Asn and Asp analysis. The adducts have been separated on a Discovery RP Amide C16 (250 mm×4.6mm, i.d.) column using a mobile phase consisting of acetonitrile (ACN) and triethylammonium (TEA) phosphate buffer (pH 3, 0.05 M) under gradient elution conditions at a flow-rate of 0.8 mL/min. Detection was set at λ=360 nm. The validation parameters such as linearity, sensitivity, accuracy, precision and specificity were found to be highly satisfactory. Linear responses were observed by placebo solutions (determination coefficient ≤0.9996). Intra-day precision (relative standard deviation, RSD) was ≤1.06% for corrected peak area and ≤0.99% for retention times (tR) without significant differences between intra- and inter-day data. Recovery studies showed good results for all examined compounds (from 97.7% to 101.5%) with RSD ranging from 0.5% to 1.3%). The high stability of derivatized compound solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of a large number of samples and consecutive chromatographic analyses by the use of an autosampler. The developed method can be considered suitable for the quality control of new and commercial products., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The use of 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde (DPD) as a pre-column derivatization reagent for HPLC (high performance liquid chromatography) analysis of octopamine (oct) and tyramine (tyr) is proposed. The compound reacts under mild conditions (2 min at ambient temperature) with primary amino groups. The derivatization conditions were optimized by considering different parameters (temperature, time and reagent concentration). The synthesized oct and tyr adducts were characterized by (1)H NMR (nuclear magnetic resonance), ESI-MS (electrospray ionization mass spectrometry), IR (infrared) and UV (ultraviolet). Derivative chromatographic separations were performed on a Sinergy Hydro-RP column (150 mm × 4.6 mm i.d.) using a mobile phase consisting of methanol and triethylammonium phosphate buffer (pH 3; 10mM) at varying composition gradient elution and at a flow rate of 0.8 mL/min. Detection was set at λ=320 nm. The obtained results were compared with those achieved by a validated direct HPLC method with detection at λ=275 nm using a Sinergy Polar-RP column (250 mm × 3 mm i.d.) by isocratic elution conditions with a mobile phase consisting of methanol/acetonitrile/sodium pentanesulphonate (SPS; pH 3; 10mM), 7.5:7.5:85 (v/v/v) at a flow rate of 0.3 mL/min. Derivatization method sensitivity proved to be ten times higher than direct method. Limit of detection of oct and tyr was 0.010 and 0.008 μg/mL, respectively. The reliability of the pre-column method was satisfactory also in terms of linearity (from 0.028 to 1.255 and 0.024 to 1.244 μg/mL for oct and tyr, respectively), precision (relative standard deviation ≤2, without significant differences between intra-day and inter-day data) and recovery (from 98.9 to 101.2%). The proposed method showed to be suitable for a reliable determination of oct and tyr traces in commercially available phytoproducts using the instrumentation usually present in any analytical laboratory., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The identification of the motor unit (MU) innervation zone (IZ) using surface electromyographic (sEMG) signals detected on the skin with a linear array or a matrix of electrodes has been recently proposed in the literature. However, an analysis of the reliability of this procedure and, therefore, of the suitability of the sEMG signals for this purpose has not been reported. The purpose of this work is to describe the intra and inter-rater reliability and the suitability of surface EMG in locating the innervation zone of the upper trapezius muscle. Two operators were trained on electrode matrix positioning and sEMG signal analysis. Ten healthy subjects, instructed to perform a series of isometric contractions of the upper trapezius muscle participated in the study. The two operators collected sEMG signals and then independently estimated the IZ location through visual analysis. Results showed an almost perfect agreement for intra-rater and inter-rater reliability. The constancy of IZ location could be affected by the factors reflecting the population of active MUs and their IZs, including: the contraction intensity, the acquisition period analyzed, the contraction repetition. In almost all cases the IZ location shift due to these factors did not exceed 4mm. Results generalization to other muscles should be made with caution., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The identification of glomerular auto-antigens in idiopathic human membranous glomerulonephritis (MGN) is a crucial step towards the definition of the mechanisms of the disease. Recent 'in vivo' studies demonstrated a heterogeneous composition of glomerular immune-deposits in MGN biopsies only a part of which have been characterized. We studied with a proteomical approach IgGs eluted from laser capture microdissected glomeruli of 8 MGN patients and showed the existence of other three immune proteins in MGN glomeruli (α-enolase, elongation factor 2 and Glycyl Aminoacyl-tRNA Synthetase). One of these, i.e. α-enolase, fulfilled all criteria for being considered an auto-antigen. Specific IgG₁ and IgG₄ reacting with podocyte α-enolase were, in fact, eluted from microdissected glomeruli and Confocal- and Immuno Electron-Microscopy showed co-localization of α-enolase with IgG₄ and C5b-9 in immune-deposits. Serum levels of anti a-enolase IgG4 were determined in 131 MGN patients and were found elevated in 25% of cases. Overall, our data demonstrate that glomerular α-enolase is a target antigen of autoimmunity in human MGN. Circulating anti α-enolase auto-antibodies can be detected in sera of a significant quota of MGN patients. Like other auto-antigens, α-enolase may be implicated in the pathogenesis of human MGN., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A sensitive and selective reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine octopamine, tyramine and Tyrosine (Tyr) in complex matrices as formulations and phytoextracts (Citrus aurantium), after pre-column derivatization with o-phthaldialdehyde (OPA) reagent. The chromatographic separations were performed at room temperature on a Phenomenex Luna C18 column using methanol and sodium acetate buffer (pH 5.5) by varying composition gradient elution as mobile phase and detected flurometrically at λ(em)=455 nm with λ(ex)=340 nm. The results obtained by the proposed method were compared with those achieved by a validated direct RP-LC method with fluorescence detection at λ(em)=310 nm with λ(ex)=275 nm, as reference method, using a Phenomenex Gemini C18 column under isocratic elution conditions with acetonitrile and sodium 1-heptanesulphonate (pH 3), as mobile phase. The higher sensitivity of the derivatization method (detection limit about 0.06 pmol) allowed the sure determination of octopamine present in traces in the examined samples. The repeatability of method (RSD) was ≤1.90% and there was no significant difference between repeatability and intermediate precision data. Recovery studies showed good results 99.5-101.3% with RSD ranging from 0.8 to 1.2%. All analyses were performed by mild conditions in absence of preliminary difficult extraction methodologies or laborious step of sample pre-treatment., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objectives: To assess whether the ability to execute flexion/extension of hands and feet, in different patterns of in-phase and anti-phase association, is influenced by the planning of the movements according to the two body sides (i.e., right vs. left) compared to the upper and lower parts of the body (i.e., hands vs. feet); and to evaluate how training of these movements influences their performance., Methods: Fifteen healthy subjects were studied while performing four different tasks involving simultaneously in-phase and anti-phase movements of the hands and feet in the parasagittal plane. Hand and foot movements were represented by wrist and ankle kinematics, measured before and after training as duration of correct execution, median frequency of movements, median ranges of motion and discrete relative phase (AEø)., Results: During baseline assessment, subjects performed the task better when instructions were given to focus on in-phase or anti-phase movements of bilateral upper and lower extremities than when instructions were focused on the movements of the left and right side. After the training these differences disappeared. During all the conditions, the duration of correct execution and the movement frequency were significantly different between the baseline and the final assessment (p ranging from 0.001 to 0.04). The AEø did not differ among the four tasks, as well as between the baseline and the final assessment., Conclusion: Combination of movements of the hands and feet is easier when planning the movements with respect to the upper or lower segments of the body rather than right and left side. These findings might have important implications when subjects have to learn novel coordination patterns as it is the case in rehabilitative and sportive activities., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: To investigate whether the morphological and functional changes typical of cell immobilization induced by free cholesterol (FC) accumulation in macrophages is related to the activity of the ATP-binding cassette transporter (ABCA1)., Methods and Results: FC loading induced actin rearrangement with ruffling and cell spreading in macrophages that normally express ABCA1, but to a significant lesser extent in ABCA1-KO mouse peritoneal macrophages (MPMs) and in normal cells upon pharmacological inhibition of ABCA1 with probucol. In ABCA1-KO MPMs and in probucol-treated J774 cell migration was inhibited to a lower extent by FC as compared to control cells. Similar results were found in stably ABCA1 knocked down J774 (ABCA1-KD-J774) obtained by RNA interference. FC accessible to cholesterol oxidase, a measure of plasma membrane FC content, was significantly higher in FC-loaded WT MPMs and control J774 than in FC-loaded ABCA1-KO MPMs, ABCA1-KD-J774 or probucol-treated J774. In parallel plasma membrane total phospholids and sphingomyelin increased after cholesterol loading in control J774 but not in ABCA1-KD-J774. In addition, apoA-I, that removes FC from ABCA1 specific pool, partially restored chemotactic response in FC-loaded control J774. No effect was observed with HDL(2) that does not interact with ABCA1. Finally, FC-induced Rac activation was more efficient in control J774 than in ABCA1-KD-J774. and was prevented by probucol and apoA-I in control J774., Conclusion: In macrophages ABCA1 activity mediates FC ability to alter plasma membrane organization, to inhibit cell migration, and to activate a Rac-mediated signaling pathway., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: Hyper-hypo tension (like Cushing's syndrome, apparent mineralocorticoid excess syndrome and Addison's disease) diagnostic laboratory requires cortisol (F) analysis. The simultaneous analysis of human saliva F and cortisone (E), the inactive F metabolite, by solid phase extraction and RP-HPLC was studied., Methods: Saliva/standard samples were C18-SPE extracted, dried and resuspended. E and F were analysed by isocratic RP-HPLC (acetonitrile/water 27/73%) and UV detection. In the morning and in the evening Salivette stimulated saliva specimens were collected from healthy volunteers., Results: The E and F calibration curve ranges were 11.0-110.0 and 5.5-55.0 nmol/l respectively. The LOD was 0.2 and 0.1 nmol/l for E and F respectively. The intra and inter assay CVs were respectively 2.7-6.6 and 5.6-7.0% for E and 5.8-7.0 and 11.7-13.1% for F. The E and F spiked saliva sample recovery was 99% and 88% respectively. Saliva specimen stability was validated. E and F saliva levels in healthy volunteers were significantly (p<0.001) higher at 8 a.m. compared with 11 p.m. (26.4+/-8.9 vs. 4.3+/-2.9 nmol/l for E; 11.1+/-4.0 vs. 2.5+/-1.5 nmol/l for F, respectively)., Conclusions: This method is suitable for periodic analyses in a clinical biochemistry laboratory for endocrinology investigation purposes, simultaneously analysing E and F levels in a saliva specimen.

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%VO(2 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.

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.

The use of 2,7-dimethyl-3,8-dinitrodipyrazolo[1,5-a:1',5'-d]pyrazine-4,9-dione as pre-column reagent for LC analysis of amino acids is proposed. The compound reacts (30 min at 68 degrees C in presence of 0.04 M triethylamine in a dimethylsulfoxide-water mixture) with primary and secondary amino function and the stable resulting adducts can be chromatographed under reversed-phase conditions and detected at lambda=280 nm. The derivatization conditions were optimized by a series of experiments. The effect of temperature, triethylamine concentration and reagent on the reaction was investigated. The yield of the glycine derivative was found to be quantitative at a reagent amino acid molar ratio of about 6 by comparison with an authentic specimen of synthesized glycine adduct. Application of the method to quality control of commercially available oral polyaminoacid formulations is described.

Endocrine diagnostic tests are dependent on the molecular characteristics of protein hormones, a property that is also intimately tied to function. The structure-function relationship is of particular importance for multifunctional protein hormones such as growth hormone (GH). For clinical diagnosis, it is imperative to understand the relationship between GH structure (and its molecular fragments) and function and their potential interaction with single or multiple receptors. The existence of a single or aggregated intact GH 22 kDa form such as the 20 kDa GH isoform has been described, but GH fragments cannot be excluded a priori. Recent advances and probable similarity of GH with other protein hormones such as natriuretic peptides (ANP, BNP and their proANP and proBNP fragments) and POMC (ACTH, beta-endorphin, etc.) lend support to the hypothesis that GH fragments should also be present. This brief review focuses on GH heterogeneity and feasible post-synthesis events. The aim of the review is to describe which GH forms/fragments have already been recognized and/or are potentially present in the circulation. The impacts of GH isoforms (namely the intact 20 and 22 kDa isoforms) and fragments thereof on quantitative measurement are discussed with reference to traditional immunoassay technology and innovative immunofunctional laboratory assays.

Understanding the relationship between growth hormone (GH) structure (its molecular fragments) and function via interaction with single or multiple receptors is of particular importance in clinical diagnostics and physiologic biochemistry. Direct and indirect actions of GH are numerous ranging from carbohydrate and lipid metabolism to growth effects at muscle and vessels. To this end, we have focused on the influence of physical exercise on GH synthesis and release into the circulation. Physical exercise is a physiological condition to which GH multifunctionality is inextricably linked and is thus important physiologically and pathologically. This review describes the potential human GH fragments with respect to protein hormone multifunctionality and the molecular regions of potential action. The intent of the review is to highlight human GH fragments and hypothesize their potential physiologic role. GH fragmentation is also reviewed in relation to the effects of physical exercise and hormone multifunctionality.

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<0.001); E: 797+/-400 nmol/l versus 408+/-252 nmol/l (p<0.001)). The 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<0.05).

Background: The insulin-like growth factor hormone (IGF-I) is an important protein hormone under investigation with physical exercise and for doping detection. Urinary IGF-I level in fact represents a relevant measurement when the postexercise proteinuria is under analysis. To verify the IGF-I level variation in the circulation and in urinary excretion in the occasion of a competition, the plasma and urine IGF-I in athletes before and after an actual competitive event were measured., Methods: Twenty well-trained cyclists took part in a competition (102 km) and concluded the intense physical exercise in approximately 2(1/2) h. Urine and blood samples were collected from each athlete 10-20 min before and at the end of the competition. Plasma and urine total IGF-I (pIGF, uIGF), total urinary proteins (uPr), and creatinine (uCr) concentrations were measured., Results: The uIGF [from 76.2+/-15.8 to 256.9+/-29.1 ng/l (p<0.001)], uPr [from 29.4+/-6.7 to 325.9+/-95.1 mg/l (p<0.005)], and uCr [from 6.3+/-1.0 to 10.0+/-0.8 mmol/l (p<0.005)] significantly increased. The pIGF was 262.6+/-14.3 and 247.3+/-11.8 microg/l before and end-exercise, respectively. A statistical correlation between uIGF and uPr was demonstrated (p<0.001). The pIGF/uIGF ratio was significantly (p<0.05) decreased comparing the end with before the competition., Conclusions: The pIGF/uIGF significantly decreased at the end, compared with before the competition, suggesting a changed uIGF excretion. This increment appeared to be increased, although not significantly, considering the ratio with uCr.

Low doses, chronic exposure to mercurial organic compounds is a worldwide health concern and could be pathogenetically relevant as co-factor in several neurodegenerative diseases. In this in vitro study we wanted to further improve our knowledge on the mechanisms of toxicity of methylmercury hydroxide (MeHgOH) in the unprimed PC12 cell line. Cell viability, mitochondrial function, redox state, and cell morphology were recorded at different time points to sequence the events leading to cell death. The lowest cytotoxic concentration and EC50 were 0.3 and 1.3 microM, respectively. 5 microM MeHgOH was fatal for 80% of the cell population after 24 h; within 1 h it caused glutathione (GSH) depletion and a partial dissipation of Deltapsim. At this concentration, reactive oxygen species (ROS) generation was only slight and delayed. After 6h more than 50% of ATP was available and caspase 3 was active. Time-lapse confocal microscopy showed that only a fraction of the cells completed apoptosis while others turned toward necrosis (necrapoptosis). Pre-incubation with N-acetylcysteine (NAC) and GSH but not Cyclosporin A rescued over 80% of the cells. These results provide experimental evidence that, in this cell model, MeHgOH triggers cell death via a primary depletion of GSH but in the absence of ROS overproduction.

Background: Insulin-like growth factor I (IGF-I), like growth hormone (GH), is excreted in urine in a smaller fraction than the concentration found in blood. Exercising subjects undergo post-exercise proteinuria. The present work aims to propose a method for urinary IGF-I analysis (uIGF-I) by defining urinary concentration in sedentary individuals and athletes before and after strenuous exercise., Methods: Urine samples were collected from 30 sedentary healthy male individuals during the morning and from 30 well-trained cyclists, before and after a competition of about 3 h (150 km). uIGF-I was measured in undiluted acidified urine by radioimmunoassay (RIA) method using a purified polyclonal rabbit antibody, human 125I-IGF-I and a second anti-rabbit antiserum. The acidification of the urine samples and the excess of IGF-II addition in the incubation medium of the assay were used to dissociate the binding and to block the interference from IGF binding proteins (IGFBPs). Urinary growth hormone (uGH), total protein (utPr) and creatinine (ucr) concentrations were also measured by immunoradiometric assay (IRMA), colorimetric and capillary electrophoresis methods, respectively., Results: The analysis range was 0-2500 ng/l (0-327 pmol/l), the intra- and inter-assay coefficients of variations (CVs) ranged from 2.3% to 7.8%, respectively. The detection limit was 0.6 pg/tube. The uIGF-I/creatinine (cr) ratio in healthy subjects was 70 +/- 8 pg/mg cr. The uIGF-I/creatinine ratio (pg/mg cr) was different (p<0.001) in athletes before vs. after competition 93 +/- 27 vs. 136 +/- 13. Athletes' [uIGF-I/total proteins] ratio (ng/mg tPr) before and post-exercise was 2.3 +/- 0.5 and 2.5 +/- 0.3, respectively., Conclusions: uIGF-I assay appears to be an effective way of monitoring IGF-I excretion. In the cyclists, in the pre-exercise state, uIGF-I was comparable with that measured in sedentary healthy individuals. In the cyclists, after strenuous exercise, the increased uIGF-I/cr and uGH/cr ratios suggested a relation with the post-exercise proteinuria. In conclusion, proteinuria physiologically obtained, such as post-exercise proteinuria, might be a new approach in IGF-I system investigation., (Copyright 2002 Elsevier Science B.V.)

Acyclovir (ACV) is an antiviral drug, which selectively inhibits replication of members of the herpes group of DNA viruses with low cell toxicity. Valaciclovir (VACV), a prodrug of ACV is usually preferred in the oral treatment of viral infections, mainly herpes simplex virus (HSV). Also other analogues such as ganciclovir and penciclovir are discussed here. The former acts against cytomegalovirus (CMV) in general and the latter against CMV retinitis. The action mechanism of these antiviral drugs is presented briefly here, mainly via phosphorylation and inhibition of the viral DNA polymerase. The therapeutic use and the pharmacokinetics are also outlined. The measurement of the concentration of acyclovir and related compounds in biological samples poses a particularly significant challenge because these drugs tend to be structurally similar to endogenous substances. The analysis requires the use of highly selective analytical techniques and chromatography methods are a first choice to determine drug content in pharmaceuticals and to measure them in body fluids. Chromatography can be considered the procedure of choice for the bio-analysis of this class of antiviral compounds, as this methodology is characterised by good specificity and accuracy and it is particularly useful when metabolites need to be monitored. Among chromatographic techniques, the reversed-phase (RP) HPLC is widely used for the analysis. C18 Silica columns from 7.5 to 30 cm in length are used, the separation is carried out mainly at room temperature and less than 10 min is sufficient for the analysis at 1.0-1.5 ml/min of flow-rate. The separation methods require an isocratic system, and various authors have proposed a variety of mobile phases. The detection requires absorbance or fluorescence measurements carried out at 250-254 nm and at lambdaex=260-285 nm, lambdaem=375-380 nm, respectively. The detection limit is about 0.3-10 ng/ml but the most important aspect is related to the sample treatment, mainly when body fluids are under examination. The plasma samples obtained from human blood are pre-treated with an acid or acetonitrile deproteinization and the supernatant after centrifugation is successively extracted before RP-HPLC injection. Capillary Electrophoresis methods are also discussed. This new analytical approach might be the expected evolution, in fact the analyses are improved with regard to time and performance, in particular coated capillary as well as addition of stabilisers have been employed. The time of analysis is shortened arriving at less than half a minute. Furthermore by using an electrochemical detection, and having a calibration linearity in the range of 0.2-20.0 ng/ml, the detection limit is 0.15 microg/ml. The measurements of acyclovir and penciclovir have been presented but in the future other related drugs will probably be available using CE methods.

The family of the atrial natriuretic peptides, proANP fragments and the active alphaANP, is strongly related to heart disease. The aim was to study in CHF subjects the relation of mdANP and NtANP with brain natriuretic peptide (BNP) and with other traditional medical parameters. Sixteen CHF patients (aged 51.9+/-13.7 years) and 16 healthy subjects age matched (50.8+/-5.9 years) were selected. Both NtANP and mdANP were higher in CHF patients than in healthy subjects (1436+/-288 vs. 288+/-22 pmol/l p<0.001 and 2305+/-383 vs. 423+/-65 pmol/l p<0.0001, respectively). BNP in CHF patients was 28.0+/-9 pmol/l (reference values 1.7+/-1.8 pmol/l). Both NtANP and mdANP demonstrated positive correlation with BNP, p<0.0001 and with left atrial end-systolic volume, p<0.05. BNP correlated with left ventricular mass, p<0.03. In conclusion, plasma NtANP and mdANP analyses are useful laboratory markers in CHF patient investigation and follow up. In particular, they could be employed as non-invasive parameters to follow up worsening of systolic dysfunction until heart transplantation is required.

Background: ProANP(1-126), the prohormone synthesized and secreted by atrial myocites, generates an ANP peptide family, the main forms of which are proANP(1-30), proANP(31-67), proANP(1-98) and proANP(99-126). These molecular circulating forms are involved in hemodynamic and electrolyte homeostasis. In cirrhotic patients, volume homeostasis is almost impaired due to abnormal sodium retention, which results in ascites formation and hemodynamic changes, including high cardiac output and low systemic vascular resistance. During liver transplantation, in the anhepatic phase, hemodynamic instability may occur because of decreased venous return due to surgical manipulation of inferior vena cava, considerable blood loss or cross-clamping. Moreover, marked hemodynamic instability is often observed at the reperfusion of the graft., Aims: The aims of present study are to investigate the changes of ANP during the perioperative phases of Orthotopic Liver Transplantation (OLTx) in end-stage cirrhotic patients., Patients and Methods: From July to September 1999, 11 cirrhotic patients undergoing to OLTx were included in the study: seven males and four females (average age 46+/-10.4 years) affected by post-alcoholic cirrhosis [Hypertension 15 (1990) 9], post-hepatitis cirrhosis [D.G. Gardner, M.C. Lapointe, B. Kovacic-Milivojevic, C.F. Deschepper, Molecular analisys and regulation of the atrial natriuretic factor gene, in: A.D. Struphers (Ed.), Frontiers in Farmacology and Therapeutics: Atrial Natriuretic Factor, Blackwell, Oxford, England, 1991, pp. 1-22], Wilson disease [Life Sci. 28 (1981) 89] and polycystic disease [Life Sci. 28 (1981) 89], autoimmune cirrhosis [Life Sci. 28 (1981) 89]. In each patient, a hemodynamic assessment was achieved using a Swan-Ganz catheter. Periferical venous samples were performed during and immediately after OLTx for the determination of ANP(1-98) and other biohumoral parameters., Results: Mean ANP(1-98) (pmol/ml mean+/-SD) basal levels resulted higher than that recorded in the group of healthy subjects. A significant correlation between 24-h post-reperfusion ANP and intra-operative RBC and RIS requirement was found (p<0.05). The basal values resulted significantly higher than that observed at phase II degrees (p<0.04) and lower than that at phase VI degrees (p<0.05); the anesthetic induction values were significantly lower than that observed at phase VI degrees (p<0.03)., Conclusions: ANP(1-98) values may represent a useful marker of hemodynamic derangements during and after OLTx. Further clinical correlations will need a larger patient basis.

The GH-related effects are primarily mediated by insulin-like growth factor I (IGF-I), a peptide hormone almost completely produced by the liver. Liver cirrhosis is usually accompanied by a fall in protein turnover. Furthermore, an important consequence of chronic liver disease (CLD) is growth hormone/insulin-like growth factor (GH/IGF) axis modification and growth failure. Nutritional status also suffers in this condition, and IGF-I has been proposed as a marker of hepatocellular dysfunction, malnutrition and survival. CLD is characterised by alterations of various clinical biochemistry laboratory parameters. Aminotransferases, bilirubin, plasma proteins, together with prothrombin time and gamma globulins, are usually examined for laboratory diagnostic and/or monitoring purposes. These traditional parameters are also used in the perioperative liver transplantation, but an early signal of graft functioning has still not been established. The aim of the present work is a review of the possibility offered by the clinical biochemistry laboratory GH/IGF investigation in the outcome of liver transplantation.

The importance of hormones on body metabolism when physical exercise is carried out has been established for a long time. Growth hormone (GH) is crucial in energy metabolism as well as in body anabolism. Recent studies have increased our knowledge of GH's mechanisms of action. In particular, insulin-like growth factor I (IGF-I), the main hormone mediating the principal GH effects and other protein structures (i.e. the binding proteins related to these two hormones), has been recognized as playing a crucial role. The biochemical aspects relating to the molecules of the GH/IGF-I axis have been described here. Furthermore, the belief that GH and IGF-I enhance performance has induced an 'abuse' of GH (and possibly of IGF-I) by competitive sports athletes and amateurs. The present study outlines the best methods available to uncover abuse, as well as a series of potential research projects to recognize doping. The review also underlines the principal variables measurable in the laboratory and summarizes published reference ranges of these parameters. These biochemical and laboratory profiles describe principal experimental approaches, with the hope that this will stimulate new ideas on the subject of detecting doping practices.

Simple and rapid reversed-phase gradient column liquid chromatography (LC) with fluorescence detection at different wavelengths was developed for the simultaneous analysis of all-trans, 13-cis, 9-cis retinoic acids, vitamin A palmitate and beta-carotene in galenicals. The assay results agreed with those obtained by an LC method with diode-array UV detection. A post-column on-line photochemical reactor (irradiation at 254 and 366 nm) was inserted between the LC column and the fluorescence detector to enhance the performance of the method. Two fluorescence spectra (photoreactor on and off) were obtained for each analyte which proved useful for the unambiguous identification of the various analytes.

Cell cycle transition defects of homozygous ataxia-telangiectasia (A-T) cells were studied by using a cell cycle flow calculation method, which evaluates the dynamics of cell cycle traverse. We compared five human lymphoblastoid cell lines (LCLs) from A-T homozygotes belonging to complementation group A (ARO, BRO, RJO) and group C (CSA, BMA) with three cell lines from healthy volunteers (KK-B2, MTB, HGL). The A-T cell lines ARO and BRO were derived from the same family. Cell growth and cell cycle traverse were followed for 72 h after X-irradiation with 1-6 Gy. LCLs from healthy volunteers immediately arrested in G1 in a dose-dependent pattern, while the A-T cells did not arrest in G1 until after 12 to 24 h. The time for the appearance of the G1 arrest of these cells was independent of complementation group. The delayed G1 arrest seen in the A-T cells paralleled a lack of induction of p53, as described by others. In respect to G2 arrest, A-T cells from complementation group C (CSA, BMA) arrested to the same extent as cells from healthy volunteers. On the other hand, the other LCLs from complementation group A arrested normally, while cells from ARO and BRO did not arrest in G2. The lack of G2 arrest in BRO cells was accompanied by unchanged cdc2p34 activity. In summary, a defective radiation-induced G1 arrest seems to be present in both complementation groups of A-T homozygotes, whereas a defective G2 arrest in not always observed. The defective G1 arrest seen in A-T cells may play an important role in tumor cell survival after exposure to therapeutic irradiation.

HPLC analyses of pharmaceutical dosage forms containing analgesics and related compounds (acetylsalicyclic acid, paracetamol, propyphenazone, caffeine and chlorpheniramine) were performed on C18 and cyano columns under reversed-phase conditions. The performance of the methods was enhanced by introducing postcolumn on-line photochemical derivatization in combination with a diode-array detection. The column effluents were subjected on-line to UV irradiation (254 nm) and the characteristic photo-induced spectral modifications were useful for the unambiguous identification of the various analgesic compounds. The proposed HPLC methods were successfully applied to the analysis of commercially available analgesic dosage forms.

A reversed phase liquid chromatographic (HPLC) method is proposed for the determination of sodium fusidate, a steroid antibiotic, in a commercial ointment using three different detectors: a) fluorescence detector, after a preliminary derivatization with 2-bromoacetyl-6-methoxynaphthalene (Br-AMN), b) UV detection and c) evaporative light scattering detector (ELSD). Fluorescent derivatization was applied directly to the diluted sample, without any purification, whilst UV and ELSD detectors involved a preliminary liquid-liquid extraction procedure to obtain accurate analysis data.

A simple difference spectrophotometric method has been developed for the selective and rapid determination of minoxidil in pharmaceutical formulations. The method is based on the characteristic modifications of the minoxidil UV spectrum induced by pH changes; a difference spectrum can be obtained and the absorbance A282.8 or the absorbance difference delta A = (A282.8-A259.2) can be used for the selective drug quantitation. As an alternative, a derivative second-order spectrophotometric method was also developed. The spectrophotometric methods were applied to the quality control of commercial minoxidil dosage forms and the results were comparable with those obtained by a reference HPLC procedure.

A sensitive reversed-phase HPLC method with fluorimetric detection was developed for the determination of salicilic acid in saliva samples. The method, involving a preliminary solid-phase extraction procedure, was found to be suitable for the determination of salivary salicilic acid concentrations following the administration of a single oral dose of some commercial acetylsalicilic acid formulations.

The use of the methyl ester of 4-(6-methoxynaphthalen-2-yl)-4-oxo-2-butenoic acid as a fluorogenic labelling reagent for the high-performance liquid chromatography (HPLC) of biologically important thiols (glutathione, cysteine, acetylcysteine, homocysteine, cysteamine, sodium 2-mercaptoethanesulphonate and thiola) was investigated. The compound reacts selectively and rapidly (10 min at ambient temperature and pH 7.5) with the thiols to give fluorescent adducts that can be separated by reversed-phase HPLC and detected fluorimetrically (lambda em = 450 nm; lambda em = 310 nm). Applications to the determination of L-cysteine and mesna in pharmaceutical formulations are described.