Plasma serotonin levels in Italian Fresian dairy cows.

The aim of this work was to investigate the metabolism of plasma serotonin or 5-hydroxytryptamine (5-HT), an important neurotransmitter, in Fresian dairy cows, a breed of zootechnical interest, using high-performance liquid chromatography with electrochemical detection. The subjects under study were at the stage of early lactation (n = 10; mean body weight 375 +/- 50 kg; average age of 3 years; body condition score 2.5), bred in a farm at an altitude of 150 m a.s.l. To evaluate animal welfare on this farm, which is closely connected to an animal's physiological status, tryptophan and cortisol levels (measured by enzyme-linked immunosorbent assay), together with levels of certain blood components [total proteins (TP), albumin, creatinine, glucose (Glu), triglycerides, phospholipids, total cholesterol, and aspartate transaminase, measured by spectrophotometry] were analyzed. The results obtained are discussed in comparison with reference values, taking into account the environmental living conditions. Measured plasma serotonin concentrations, which were lower than values reported for Brown Swiss dairy cows of a comparable age and diet, appeared to be affected by breed, temperature, blood sampling season, and altitude. Additional differences between the levels of plasma tryptophan, the amino acid precursor of serotonin, of the two breeds were comparable. Negative correlations between plasma tryptophan and plasma cortisol levels (r = -0.83, P < 0.005), plasma serotonin and plasma TP levels (r = -0.72, P < 0.05), or Glu levels (r = -0.77, P < 0.05) highlight the existence of a stress condition, which is connected to an energetic deficit related to lactation.

Differential effect of growth factors on hyaluronan synthase gene expression in fibroblasts exposed to oxidative stress.

The aim of this study was to evaluate how growth factors (PDGF-BB, EGF, and TGF-1beta) modulate hyaluronan synthase (HAS) activities in normal or stressed cultured human skin fibroblasts. The effects of concomitant treatment with cytokines and FeSO4 plus ascorbate on HAS mRNA expression, protein synthesis, and hyaluronic acid (HA) concentrations were also studied. Treatment of fibroblasts with growth factors up-regulated HAS gene expression and increased HAS enzymes and HA production. PDGF-BB induced HAS mRNA expression, protein synthesis, and HA production more efficiently than EGF and TGF-1beta. EGF was less effective than TGF-1beta. In addition, TGF-1beta reduced the expression and synthesis of HAS3, while PDGF-BB and EGF had the opposite effect. Concomitant treatment with growth factors and the oxidant was able to further increase HAS mRNA expression, once again with the exception of HAS3 with TGF-1beta. HAS protein synthesis was reduced, while HA levels were unaffected in comparison to those obtained from exposure to FeSO4 plus ascorbate alone. In conclusion, although growth factors plus the oxidant synergistically induced HAS mRNA expression in part, enzyme production was not correlated with this increase. Moreover, the increase in HAS mRNA levels was not translated into a consequent rise in HA concentration.

Chondroitin sulphate: antioxidant properties and beneficial effects.

Most biological molecules exhibit more than one function. In particular, many molecules have the ability to directly/indirectly scavenge free radicals and thus act in living organisms as antioxidant. During oxidative stress, the increase of these molecules levels seems to be a biological response that in synergism with the other antioxidant defence systems may protect cells from oxidation. Among these structures, chondroitin sulphate is a biomolecule which has increasingly focused the interest of many research groups due to its antioxidant activity. This review briefly summarises the action of chondroitin sulphate in reducing molecular damage caused by free radicals and associated oxygen reactants.

Improved high-performance liquid chromatographic method to estimate aminosugars and its application to glycosaminoglycan determination in plasma and serum.

An improved isocratic high-performance liquid chromatography (HPLC) method for the analysis of L-(-)-fucose. D-(+)-galactosamine, D-(+)-glucosamine, D-(+)-galactose, obtained by hydrolysis of glycosaminoglycans (GAGs) and D-(+)-glucose and D-(+)-mannose is described. The presence in circulation of GAGs, acid polysaccharide sequences of alternate monosaccharide units, aminosugar and uronic acid (galactose in keratan sulfate), has been measured in terms of their sugar components. To evaluate concentration of these circulating sugars we considered blood samples obtained from healthy humans. Plasma or serum was filtered through weak anion-exchange Ecteola-cellulose either untreated or after mild alkaline treatment. GAGs adhering to resin were recovered by salt elution, and desalted on Bio-Gel P-2 resin. GAG fractionation by charge was carried out on a strong anion exchanger. GAG composition was evaluated in terms of galactose and aminosugars, measured in HPLC by the proposed procedure using anion-exchange resin and pulsed amperometric detection. The mobile phase consisted of 0.02 M NaOH and elution was carried out at flow-rate of 1.0 ml/min. The amperometric detector was set as follows: t1 (0.5 s), E1 (+0.1 V); t2 (0.09 s), E2 (+0.6 V); t3 (0.05 s), E3 (-0.6 V). The analysis required 14 min. Calibration standard curves for the six analytes were linear from 0.25 to 40 microM. RSD values for intra- and inter-day variabilities were < or = 5.3% at concentrations between 0.25 and 40 microM. Accuracy, expressed as percentage error, ranged from - 16 to 14%. The method was specific and sensitive with quantitation limits of 1 pmol for L-(-)-fucose, D-galactosamine and D-glucosamine, 3 pmol for D-(+)-galactose and D-(+)-glucose and 5 pmol for D-(+)-mannose. The results of the assay showed higher GAG concentrations in serum than in plasma.

Factors affecting glycosaminoglycan concentration in normal human plasma.

Acid Glycosaminoglycans (GAGs) were measured, in terms of hexuronic acid, following alkaline treatment and Ecteola chromatography, in plasma obtained from healthy volunteers, blood-donors, amateur soccer players and university students, and from hospitalized subjects at the end of their convalescence. Diurnal variations of plasma GAG concentration, with significant decrease during the morning, were obtained in students and patients, suggesting hormonal influences. Furthermore, moderate modifications of plasma GAG concentration were observed in students following cyclo ergometer exercise which were consistent in each subject with cortisol mediated changes. However, the absolute value of plasma GAG concentration appears to be depending on the physical training of the subject, being significantly higher in the soccer players and in the blood-donors than in the other groups of subjects, chiefly composed of sedentary individuals. The intramuscular connective tissue is then suggested to represent a main site of origin of plasma GAGs.

Characteristics of the interactions between acid glycosaminoglycans and proteins in normal human plasma as revealed by the behaviour of the protein-polysaccharide complexes in ultrafiltration and chromatographic procedures.

Acid glycosaminoglycans were isolated from normal human plasma: (a) following fractionation of plasma (with protease inhibitors) on Sephadex G-200; (b) by ultrafiltration through membranes with retention of molecules above 50 kDa, with and without previous addition of NaCl, Triton X-100, urea, or guanidine HCl; (c) by filtering on Ecteola-cellulose either untreated plasma or after treatment with NaCl, urea, Triton X-100, papain or NaOH. More than 95% of plasma glycosaminoglycans interact with plasma proteins to give complexes that exhibit reproducible behaviour on Sephadex G-200 and are retained by ultrafiltration membranes, which the 12-20 kDa polysaccharide chains do filter. High charge plasma glycosaminoglycans show ionic interactions with proteins, while low charge glycosaminoglycan interactions are resistant to Ecteola charged groups, to 0.5% Triton and 4 M urea, while not to 4 M guanidine HC1. Some glycosaminoglycan-protein complexes appear resistant to proteolysis, suggesting that they may originate from lymphocytes. The simple method utilized for plasma GAG measurement may represent an useful tool in clinical practice.

Glycosaminoglycan concentrations in horse plasma and serum. Differences with other animal species and identification of affecting factors.

1. The measured values of acid glycosaminoglycan (GAG) concentration in plasma or in serum show significant differences between trained and untrained horses and among sedentary horses and other animal species (cattle, rabbit, sheep). 2. Diurnal variations in serum GAG levels are reported (cattle), and changes in plasma GAG concentrations after road transport (horses) and in late pregnancy (mares, cows), while sex, age and breed do not affect them.