Paolo Spinella

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.

Effect of licorice on the reduction of body fat mass in healthy subjects

The history of licorice, as a medicinal plant, is very old and has been used in many societies throughout the millennia. The active principle, glycyrrhetinic acid, is responsible for sodium retention and hypertension, which is the most common side-effect. We show an effect of licorice in reducing body fat mass. We studied 15 normalweight subjects (7 males, age 22–26 yr, and 8 females, age 21–26 yr), who consumed for 2 months 3.5 g a day of a commercial preparation of licorice. Body fat mass (BFM, expressed as percentage of total body weight, by skinfold thickness and by bioelectrical impedance analysis, BIA) and extracellular water (ECW, percentage of total body water, by BIA) were measured. Body mass index (BMI) did not change. ECW increased (males: 41.8±2.0 before vs 47.0±2.3 after, p<0.001; females: 48.2±1.4 before vs 49.4±2.1 after, p<0.05). BFM was reduced by licorice: (male: before 12.0±2.1 vs after 10.8±2.9%, p<0.02; female: before 24.9±5.1 vs after 22.1±5.4, p<0.02); plasma renin activity (PRA) and aldosterone were suppressed. Licorice was able to reduce body fat mass and to suppress aldosterone, without any change in BMI. Since the subjects were consuming the same amount of calories during the study, we suggest that licorice can reduce fat by inhibiting 11β-hydroxysteroid dehydrogenase Type 1 at the level of fat cells.

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.

GH/IGF system, cirrhosis and liver transplantation

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.

IGF-I/IGFBP system: metabolism outline and physical exercise.

The GH/IGF-I system plays a well-known hormonal role and its effects, mainly anabolic and insulin-sensitizing, are mediated through endocrine as well as paracrine/autocrine mechanisms. This system includes the binding proteins, namely GH binding proteins and IGF-I binding proteins (IGFBP). As expected, this axis plays a key role in organism modification in consequence of a physical exercise. Physical activity, training, and exercise capacity chiefly involve anabolism process modifications of various tissues, in particular muscular adjustments. Numerous investigators found a correlation among the level of exercise tolerance, muscle strength or walking speed and IGF-I/IGFBP-3 concentrations. However, also inverse and absent correlations between circulating IGF-I concentrations and acute or chronic exercise responses have been reported. IGF-I is generally accepted as an important GH mediator with metabolic effects, through both endocrine and paracrine or autocrine mechanisms. GH is the main regulator of the hepatic synthesis of IGF-I and IGFBP-3, which is the most abundant IGF carrier in human plasma. Recently, it has been shown that the physical exercise stimulatory impact on skeletal muscles is mediated through an increased local IGF-I synthesis with an IGFPB involvement. An absent association of exercise performance and circulating IGF-I may indicate that exercise will exert muscle strength by predominately locally derived paracrine or autocrine mediators rather than endocrine circulating IGF-I. The present review considers the general aspects of the IGF/IGFPB system and the role of the IGF/IGFPB system in relation to physical exercise (type, duration, etc.) taking into account the training aspects.

Epigallocatechin-3-gallate reduces inflammation induced by calcium pyrophosphate crystals in vitro

Although osteoarthritis (OA) is defined as a cartilage disease, synovitis involving mononuclear cell infiltration and overexpression of proinflammatory mediators is common in early and late OA. Calcium crystals deposition is thought to be a factor that likely contributes to synovial inflammation. In recent years, significant interest has emerged in the beneficial health effects attributed to the green tea polyphenols and in particular to epigallocatechin-3-gallate (EGCG). It has been demonstrated that some of the actions of EGCG are linked to its ability to interfere with cell membranes. The objective of this study was to evaluate the influence of EGCG in some inflammatory aspects of OA and whether EGCG is able to interfere with membrane organization. We assessed the effect of EGCG on the production of proinflammatory cytokines and chemokines released by human fibroblast-like synoviocytes (FLS) and THP-1 cells stimulated with calcium pyrophosphate (CPP) crystals in presence of methyl-β-cyclodextrin (MβCD), a cholesterol-removing agent that disturbs lipid raft structures. The chemotactic effect of culture supernatants was also evaluated. EGCG inhibited interleukin (IL)-1β, transforming growth factor beta, IL-8, and chemokine (C–C motif) ligand 2 (CCL2) release by stimulated FLS and/or THP-1 cells in a dose-dependent manner. Supernatants of CPP-stimulated cells induced the migration of neutrophils and mononuclear cells which decreased in a dose-dependent manner in the presence of EGCG. EGCG increased cell viability when added to THP-1 cells treated with MβCD. Furthermore, MβCD enhanced the inflammatory response to CPP crystals increasing IL-8 and CCL2 secretion which was inhibited by EGCG in a dose-dependent manner. This study showed that EGCG is able to reduce the inflammatory response induced by CPP crystals in vitro. The identification of EGCG as dietary supplement capable of affording protection or modulating the inflammatory response to CPP crystals may have important implications in the prevention and treatment of OA and crystal-related arthropathies.

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.

Free carnitine and acetyl carnitine plasma levels and their relationship with body muscular mass in athletes

SummaryThe purpose of the present study was to investigate the relationship between plasma carnitine concentration and body composition variation in relation to muscular and fat masses since there is no experimentally proved correlation between plasma carnitine and body masses. We used bioelectric impedance analysis (BIA), to determine body composition and to have a complete physical fitness evaluation. The post-absorptive plasma free carnitine and acetyl carnitine plasma levels, body composition as Fat-Free Mass (FFM) and Fat Mass (FM) in kg, as well as in percent of body mass, were analysed in 33 healthy subjects. A significant negative correlation was found between plasma acetyl carnitine and FFM in weight (kg) as well as in percent of body mass (respectively p < 0.0001; p < 0.01); a significant positive correlation was found only between FM in percent and plasma acetyl carnitine (p < 0.01). The observed negative correlation between plasma acetyl carnitine and muscular mass variation might reflect an oxidative metabolic muscle improvement in relation to muscular fat free mass increment and might be evidence that muscle metabolism change is in relation to plasma acetyl carnitine concentration.