Concentration and Composition of Serum and Plasma Glycosaminoglycans in Domestic Animal Species

Acid glycosaminoglycans (GAGs) were isolated from serum and/or plasma of some domestic animals and the composition of the isolated GAG mixtures were studied. Mean values of total GAG concentration, in terms of hexuronic acid, ranged from a maximum of about 12 mg/l in serum of calves, trained horses and sheep to about 9 mg/l in serum of cows and donkeys and in plasma of trained horses to about 6.5 mg/l in sedentary horse serum and rabbit plasma to a minimum of 4 mg/l in dog serum and sedentary horse plasma. Statistically significant differences in total GAG concentrations (P < 0.0005) were found in horses between plasma and serum and also between sedentary and trained subjects. Chondroitin sulphate was the main component in serum and plasma GAG mixtures, accounting for 81–84% of total GAGs in the examined animals, except in cow serum (72%), trained horse plasma (75.5%) and sheep serum (87%). Keratan sulphate-like structures, measured as galactose, ranged from 12% in sheep serum to 17% in cattle serum. Fucose was associated with galactose in GAG fractions, which supports the hypothesis that articular cartilage is among the sites of origin of circulating GAGs.     

A promising 31P NMR-multivariate analysis approach for the identification of milk phosphorylated metabolites and for rapid authentication of milk samples

A fast and reliable method for the identification of milk from different mammalians was developed by using ³¹P NMR metabolite profile of milk serum coupled to multivariate analysis (PCA and classification models UNEQ, SIMCA and K-NN). Ten milk samples from six different mammalians, relevant to human nutrition (human, cow, donkey, mare, goat, sheep), were analyzed and eight monophosphorylated components were identified and quantified: phosphocreatine (PCr), glycerophosphorylcholine (GPC), glycerophosphorylethanolamine (GPE), N-acetylglucosamine-1-phosphate (NAcGlu-1P), lactose-1-phosphate (Lac-1P), galactose-1-phosphate (Gal-1P), phosphorylcholine (PC), glucose-6-phosphate (Glu-6P). PCA showed interesting clustering based on the animal genus. K-NN can be successfully used to discriminate between donkey and cow samples while UNEQ class-modeling resulted more suitable for compliance verification. Results confirm the natural variability of milk samples among different species. These data highlight the great potentials of NMR/multivariate analysis combined method in the rapid analysis of phosphorylated milk serum metabolites for milk origin assessment and milk adulteration detection.     

Modified Bacillus thuringiensis Toxins and a Hybrid B. thuringiensis Strain Counter Greenhouse-Selected Resistance in Trichoplusia ni

Resistance of greenhouse-selected strains of the cabbage looper, Trichoplusia ni, to Bacillus thuringiensis subsp. kurstaki was countered by a hybrid strain of B. thuringiensis and genetically modified toxins Cry1AbMod and Cry1AcMod, which lack helix α-1. Resistance to Cry1AbMod and Cry1AcMod was >100-fold less than resistance to native toxins Cry1Ab and Cry1Ac.Insecticidal proteins from Bacillus thuringiensis are used widely for pest control, but evolution of resistance by pests can reduce their efficacy (3, 4, 6, 14). Resistance to B. thuringiensis toxins has been reported in field populations of four species of Lepidoptera, one species in response to sprays (3, 14) and three species in response to transgenic crops (10, 15, 16). Here, we focus on understanding and countering resistance to sprays of Bacillus thuringiensis subsp. kurstaki that evolved in commercial greenhouse populations of the cabbage looper, Trichoplusia ni (7, 17).We compared responses to single toxins and formulations of B. thuringiensis by two resistant strains (GipBtR and GlenBtR) and two related susceptible strains (GipS and GlenS) of T. ni. All four strains were started by the collection of larvae in 2001 from commercial greenhouses near Vancouver in British Columbia, Canada (7). Resistance evolved in the greenhouses in response to repeated sprays of DiPel (7), a formulation of B. thuringiensis subsp. kurstaki strain HD1 containing Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa (9). Previously reported concentrations required to kill 50% of larvae (LC₅₀s) indicated that, relative to a susceptible laboratory strain, initial resistance to DiPel was 113-fold in the Gip population (labeled T2c in reference 7) and 24-fold in the Glen population (labeled P5 in reference 7).We reared larvae on a wheat germ diet (5) at 26°C on a light-to-dark schedule of 16 h:8 h. GipS and GlenS were reared on diet without B. thuringiensis toxins, which allowed resistance to decline (7). To maintain resistance, GipBtR and GlenBtR were reared each generation on a diet treated with 5 or 10 mg of DiPel WP (Abbott Laboratories, Ontario, Canada) per milliliter of diet (7). In bioassays, groups of five third-instar larvae were put in 60-ml plastic cups containing diet, and mortality was assessed after 3 days by gently probing larvae for movement.We used diet overlay bioassays to evaluate the toxicity to GipBtR and GipS of the protoxin forms of Cry1Ab, Cry1Ac, Cry1AbMod, and Cry1AcMod produced in B. thuringiensis strains (12). Cry1AbMod and Cry1AcMod are genetically engineered variants of Cry1Ab and Cry1Ac, respectively, each lacking 56 amino acids from the amino-terminal region, including helix α-1 (12). An 80-μl aliquot containing distilled water and toxin was dispensed evenly over the surfaces of 2 ml of diet (a mean surface area of 7.1 cm²) and allowed to dry. Fifty to 200 larvae from each strain were tested at five to eight concentrations of each toxin.We used diet incorporation bioassays (7) to evaluate the toxicities of DiPel and Agree WG (Certis, Columbia, MD) to GipS, GipBtR, GlenS, and GlenBtR. Agree is a formulation of hybrid strain GC91, which was created from the conjugation-like transfer of a plasmid from B. thuringiensis subsp. kurstaki strain HD191 into B. thuringiensis subsp. aizawai strain HD135, and it contains Cry1Ac, Cry1C, and Cry1D (1, 8). DiPel and Agree were diluted in distilled water and mixed into diet (7). Twenty-five to 50 larvae from each strain were tested at six to seven concentrations of DiPel and Agree.We used probit analysis (13) to estimate the LC₅₀s and their 95% fiducial limits (FL), as well as the slopes of concentration-mortality lines and their standard errors. The mortality of larvae fed treated diet was not adjusted for the mortality of control larvae on untreated diet, because the control mortality was low (mean, 3.6%; range, 0 to 16%). LC₅₀s with nonoverlapping 95% FL are significantly different. Resistance ratios were calculated as the LC₅₀ of a resistant strain (GipBtR or GlenBtR) divided by the LC₅₀ of its susceptible counterpart (GipS or GlenS).The genetically modified toxins Cry1AbMod and Cry1AcMod were much more effective than the native toxins Cry1Ab and Cry1Ac against larvae of T. ni from the resistant GipBtR strain (Table ​(Table1).1). Resistance ratios of GipBtR were 580 for Cry1Ab and 1,400 for Cry1Ac but only 5.5 for Cry1AbMod and 9.3 for Cry1AcMod (Table ​(Table1).1). Against GipBtR, the LC₅₀ was 53-fold higher for Cry1Ab than for Cry1AbMod and 11-fold higher for Cry1Ac than for Cry1AcMod (Table ​(Table1).1). Against GipS, however, the LC₅₀ was 2-fold higher for Cry1AbMod than for Cry1Ab and 14-fold higher for Cry1AcMod than for Cry1Ac (Table ​(Table11).

TABLE 1.

Responses of resistant (GipBtR and GlenBtR) and susceptible (GipS and GlenS) strains of T. ni to native toxins (Cry1Ab and Cry1Ac), modified toxins (Cry1AbMod and Cry1AcMod), and formulations (DiPel and Agree)

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 -(−)-fucose, -(+)-galactosamine, -(+)-glucosamine, -(+)-galactose, obtained by hydrolysis of glycosaminoglycans (GAGs) and -(+)-glucose and -(+)-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 μM. RSD values for intra- and inter-day variabilities were ≤5.3% at concentrations between 0.25 and 40 μM. Accuracy, expressed as percentage error, ranged from −16 to 14%. The method was specific and sensitive with quantitation limits of 1 pmol for -(−)-fucose, -galactosamine and -glucosamine, 3 pmol for -(+)-galactose and -(+)-glucose and 5 pmol for -(+)-mannose. The results of the assay showed higher GAG concentrations in serum than in plasma.     

Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment

Many findings demonstrated that Glycosaminoglycans (GAGs) and Proteoglycans (PGs) possess antioxidant activity. Collagen-induced arthritis (CIA) is an experimental animal model similar to human rheumatoid arthritis (RA) in which free radicals are involved. Sodium salicylate can be used as a chemical trap for hydroxyl radicals (OH •), the most damaging reactive oxygen species (ROS), yielding 2,5-dihydroxybenzoic acid), (2,5-DHBA) and 2,3-dihydroxybenzoic acid (2,3-DHBA). The measurement of these two acids in the plasma allows to indirectly assess the production of OH • radicals. The aim of the study was to investigate the effect of hyaluronic acid (HYA) (30 mg/kg i.p.) or chondroitin-4-sulphate (C4S) (30 mg/kg i.p.), on free radical production in Lewis rats subjected to CIA. After the immunization with bovine collagen type II in complete Freund's adjuvant, rats developed an erosive hind paw arthritis, that produced high plasma OH • levels assayed as 2,3-DHBA and 2,5-DHBA, primed lipid peroxidation, evaluated by analyzing conjugated dienes (CD) in the articular cartilage; decreased the concentration of endogenous vitamin E (VE) and catalase (CA) in the joint cartilage; enhanced macrophage inflammatory protein-2 (MIP-2) serum levels and increased elastase (ELA) evaluated as an index of activated leukocyte polymophonuclear (PMNs) accumulation in the articular joints. The administration of HYA and C4S starting at the onset of arthritis (day 11) for 20 days, limited inflammation and the clinical signs in the knee and paw, reduced OH • production, decreased CD levels, partially restored the endogenous antioxidants VE and CA, reduced MIP-2 serum levels and limited PMNs infiltration. The results indicate that the GAGs HYA and C4S significantly reduce free radical production in CIA and could be used as a tool to investigate the role of antioxidants in RA.     

Ubiquinol and a Coenzyme Q Reducing System Protect Platelet Mitochondrial Function of Transfusional Buffy Coats from Oxidative Stress

The conditions under which Coenzyme Q (CoQ) may protect platelet mitochondrial function of transfusional buffy coats from aging and from induced oxidative stress were investigated. The Pasteur effect, i.e. the enhancement of lactate production after inhibition of mitochondrial respiratory chain, was exploited as a marker of mitochondrial function as it allows to calculate the ratio of mitochondrial ATP to glycolytic ATP. Reduced CoQ 10 improves platelet mitochondrial function of transfusional buffy coats and protects the cells from induced oxidative stress. Oxidized CoQ is usually less effective, despite the presence, shown for the first time in this study, of quinone reductase activities in the platelet plasma membranes. The addition of a CoQ reducing system to platelets is effective in enhancing the protection of platelet mitochondrial function from the oxidative stress. The results support on one hand a possibility of protection of mitochondrial function in aging by exogenous CoQ intake, on the other a possible application in protection of transfusional buffy coats from storage conditions and oxidative deterioration.     

The antioxidant and antifibrogenic effects of the glycosaminoglycans hyaluronic acid and chondroitin-4-sulphate in a subchronic rat model of carbon tetrachloride-induced liver fibrogenesis

Hepatic fibrosis involves the interplay of many factors including reactive oxygen species. Recent reports described antioxidant properties of glycosaminoglycans (GAGs). Since several findings have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) may act as antioxidant molecules, the aim of this research was to evaluate the antioxidant effects of HYA and C4S treatment in a rat model of liver fibrosis. The effect on tissue inhibitors of metalloproteinases (TIMPs) was also studied. Liver fibrosis was induced in rats by eight intraperitoneal injections of CCl4, twice a week for 6 weeks. HYA or C4S alone (25 mg/kg) or HYA and C4S in combination (12.5 + 12.5 mg/kg) were administered daily by the same route during the 6 weeks. At the end of the 6-week treatment period (24 h after the last dose of GAGs), the following parameters were evaluated: (1) serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as index of hepatic cell disruption; (2) hepatic conjugated dienes (CD), as index of lipid peroxidation; (3) hepatic TIMPs activity and expression; (4) hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, as index of endogenous defences; (5) hepatic hydroxyproline, as index of collagen deposition. CCl4-induced liver fibrosis enhanced lipid peroxidation and TIMPs activation, increased ALT and AST, depleted antioxidants SOD and GPx, and caused collagen deposition in liver tissue. Treatment with GAGs, especially when in combination, successfully reduced ALT and AST rise, lipid peroxidation by evaluating conjugated dienes, TIMPs activation and mRNA expression, partially restored SOD and GPx activities, and limited collagen deposition in the hepatic tissue. The data obtained showed that these molecules were able to limit hepatic injury induced by chronic CCl4 intoxication and especially limited liver fibrosis. They also confirm that HYA and C4S may exert antioxidant mechanism, while reduction of TIMPs expression suggests that GAGs may influence MMPs and TIMPs imbalance in liver fibrosis.