Enrico Cappellin

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.

Plasma lactate, GH and GH-binding protein levels in exercise following BCAA supplementation in athletes

Summary. Branched chain amino acids (BCAA) stimulate protein synthesis, and growth hormone (GH) is a mediator in this process. A pre-exercise BCAA ingestion increases muscle BCAA uptake and use. Therefore after one month of chronic BCAA treatment (0.2 g kg−1 of body weight), the effects of a pre-exercise oral supplementation of BCAA (9.64 g) on the plasma lactate (La) were examined in triathletes, before and after 60 min of physical exercise (75% of VO2max). The plasma levels of GH (pGH) and of growth hormone binding protein (pGHBP) were also studied. The end-exercise La of each athlete was higher than basal. Furthermore, after the chronic BCAA treatment, these end-exercise levels were lower than before this treatment (8.6 ± 0.8 mmol L−1 after vs 12.8 ± 1.0 mmol L−1 before treatment; p < 0.05 [mean ± std. err.]). The end-exercise pGH of each athlete was higher than basal (p < 0.05). Furthermore, after the chronic treatment, this end-exercise pGH was higher (but not significantly, p = 0.08) than before this treatment (12.2 ± 2.0 ng mL−1 before vs 33.8 ± 13.6 ng mL−1 after treatment). The end-exercise pGHBP was higher than basal (p < 0.05); and after the BCAA chronic treatment, this end-exercise pGHBP was 738 ± 85 pmol L−1 before vs 1691 ± 555 pmol L−1 after. pGH/pGHBP ratio was unchanged in each athlete and between the groups, but a tendency to increase was observed at end-exercise.The lower La at the end of an intense muscular exercise may reflect an improvement of BCAA use, due to the BCAA chronic treatment. The chronic BCAA effects on pGH and pGHBP might suggest an improvement of muscle activity through protein synthesis.

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

Plasma atrial natriuretic peptide (ANP) fragments proANP (1–30) and proANP (31–67) measurements in chronic heart failure: a useful index for heart trasplantation?

The family of the atrial natriuretic peptides, proANP fragments and the active αANP, 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.

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.

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.

Physiological and clinical implications of proANP(1-98) circulating levels in the perioperative phase of liver transplantation.

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.

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.