Urinary insulin-like growth factor-I measurement in an actual sport competition, an additional approach in laboratory antidoping tests

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

Asialo-transferrin: Biochemical aspects and association with alcohol abuse investigation

Asialo-human transferrin (asialo-hTf) is a glycoform of the human serum protein transferrin characterized by the lack of the sialic acid (SA) terminal unit. It is known that glycosylation micro-heterogeneity and the presence of SA are strongly involved in protein functioning and pathophysiological activities. Some hTf glycoforms are valuable biomarkers for the detection of both genetic defects of glycosylation and/or sialoform distribution changes. The detection of the carbohydrate deficient transferrin (CDT) glycoforms is currently a widely employed method for the diagnosis of chronic alcohol abuse. The physiological significance of asialo-hTf is still unclear, despite its important biological implications. The current knowledge suggests that asialo-hTf may be involved in regulation of iron transport and release at the hepatic level, which, consequently, could strongly be affected by alcohol consumption. For these reasons, a deeper understanding of asialo-hTf structure and its physiological role is required, and an improved method of its analysis would favor the detection of both chronic abuse and other habits of alcohol intake and/or misuse. Thus, suitable analytical methods possessing higher sensitivity and specificity in comparison with the currently available techniques are certainly recommended. The present review summarizes the studies on asialo-hTf structure, roles, and detection techniques mainly in relation to its possible use as a potentially additional useful biomarker of alcohol abuse, and underlines its prospective value as a forensic and diagnostic tool

Finger-prick dried blood spot and capillary electrophoresis: a challenge for alcohol abusers screening analysis

Introduction and aim Finger-prick related DBS (fpDBS) is a new and innovative specimen collection in clinical practice to develop a screening method for forensic toxicology study [1].Despite this, still very limitedly employed is the fpDBS specimens for capillary electrophoresis (CE) analyses. Transferrin glycoforms are a suitable parameter in alcohol abuse investigation and in particular the carbohydrate deficient transferrin (CDT), defined as the percentage distribution of less glycosilated transferrin, is increased after chronic sustained alcohol intake [2]. The aim of this study was to verify if the use of fpDBS could be suitable for CDT screening CE analysis. Method Human serum specimens were collected from volunteer subjects. fpDBS sample collection was performed by pricking the little finger, absorbing the capillary blood drops on DBS cards and letting it to air dry. Once dried, samples required a treatment, which included three easy steps: A) sample resuspension from fpDBS with an acid solution (to remove interfering circulating proteins, mainly haemoglobin); B) removing the paper from the sample solution and pH check (3<pH<4); C) sample neutralization (pH 7-8). The final sample was centrifuged and the supernatant was diluted with an iron rich solution before CE analysis. The CE running buffer was borate (pH 7.9) with diaminobutane (7.5 mmol/L), and the separations were performed within a 30 \u3bcm i.d. uncoated fused-silica capillary (length 60cm) using a constant voltage (30kV) and detection at 200 nm. The study was executed analysing fpDBS and parallel serum samples collected from each investigated subject and %CDT levels were measured using both high performance liquid chromatography (HPLC) and CE techniques. Results and conclusion The comparison between fpDBS and parallel serum CDT level, analysed by the traditional CE and HPLC methods, demonstrated significant correlation r2>0.85. In addition, statistical analysis confirmed that the concentration differences measured in DBS specimens were not relevant. Our results demonstrate how the acid treatment allows analysis in CE despite the small volumes and the large amount of various interfering compounds of whole blood. Therefore, the CE technique use coupled to the fpDBS procedure for CDT analysis expresses a simplified and inexpensive tool designed for use in population screening. In conclusion, the fpDBS CE procedure appear suitable in the forensic alcohol abuse investigations. References [1] CP Stove, AS Ingels, PM De Kesel, WE Lambert. Crit Rev Toxicol. 42 (2012) 230-43. [2] JR Delanghe, ML De Buyzere. Clin Chim Acta. 406 (2009) 1-7

Effects of acute, heavy-resistance exercise on urinary peptide hormone excretion in humans

IF(2007): 1.741To examine physical exerciserelated changes in urinary excretion of protein/peptide hormones and to correlate modifications with the general increase in postexercise proteinuria, urine Cpeptide, insulin and insulinlike growth factorI (IGFI) and their plasma concentrations were measured. Plasma and urinary Cpeptide, insulin and IGFI before (B-ex) and at the end (E-ex) of physical exercise (a 2.5-hour competition, 102 km) were analysed in 20 young cyclists. At Eex compared with Bex, concentration of urinary Cpeptide decreased slightly but significantly (21.3 +/- 2.7 vs. 13.5 +/- 1.7 nmol/l), but urinary insulin and urinary IGFI concentrations significantly increased at Eex (92.5 +/- 4.2 vs. 131.4 +/- 15.7 pmol/l and 10.0 +/- 2.1 vs. 33.6 +/- 3.8 pmol/l, respectively). Plasma insulin and plasma C-peptide significantly decreased, whereas plasma IGFI was unchanged. Urinary concentrations of total proteins and creatinine significantly increased. Both E-ex urinary C-peptide/urinary protein and urinary C-peptide/urinary creatinine ratios were significantly reduced. The correlation between C-peptide and insulin in plasma was confirmed at Bex as well as E-ex, but in urine only at Bex. An increased renal tubular reabsorption of C-peptide at the end of exercise might be suggested, but the expected values considering creatinine excretion were almost three times less. The E-ex urinary insulin concentration was higher than expected, considering the circulation levels, but lower when compared with the expected concentration considering creatinine excretion. Physical exercise proteinuria, related to an increased protein filtration and a saturation of the mechanisms responsible for the reabsorption, does not appear similar for all peptide hormone

CORRELATIONS OF GROWTH HORMONE (GH) AND INSULIN-LIKE GROWTH FACTOR I (IGF-I): EFFECTS OF EXERCISE AND ABUSE BY ATHLETES

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 land 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. (C) 2001 Elsevier Science B.V. All rights reserved

The use of finger-prick dried blood spots (fpdbs) and capillary electrophoresis for carbohydrate deficient transferrin (cdt) screening in forensic toxicology

Continued progress in chronic alcohol abuse investigation requires the development of less invasive procedures for screening purposes. The application of finger-prick and related dried blood spots (fpDBS) for CDT detection appears suitable for this aim. Therefore, the goal of this project was to develop a screening method for CDT using fpDBS with capillary electrophoresis (CZE) analysis.Blood samples prepared by finger-prick were placed on DBS cards and left to air dry; each dried fpDBS disc was shredded into small pieces and suspended in acid solution (60 \u3bcL of HCl 120 mmol/L). After centrifugation (10 min at 1500g) the collected sample was adjusted to pH 3.5. After an overnight incubation, the pH was neutralized and an iron rich solution was added. After 1 hour, CZE analysis was carried out. A group of 47 individuals was studied. Parallel serum samples collected from each investigated subject and the %CDT for each sample was measured using high performance liquid chromatography (HPLC) and CZE techniques. The fpDBS transferrin sialo isoform electropherograms were similar to those obtained with serum. Moreover, fpDBS CZE CDT percentage levels demonstrated significant statistical correlation with those obtained from serum for both HPLC and CZE %CDT (p< 0.01; r(2) = 0.8913 and r(2) = 0.8976 respectively), with %CDT from 0.8% to 13.7% for fpDBS and from 0.7% to 12.7% for serum. The newly developed fpDBS procedure for CDT analysis provides a simple and inexpensive tool for use in population screening. This article is protected by copyright. All rights reserved

Development and preliminary use of CDT analysis on dried blood spot (DBS) in forensic and administrative contexts

BACKGROUND-AIM Carbohydrate Deficient Transferrin (CDT) defines two minor glycoforms of transferrin (asialo and disialo-tansferrin), characterized by a reduced glycosylation degree, whose serum concentration increases after chronic sustained alcohol intake (60-80 g per day for at least 10 days). The use of finger-prick and related dried blood spots (fpDBS) is an innovative tool for blood sample collection in clinical and forensic toxicology. The aim of this work was to develop a screening method for CDT analysis based on the use of fpDBS coupled with capillary electrophoresis. METHODS Capillary blood drops collected by finger-prick were placed on DBS cards and left to air dry. Each dried fpDBS disc was sliced and suspended in acid solution. After centrifugation the sample pH was adjusted by 120 mmol HCl to pH 3-4. After overnight incubation the sample pH was neutralized and an iron rich solution was added. The resulting sample was analyzed by a validated CE method. The CDT level was expressed as %CDT (%ratio of disialo-Tf on total transferrin). The blood samples were obtained from volunteers of the forensic toxicology laboratory and from subjects submitted to blood testing for mandatory toxicological investigations. The DBS were analyzed in parallel with the sera of each investigated subject, using HPLC and CE techniques. The %CDT cut-offs used for the study were 1.80% and 1.90% for CE and HPLC, respectively. RESULTS The observed fpDBS transferrin glycoform CE patterns were comparable with serum CE CDT patterns. Moreover, a statistical correlation was demonstrated of fpDBS CDT percentage levels with both HPLC and CE % CDT (p< 0.01). This correlation was confirmed also by Passing-Bablok tests and Bland Altman test. The cut off proposed for this %CDT screening method was 1.6% demonstrating a sensitivity and specificity of about 75% and 90%, respectively. These data were calculated comparing %CDT by fpDBS CE vs serum HPLC, the latter considered the reference method. CONCLUSIONS The results of the study, even if preliminary, showed that fpDBS procedure coupled with CE for CDT analysis could express a simplified and inexpensive tool designed for use in population screening

Thanatochemistry at the crime scene: a microfluidic paper-based device for ammonium analysis in the vitreous humor

Most of the on-site approaches for inferring of the post-mortem interval are still based on observative data from the direct body inspection, whereas, objective and quantitative analyses, such as potassium in the vitreous humor, are require laboratory instrumentation and skilled personnel. The present paper presents a simple and low cost analytical method suitable for use at the crime scene for inferring the time since death. The method uses a microfluidic paper-based device (\u3bcPAD) for the determination of ammonium in the vitreous humor (VH) based on the selective interaction between the ammonium and the Nessler's reagent. The color change was measured in terms of "RGB distance" by using a simple and free smartphone application. The optimized device showed a limit of detection of 0.4\u202fmmol\u202fL-1, with between days precision less than 9.3% expressed as relative standard deviation, and accuracy between days from 94.5% to 104.5%. The selectivity of the Nessler's reaction was tested towards the main vitreous humor compounds, and no significant interferences were found. This paper-based analytical device was successfully used for the determination of ammonium ion in VH samples from forensic autopsies. The results obtained with the proposed method, although for a limited number of cases (n\u202f=\u202f25), showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover, in order to make the evaluation of results as simple as possible, a direct correlation between the color intensity, expressed as RGB distance, and the post-mortem interval was studied and a significant correlation was found (R2\u202f&gt;\u202f0.78). In conclusion, the present preliminary study showes that the proposed device could be an additional tool to the traditional methods for a more accurate, although still presumptive, estimation of the time of death directly at the crime scene

A 3D Origami Paper-Based Microfluidics Device for Creatine Analysis in Urine: A Disposable Tool for Identifying Urine Sample Adulteration by Dilution

The attendees will be receive detailed information on the use of a novel tech to control the integrity of urine samples using paper-based microfluidics technology. In the present case this technique will be applied to the analysis of creatinine, the most common analyte to control urine adulteration by dilution. Because of the low cost and portability of the developed devices, this method has a potential application out-side the specialized laboratories and particularly to control urine integrity immediately at the site of collection in doctor’s offices, in small laboratories and in occupational medicine centers. The relevance of the problem of sample tampering is well-known in forensic toxicology and sample dilution is the most used method to cheat toxicological controls. The prevalence of this phenomenon with a long history in analytical toxicology, is reported also in recent papers [1–3]. Among the criteria to assess urine integrity by ex-cluding dilution, the quantification of creatinine probably represents the most popular method. Although the majority of analytical methods offer adequate sensitivity and specificity, at present creatinine anal-ysis requires laboratory instrumentation which hinders the possibility of direct test of urine at the collection site. This hinders the immediate interaction with the urine donor, which could be important to prevent claims of post-collection counterfeiting. Since the first introduction by Whitesides et al. [4], the use of paper-based microfluidic devices (µPADs) for the development of chemical sensors has been extensively reported [5]. Among several approaches for producing µPADs, the use of commercial wax printers proved to be inexpensive and straightforward in the fabrication of the device. The procedure is based on two steps: (i) patterning chromatography paper into hydrophilic channels by fabricating hydrophobic barriers, and (ii) addition of the reagent to the hydrophilic portion of the paper sup-port. The sample is driven through the reagent zone as results of the wicking capacity of the paper without ex-ternal assistance. This approach does not require highly qualified personnel nor expensive instrumentation. Al-so, it can be performed on-site, enabling a prompt analytical response also in less-equipped environments. The advantages of µPADs have provided forensic science with reliable tools to face different forensic issues. On the grounds of the above considerations, the goal of the present work was to develop a low-cost device able to provide a rapid and sensitive colorimetric detection of creatinine in urine samples. This presentation will provide details of the developed procedure which was conceived as a first-line screening potentially to be con-firmed with laboratory instrumentation. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The test was based on three specific reactions for the detection of creatinine using picric acid, 3,5-dinitrobenzoic acid and Nessler’s reagent. The urine sample is transferred without any treatment directly onto the hydrophilic portion of the paper, and colorimetric reactions are developed in few minutes. The color change is measured in terms of "RGB dis-tance" by using a simple and free software for smartphone cameras. The device was also validated for quantita-tive determinations in terms of accuracy and precision. The optimized method was tested on real urine samples (n=53) using as reference a clinical chemistry method performed on immunoassay instrument. In conclusion, the perspective usefulness of paper-based microfluidics as a low-cost and easy to use technique in forensic toxicology will be presented with a specific focus on its possibilities of on-site analysis to prevent urine adulteration. Keywords Microfluidic paper-based devices (µPADs); Urine adulteration; forensic toxicology References [1] B. Holden, E.A. Guice, An Investigation of Normal Urine with a Creatinine Concentration Under the Cutoff of 20 mg/dL for Specimen Validity Testing in a Toxicology Laboratory , J. Forensic Sci. 59 (2014) 806–810. doi:10.1111/1556-4029.12386. [2] S.Y. Lin, H.H. Lee, J.F. Lee, B.H. Chen, Urine specimen validity test for drug abuse testing in workplace and court settings, J. Food Drug Anal. 26 (2018) 380–384. doi:10.1016/j.jfda.2017.01.001. [3] T. Arndt, Urine-creatinine concentration as a marker of urine dilution: Reflections using a cohort of 45,000 samples, Forensic Sci. Int. 186 (2009) 48–51. doi:10.1016/j.forsciint.2009.01.010. [4] A.W. Martinez, S.T. Phillips, M.J. Butte, G.M. Whitesides, Patterned paper as a platform for inexpensive, low-volume, portable bioassays., Angew. Chem. Int. Ed. Engl. 46 (2007) 1318–20. doi:10.1002/anie.200603817. [5] D.M. Cate, J.A. Adkins, J. Mettakoonpitak, C.S. Henry, Recent developments in paper-based microfluidic devices, Anal Chem. 87 (2015) 19–41. doi:10.1021/ac503968p

An origami microfluidic paper device for on-site assessment of urine tampering. First use of Nessler's reagent for the colorimetric determination of creatinine

The relevance of the problem of urine tampering is well-known in forensic toxicology, with sample dilution being the most used method to cheat toxicological controls. Among the criteria to assess urine integrity, the quantification of creatinine probably represents the most popular method. The present paper presents a simple and low-cost analytical device for on-site creatinine determination as first-line screening for urine dilution. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The device included three colorimetric reactions based on picric acid (PA-based reagent), 3,5-dinitrobenzoic acid (DNBA-based reagent), and Nessler’s reagent. The last one, to the best of our knowledge, has never been used before for creatinine determination. In order to assure the highest ease and economy of operation, the color detection and data processing were performed using a built-in smartphone camera and the associated software. The optimized device showed a detection limit of 0.02 g/L. The proposed method was used for the qualitative screening for urine dilution of 48 samples, showing a diagnostic sensitivity and specificity for PA-based, DNBA-based and Nessler’s reagent of 83.3%-80.0%, 72.2%-70.0%, and 100.0%-93.3% respectively, versus reference enzymatic method adopting a cut-off of 0.2 g/L. In conclusion, the present preliminary study shows that the proposed device could be a useful tool for on-site screening for urine tampering at the time of sample collection for toxicological testing