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.

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 (µPAD) 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 mmol L-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 = 25), 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 > 0.78). 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.

Development of a low cost gas diffusion device for ammonia detection in the vitreous humor and its preliminary application for estimation of the time since death.

A simple and low cost analytical device is described for the determination of ammonium in the vitreous humor suitable for inferring the post mortem interval in forensic cases. The device is based on ammonia formation from ammonium ion by means of NaOH addition to the vitreous humor sample and its detection with a pH chemical indicator in the gas phase above the vitreous humor sample. From the gas phase, ammonia diffuses through a polymeric membrane and it is trapped and detected with a droplet of pH indicator thymol blue. The color change of the droplet is measured using a smartphone camera. Under optimal conditions, the device showed a limit of detection of 0.2mM, with between days precision of ≤ 15% expressed as relative standard deviation, and an accuracy between days from 88.3% to 114.5%. This homemade gas diffusion analytical device was successfully used for the determination of ammonia in vitreous humor samples from forensic autopsies. The results obtained with the proposed method, although for a limited number, showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover a significant correlation was also found between the results of the present method and the time elapsed since death by a simple evaluation of the color intensity. In conclusion, this preliminary study showed that the proposed device, after adequate validation, could be a promising tool for a presumptive estimation of the time since death directly at the crime scene.

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 carbohydrate deficient transferrin (CDT) detection appears suitable for this aim. Therefore, the goal of this project was to develop a screening method for CDT using fpDBS with 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 µL of HCl 120 mmol/L). After centrifugation (10 min at 1500 × g), the collected sample was adjusted to pH 3.5. After an overnight incubation, the pH was neutralised and an iron rich solution was added. After 1 h, CZE analysis was carried out. A group of 47 individuals was studied. Parallel serum samples were collected from each investigated subject and the %CDT for each sample was measured using 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; r2 = 0.8913 and 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.

Human saliva total protein levels by AV17 pigment based analysis: validation, stability and short-term variation studies.

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 microL) 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.

Cortisol assays and diagnostic laboratory procedures in human biological fluids.

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.

Human saliva cortisone and cortisol simultaneous analysis using reverse phase HPLC technique.

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.

Effects of two different types of exercise on GH/IGF axis in athletes. Is the free/total IGF-I ratio a new investigative approach?

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.

Saliva specimen: a new laboratory tool for diagnostic and basic investigation.

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.

Growth hormone isoforms and segments/fragments: molecular structure and laboratory measurement.

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.

Growth hormone isoforms, segments/fragments: does a link exist with multifunctionality?

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.

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.

Effect of prolonged physical exercise on urinary proANP1-30 and proANP31-67.

Dynamic exercise strongly affects atrial natriuretic peptides (ANP), in particular the mature bioactive alphaANP and the proANP fragments, namely proANP1-98, proANP1-30 and proANP31-67. The proANPs influence kidney functions and their plasma levels increase after physical exercise. We measured urinary proANP1-30 and proANP31-67 levels before and at the end of physical exercise in 28 well-trained male cyclists. For the first time, the proANP1-30 and proANP31-67 urinary levels in athletes before and at the end of a prolonged agonistic bicycle race were measured. Urinary creatinine and total proteins were also measured. The urinary proANP31-67, creatinine and total protein levels were significantly higher at the end of exercise than before. In contrast, proANP1-30/protein and proANP31-67/protein ratios decreased after exercise. Even proANP1-30/creatinine and proANP31-67/creatinine ratios were lower after exercise. A significant correlation between proANP1-30 and proANP31-67 urinary levels at the end of exercise was found. The proANP31-67/creatinine ratio before and after exercise also showed a significant correlation. The variation of urinary proANP fragments confirmed their possible role in physical exercise. In particular, it could be interpreted as a response of the body or kidney to renal impairment occurring during exercise.

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

To examine physical exercise-related changes in urinary excretion of protein/peptide hormones and to correlate modifications with the general increase in post-exercise proteinuria, urine C-peptide, insulin and insulin-like growth factor-I (IGF-I) and their plasma concentrations were measured. Plasma and urinary C-peptide, insulin and IGF-I before (Bex) and at the end (Eex) of physical exercise (a 2.5-hour competition, 102 km) were analysed in 20 young cyclists. At Eex compared with Bex, concentration of urinary C-peptide decreased slightly but significantly (21.3 +/- 2.7 vs. 13.5 +/- 1.7 nmol/l), but urinary insulin and urinary IGF-I 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 IGF-I was unchanged. Urinary concentrations of total proteins and creatinine significantly increased. Both Eex 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 Eex, 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 Eex 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 hormones.

The measurement of insulin-like growth factor-I (IGF-I) concentration in random urine samples.

The aim of the present study was to assess a suitable expression of the urinary concentration of a protein/ peptide hormone such as insulin-like growth factor-I (IGF-I), measured in the urine of healthy individuals when the specimen collection is executed randomly. One hundred and twenty male subjects were divided by age into four groups, namely healthy sedentary young (SYA) and older (SOA) adults, older (OC) and young (YC) children. In a single urine specimen, randomly collected during the morning from each individual, total urinary IGF-I was measured by immunoradiometric method, and urinary creatinine (uCr) and total proteins (utPr) were measured by capillary electrophoresis and spectrophotometric methods, respectively. The urinary IGF-I concentrations were not significantly different in all groups investigated and they were (mean +/- SD): 82.7 +/- 82.8 ng/l, 103.5 +/- 83.3 ng/l, 80.4 +/- 64.4 ng/l in OC, SYA and SOA, respectively; only in the YC group there was a tendency to higher values (125.2 +/- 93.2 ng/l) compared with the other groups. utPr ranged from 26 to 40 mg/l and did not demonstrate significant differences between groups. The urinary IGF-I correlated with uCr and utPr, and statistical significance was observed in all measurements. The measurement of urinary IGF-I in random urine and its ratio to utPr is an innovative, useful way of investigation of urinary protein/peptide hormones.

Urinary insulin-like growth factor I in athletes, before and after physical exercise, and in sedentary subjects.

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.