Elio Franco De Palo

A rapid urine creatinine assay by capillary zone electrophoresis

Using capillary zone electrophoresis, the urine creatinine (uCr) assay was validated in extemporaneous diluted urine, both in healthy subjects and athletes, with the uCr concentration as a reference value to compare excretion rates of other metabolites in the same samples. The electrokinetic sample injection was carried out at 10 kV per 10 s; UV absorbance detection was at 254 nm. Using standard samples, the creatinine migration mean time in 100 mmol/L acetate buffer, pH 4.4, was 3.3 ± 0.2 min; the repeatability for absolute migration mean time was 0.6% and peak height repeatability was 2.9%. The correlation coefficient of the standard curve was r = 0.999 and the detection limit was 23.1 μmol/L. Intra‐ and interassay coefficients of variation (CV) were 3.0 and 3.6%, respectively; recovery was 99 ± 3% and linearity was r = 0.98. Normal urine samples were diluted 1:80 in run buffer. The present CE urine creatinine assay showed a good correlation with HPLC and with Jaffé methods (r = 0.98 and r = 0.97, respectively; p < 0.0001). The uCr in the morning urine samples of 34 healthy males (M), 38 healthy females (F), and 83 male athletes (A) was 10.4 ± 6.1 mmol/L, 10.8 ± 8.1 mmol/L and 13.2 ± 6.5 mmol/L, respectively. The uCr difference (p < 0.02) between M and A and a correlation (p < 0.05) with age in A were observed.

Effects of Acute, Heavy-Resistance Exercise on Urinary Peptide Hormone Excretion in Humans

Abstract 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. Clin Chem Lab Med 2003; 41(10):13081313

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

Abstract 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.

Chiral separation and determination of ketamine and norketamine in hair by capillary electrophoresis.

Ketamine, traditionally available as racemic mixture, has recently become available in the form of the single S-enantiomer, due to its higher anaesthetic potency associated with faster recovery times. The different pharmaceutical forms and the different pharmacodynamics of the two enantiomers imply the need for a chiral method, since most available analytical methods for biological matrices are not enantioselective. The method herein showed consists of simple capillary zone electrophoresis (CZE) for the chiral separation of ketamine and its major metabolite, norketamine, in hair specimens. After liquid-liquid extraction, the samples were electrokinetically injected and analysed in CE (running buffer: 15mM Tris phosphate pH 2.5, containing HS-γ-CDs, 0.1%, w/v). A complete separation of both racemic ketamine and norketamine in the respective enantiomers was obtained in less than 10minutes. Limit of detection (LOD) and limit of quantification (LOQ) were 0.08ng/mg and 0.25ng/mg, respectively. Percent recovery varied from 49% to 91% for all four enantiomers. Matrix effect on spiked hair samples demonstrated values ranging from 63% to 119%. Linearity was estimated using a calibration curve consisting of five concentration levels for each enantiomer (0.5-8.0ng/mg); the regression coefficients (R(2)) of weighted (1/x(2)) linear regression were all >0.988. The method is suitable for the analysis of real-world hair samples in order to investigate ketamine chronic abuse and to discriminate between the type of abused drug, either single enantiomer or racemic drug.

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 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.

Natriuretic peptide fragments as possible biochemical markers of hypertension in the elderly

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

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

Abstract Dynamic exercise strongly affects atrial natriuretic peptides (ANP), in particular the mature bioactive αANP 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.

A new sample treatment for asialo-Tf determination with capillary electrophoresis: an added value to the analysis of CDT

BACKGROUND AND AIM The non-glycosylated glycoform of transferrin (Tf), known as asialo-Tf, was not selected (in favor of disialo-Tf) as the measurand for the standardization of carbohydrate deficient transferrin (CDT) determination because of a lower diagnostic sensitivity provided with the currently available analytical procedures for sera. However, asialo-Tf could provide an additional value to disialo-Tf in the CDT analysis employed in forensic toxicology contexts. The present work aimed at developing an easy sample preparation based on PEG precipitation in order to improve the detectability of asialo-Tf in capillary electrophoresis (CE). METHODS Equal volumes (35 μL) of serum and of 30% PEG-8000 were mixed and briefly vortexed. After centrifugation, the supernatant was iron saturated with a ferric solution (1:1, v/v). The mixture was analyzed in CE for asialo-Tf and disialo-Tf determination. RESULTS PEG-8000 precipitation allowed the improvement of the baseline in the electropherograms in terms of interferences reduction particularly in the asialo-Tf migration region. The detection of asialo-Tf was possible in 89% of samples with disialo-Tf above the cut-off limit, whereas only 16% of them showed asialo-Tf by employing the traditional sample preteatment. CONCLUSIONS Asialo-Tf represents an additional value to disialo-Tf as a biomarker of alcohol abuse in forensic toxicology.

Terbium chelation, a specific fluorescent tagging of human transferrin. Optimization of conditions in view of its application to the HPLC analysis of carbohydrate-deficient transferrin (CDT)

AbstractTransferrin (Tf) is the major iron-transporting protein in the human body and, for this reason, has been extensively studied in biomedicine. This protein undergoes a complex glycosylation process leading to several glycoforms, some of which are important in the diagnosis of alcohol abuse and of congenital glycosylation defects under the collective name of carbohydrate-deficient transferrin (CDT). Exploiting the Tf ability to bind not only iron but also other ions, specific attention has been devoted to binding activity towards Tb3+, which was reported to greatly enhance its intrinsic fluorescence upon the interaction with Tf. However, the structural properties of the Tb3+-Tf complex have not been described so far. In the present work, the formation of the Tf-Tb3+ complex has been investigated by the employment of several biophysical techniques, such as fluorescence resonance energy transfer (FRET), “native” mass spectrometry (MS), and near-UV circular dichroism (CD). Each method allowed the detection of the Tf-Tb3+ complex, yielding a specific signature. The interaction of Tb3+ with Fe3+-free Tf (apoTf) has been described in terms of stoichiometry, affinity, and structural effects in comparison with Fe3+. These experiments led to the first direct detection of the Tf-Tb3+ complex by MS, indicating a 1:2 stoichiometry and allowing the investigation of structural effects of metal binding. Either Tb3+ or Fe3+ binding affected protein conformation, inducing structural compaction to a similar extent. Nevertheless, near-UV CD and pH-dependence profiles suggested subtle differences in the coordination of the two metals by Tf side chains. Experimental conditions that promote complex formation have been identified, highlighting the importance of alkaline pH and synergistic ions, such as carbonate. On the basis of these studies, sample pretreatment, separation, and detection conditions of a high-performance liquid chromatographic method for CDT analysis are optimized, achieving relevant increase (by a factor of ∼3) of analytical sensitivity. Graphical abstractSchematic representation of HPLC-separated transferrin glycoforms detected by fluorescence emission of the terbium ions bound to the protein.