Impact-induced muscle damage and urinary pterins in professional rugby: 7,8-dihydroneopterin oxidation by myoglobin.

Muscle damage caused through impacts in rugby union is known to increase oxidative stress and inflammation. Pterins have been used clinically as markers of oxidative stress, inflammation, and neurotransmitter synthesis. This study investigates the release of myoglobin from muscle tissue due to force-related impacts and how it is related to the subsequent oxidation of 7,8-dihydroneopterin to specific pterins. Effects of iron and myoglobin on 7,8-dihydroneopterin oxidation were examined in vitro via strong cation-exchange high-performance liquid chromatography (SCX-HPLC) analysis of neopterin, xanthopterin, and 7,8-dihydroxanthopterin. Urine samples were collected from 25 professional rugby players pre and post four games and analyzed for myoglobin by enzyme-linked immunosorbent assay, and 7,8-dihydroneopterin oxidation products by HPLC. Iron and myoglobin oxidized 7,8-dihydroneopterin to neopterin, xanthopterin, and 7,8-dihydroxanthopterin at concentrations at or above 10 µM and 50 µg/mL, respectively. All four games showed significant increases in myoglobin, neopterin, total neopterin, biopterin, and total biopterin, which correlated between each variable (P < 0.05). Myoglobin and iron facilitate 7,8-dihydroneopterin oxidation to neopterin and xanthopterin. In vivo delocalization of myoglobin due to muscle damage may contribute to oxidative stress and inflammation after rugby. Increased concentrations of biopterin and total biopterin may indicate production of nitric oxide and monoamine neurotransmitters in response to the physical stress.

Assessing the Effectiveness of Selected Biomarkers in the Acute and Cumulative Physiological Stress Response in Professional Rugby Union through Non-invasive Assessment.

Rugby union is a sport involving high force and frequency impacts making the likelihood of injury a significant risk. The aim of this study was to measure and report the individual and group acute and cumulative physiological stress response during 3 professional rugby games through non-invasive sampling. 24 professional rugby players volunteered for the study. Urine and saliva samples were collected pre and post 3 matches. Myoglobin, salivary immunoglobulin A, cortisol, neopterin and total neopterin (neopterin+7,8-dihydroneopterin) were analysed by high performance liquid chromatography or enzyme linked immunosorbent assay. Significant increases in cortisol, myoglobin, neopterin and total neopterin when urine volume was corrected with specific gravity were observed (p<0.05). Significant decreases in salivary immunoglobulin A concentration were observed for games 1 and 2 while secretion rate decreased after games 2 and 3. Significant decreases were seen with the percent of 7,8-dihydroneopterin being converted to neopterin following games 2 and 3. The intensity of 3 professional rugby games was sufficient to elicit significant changes in the physiological markers selected for our study. Furthermore, results suggest the selected markers not only provide a means for analysing the stress encountered during a single game of rugby but also highlight the unique pattern of response for each individual player.

Spectral CT of carotid atherosclerotic plaque: comparison with histology.

OBJECTIVE: To distinguish components of vulnerable atherosclerotic plaque by imaging their energy response using spectral CT and comparing images with histology. METHODS: After spectroscopic calibration using phantoms of plaque surrogates, excised human carotid atherosclerotic plaques were imaged using MARS CT using a photon-processing detector with a silicon sensor layer and microfocus X-ray tube (50 kVp, 0.5 mA) at 38-µm voxel size. The plaques were imaged, sectioned and re-imaged using four threshold energies: 10, 16, 22 and 28 keV; then sequentially stained with modified Von Kossa, Perl's Prussian blue and Oil-Red O, and photographed. Relative Hounsfield units across the energies were entered into a linear algebraic material decomposition model to identify the unknown plaque components. RESULTS: Lipid, calcium, iron and water-like components of plaque have distinguishable energy responses to X-ray, visible on spectral CT images. CT images of the plaque surface correlated very well with histological photographs. Calcium deposits (>1,000 µm) in plaque are larger than iron deposits (<100 µm), but could not be distinguished from each other within the same voxel using the energy range available. CONCLUSIONS: Spectral CT displays energy information in image form at high spatial resolution, enhancing the intrinsic contrast of lipid, calcium and iron within atheroma. KEY POINTS: Spectral computed tomography offers new insights into tissue characterisation. Components of vulnerable atherosclerotic plaque are spectrally distinct with intrinsic contrast. Spectral CT of excised atherosclerotic plaques can display iron, calcium and lipid. Calcium deposits are larger than iron deposits in atheroma. Spectral CT may help in the non-invasive detection of vulnerable plaques.

Potential to inhibit growth of atherosclerotic plaque development through modulation of macrophage neopterin/7,8-dihydroneopterin synthesis.

The rise in plasma neopterin observed with increasing severity of vascular disease is a strong indicator of the inflammatory nature of atherosclerosis. Plasma neopterin originates as the oxidation product of 7,8-dihydroneopterin secreted by gamma-interferon stimulated macrophages within atherosclerotic plaques. Neopterin is increasingly being used as a marker of inflammation during clinical management of patients with a range of disorders including atherosclerosis. Yet the role of 7,8-dihydroneopterin/neopterin synthesis during the inflammatory process and plaque formation remains poorly understood and controversial. This is partially due to the unresolved role oxidants play in atherosclerosis and the opposing roles of 7,8-dihydroneopterin/neopterin. Neopterin can act as pro-oxidant, enhancing oxidant damage and triggering apoptosis in a number of different cell types. Neopterin appears to have some cellular signalling properties as well as being able to chelate and enhance the reactivity of transition metal ions during Fenton reactions. In contrast, 7,8-dihydroneopterin is also a radical scavenger, reacting with and neutralizing a range of reactive oxygen species including hypochlorite, nitric oxide and peroxyl radicals, thus protecting lipoproteins and various cell types including macrophages. This has led to the suggestion that 7,8-dihydroneopterin is synthesized to protect macrophages from the oxidants released during inflammation. The oxidant/antioxidant activity observed in vitro appears to be determined both by the relative concentration of these compounds and the specific chemistry of the in vitro system under study. How these activities might influence or modulate the development of atherosclerotic plaque in vivo will be explored in this review.

Protection of U937 cells from free radical damage by the macrophage synthesized antioxidant 7,8-dihydroneopterin.

Interferon-gamma stimulation of human macrophages causes the synthesis and release of neopterin and its reduced form 7,8-dihydroneopterin (7,8-NP). The purpose of this cellular response is undetermined but in vitro experiments suggests 7,8-NP is an antioxidant. We have found 7,8-NP can protect monocyte-like U937 cells from oxidative damage. 7,8-NP inhibited ferrous ion and hypochlorite mediated loss of cell viability. Fe++ mediated lipid peroxidation was effectively inhibited by 7,8-NP, however, no correlation was found between peroxide concentration and cell viability. Hypochlorite was scavenged by 7,8-NP, preventing the loss of cell viability. 7,8-NP was less effective in inhibiting H2O2-mediated loss of cell viability with significant inhibition only occurring at high 7,8-NP concentrations. Analysis of cellular protein hydrolysates showed none of the oxidants caused the formation of any protein bound DOPA or dityrosine but did show 7,8-NP prevented the loss of cellular tyrosine by HOCl. Our data suggests macrophages may synthesize 7,8-NP for antioxidant protection during inflammatory events in vivo.

Protection of erythrocytes by the macrophage synthesized antioxidant 7,8 dihydroneopterin.

Neopterin and the reduced form, 7,8-dihydroneopterin (78NP) are pteridines released from macrophages when stimulated with gamma-interferon in vivo. The role of 78NP in inflammatory response is unknown though neopterin has been used clinically as a marker of immune cell activation, due to its very fluorescent nature. Using red blood cells as a cellular model, we demonstrated that micromolar concentrations can inhibit or reduce red blood cell haemolysis induced by 2,2'-azobis(amidinopropane)dihydrochloride (AAPH), hydrogen peroxide, or hypochlorite. One hundred microM 78NP prevented HOCl haemolysis using a high HOCl concentration of 5 micromole HOCl/10(7) RBC. Fifty microM 78NP reduced the haemolysis caused by 2 mM hydrogen peroxide by 39% while the same 78NP concentration completely inhibited haemolysis induced by 2.5 mM AAPH. Lipid peroxidation levels measured as HPLC-TBARS were not affected by addition of 78NP. There was no correlation between lipid oxidation and cell haemolysis suggesting that lipid peroxidation is not essential for haemolysis. Conjugated diene measurements taken after 6 and 12 hour exposure to hydrogen peroxide support the TBARS data. Gel electrophoresis of cell membrane proteins indicated 78NP might inhibit protein damage. Using dityrosine as an indicator of protein damage, we demonstrated 200 microM 78NP reduced dityrosine formation in H(2) O(2) /Fe(++) treated red blood cell ghosts by 30%. HPLC analysis demonstrated a direct reaction between 78NP and all three oxidants. Two mM hydrogen peroxide oxidised 119 nM of 78NP per min while 1 mM AAPH only oxidised 50 nM 78NP/min suggesting that 78NP inhibition of haemolysis is not due to 78NP scavenging the primary initiating reactants. In contrast, the reaction between HOCl and 78NP was near instant. AAPH and hydrogen peroxide oxidised 78NP to 7,8-dihydroxanthopterin while hypochlorite oxidation produced neopterin. The cellular antioxidant properties of 78NP suggest it may have a role in protecting immune cells from free radical damage during inflammation.

A comparison of plasma vitamin C and E levels in two Antarctic and two temperate water fish species.

Antarctic fish have a high polyunsaturated lipid content and their muscle cells have a high mitochondria density suggesting that Antarctic fish are under greater oxidative stress than temperate water fish. To test this hypothesis, the plasma concentrations of the antioxidant vitamins E and C were measured in two Antarctic fish species, Pagothenia borchgrevinki and Trematomus bernacchii, and compared with the plasma concentrations of these vitamins in two New Zealand temperate water fish species, blue cod (Parapercis colias) and banded wrasse (Notolabrus fucicola). Neither vitamin is known to be synthesised in fish and so must be obtained from the diet. The plasma from both Antarctic fish species had vitamin E concentrations five to six times higher than those found in the two temperate water fish species. However, significantly higher levels of vitamin C were only found in the plasma of T. bernacchii, a benthic Antarctic fish. The average level of vitamin C in the plasma of the cryopelagic P. borchgrevinki was approximately one-third that of T. bernacchii. The T. bernacchii plasma yielded a high range of vitamin C values, possibly reflecting differences in nutritional status among the animals captured. No beta-carotene was found in any of the fish plasma samples studied. The data suggest that even though Antarctic fish live at -1.5 degrees C they may be exposed to greater metabolic stress from free radical mediated oxidation than temperate water species.

Factors affecting resistance of low density lipoproteins to oxidation.

Oxidation resistance (OR) of low density lipoproteins (LDL) is frequently determined by the conjugated diene (CD) assay, in which isolated LDL is exposed to CU2+ as prooxidant in the range of 1-10 microM. A brief review on major findings obtained with this assay will be given. A consistent observation is that vitamin E supplements or oleic acid-rich diets increase OR. Oxidation indices measured by the CD assay and effects of antioxidants very significantly depended on the Cu2+ concentration used for LDL oxidation. For medium and high Cu2+ concentrations, the relationship between lag time and propagation rate can be described by a simple hyperbolic saturation function, which has the same mathematical form as the Michaelis-Menten equation. At medium and high Cu2+ concentrations (0.5 to 5 microM), vitamin E increases lag time in a dose-dependent manner. The increase is higher for 0.5 microM Cu2+ as compared to 5 microM. At low Cu2+ concentrations (0.5 microM or less), the mechanism of LDL oxidation changes. Significant oxidation occurs in a preoxidation phase, which commences shortly after addition of Cu2+. Preoxidation is not inhibited by vitamin E. It is concluded that much additional work is needed to validate the importance of oxidation indices derived from CD and similar assays.

Effect of temperature and phase transition on oxidation resistance of low density lipoprotein.

The study of the effect of temperature on the kinetics of low density lipoprotein (LDL) oxidation was carried out by measuring the conjugated diene (CD) versus time curves at a fixed LDL concentration (0.1 microM) and at different Cu2+ concentrations (0.5-10 microM) in a wide temperature range, from 10 degrees C to 45 degrees C. The core melting point of the LDL determined with differential scanning calorimetry was 31.1 degrees C. We have demonstrated that temperature exerts a clear effect in the Cu(2+)-mediated LDL oxidation, with a strong decrease in lag time and a notable increase in the rate of propagation. This temperature dependence of lag time and rate of propagation fully obeys the Arrhenius law, suggesting that the core melting point of the LDL has no or only a minor effect on these oxidation indices. The Arrhenius plots of the binding of Cu2+ to LDL, measured by K, gave two breaks suggesting that this value is affected by the core transition of the LDL as well as by structural changes at around 15 degrees C. The mean activation energy during rate of initiation was 13.5 kcal/mol and tended to decrease with increasing Cu2+ concentration. The activation energy in the propagation phase was 10.6 kcal/mol and was independent of Cu2+ concentration. In this work we have also shown that the CD method can be conducted with high reproducibility and that a sucrose-supplemented plasma frozen at -80 degrees C can be used as a source of LDL with an unvarying vitamin E content and reproducible oxidation properties.

7,8 Dihydroneopterin inhibits low density lipoprotein oxidation in vitro. Evidence that this macrophage secreted pteridine is an anti-oxidant.

Neopterin and its reduced form, 7,8 dihydroneopterin are pteridines released from macrophages and monocytes when stimulated with interferon gamma in vivo. The function of this response is unknown though there is an enormous amount of information available on the use of these compounds as clinical markers of monocyte/macrophage activation. We have found that in vitro 7,8-dihydroneopterin dramatically increases, in a dose dependent manner, the lag time of low density lipoprotein oxidation mediated by Cu++ ions or the peroxyl radical generator 2,2'-azobis (2-amidino propane) dihydrochloride (AAPH). 7,8-Dihydroneopterin also inhibits AAPH mediated oxidation of linoleate. The kinetic of the inhibition suggests that 7,8-dihydroneopterin is a potent chain breaking antioxidant which functions by scavenging lipid peroxyl radicals. No anti-oxidant activity was observed in any of the oxidation systems studied with the related compounds neopterin and pterin.

Low density lipoprotein is saturable by pro-oxidant copper.

The oxidative resistance of low density lipoprotein (LDL) can be experimentally described by the length of time during which no significant lipid peroxidation is observed in a pro-oxidant environment. This period of inhibited oxidation, termed the 'lag phase', is partially due to the radical scavenging reactions of the anti-oxidants contained in the LDL particle. We have shown that the LDL lag time decreases with increasing copper concentration, leveling out at a relatively high copper-to-LDL ratio. This behaviour demonstrates the existence of a finite number of saturable pro-oxidant copper binding sites within the LDL particle. The relationship is described by the equation, lag time = [Cu]-1.K.tmin+tmin where the constant, K, is the negative reciprocal of the x-axis intercept of the graphed function, and tmin is given by the y-axis intercept. By this definition of the constant, K is the amount of copper that will produce a lag time of twice tmin, while tmin is the minimum time a particular LDL will resist oxidation at a maximum copper concentration.

Hypochlorite oxidation causes cross-linking of Lp(a).

When purified low density lipoprotein (LDL) or lipoprotein(a) (Lp[a]) was oxidized in vitro using concentrations of hypochlorite (50-500 microM) which might be achieved by activated neutrophils in vivo, high molecular weight species were observed on SDS polyacrylamide gels. The reaction was concentration-, temperature- and time-dependent. The high molecular weight apoprotein complexes were resistant to heating in SDS and DTT, suggesting covalent, but non-disulfide bond, cross-linking. Negligible amounts of lower molecular weight degradation products were formed. Bityrosine formation, measured by fluorescence and HPLC analysis, was found to increase with the amount of hypochlorite added. However, the molar concentration of bityrosine could not account for cross-linking, even if it was assumed that every bityrosine was intermolecular. Hypochlorite-oxidized Lp(a) and LDL were both effective as ligands for loading mouse peritoneal macrophages in vitro. We conclude that hypochlorite produced in inflammatory reactions might be important in the generation of atherogenic forms of lipoproteins.

Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins.

Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.

Free radical and enzymatic mechanisms for the generation of protein bound reducing moieties.

We have previously shown that exposure of many proteins, and free aromatic amino acids (particularly tyrosine) to free radical fluxes generates a stable activity capable of reducing protein bound and free transition metal ions. Here we define the capacity of several radical generating systems (gamma irradiation of water, UV irradiation, metal-dependent sugar autoxidation and Haber-Weiss systems) to produce protein-bound reducing moieties (PBRedM), and also reducing derivatives of tyrosine. Under the defined conditions of the gamma radiolysis system, reductive activity was generated under both oxic and anoxic irradiations and specific gassing regimes as well as the inclusion of specific radical scavengers established that hydroxyl radicals were responsible. When BSA was irradiated anoxically in the presence of formate a reductive activity related to the exposure of protein thiol groups was generated; all other reductive activities we detected were not thiol-related. Incubations of tyrosinase with BSA or insulin also generated reductive activity. All the conditions we have studied can convert tyrosine into DOPA and we suspect that protein-bound DOPA is the main reductive activity generated on proteins.

The participation of nitric oxide in cell free- and its restriction of macrophage-mediated oxidation of low-density lipoprotein.

The potential role of nitric oxide radical (NO .) in macrophage-mediated oxidation and conversion of human low density lipoprotein (LDL) to a high-uptake form was examined by exposing LDL to aerobic solutions of either NO . or 3-morpholino-sydnonimine-hydrochloride (SIN-1, a compound that spontaneously forms NO . and superoxide anion radical) or to mouse peritoneal macrophages in the presence and absence of modulators of cellular NO . synthesis. Incubation with NO . alone caused oxidation of LDL's ubiquinol-10 and accumulation of small amounts of lipid hydroperoxides, but failed to form any high-uptake ligand for endocytosis by macrophages and did not alter the LDL particle charge or the integrity of apoB. Exposure of LDL to SIN-1 resulted in complete consumption of all antioxidants and substantial formation of lipid hydroperoxides, but again had little effect on the lipoprotein particle charge or generation of high-uptake form. Preincubation of macrophages with interferon-gamma increased the cells ability to generate reactive nitrogen metabolites. The extent of cell-mediated oxidation of LDL and the generation of high-uptake LDL was substantial in resident cells in which NO . synthesis was barely detectable, depressed in cells active in NO . synthesis and restored when NO . synthesis was suppressed by the arginine analogue, NMMA. These results suggest that, while together with superoxide anion radical, NO . can oxidize LDL, its synthesis is not required for macrophage-mediated oxidation of LDL in vitro; rather it exerts a protective role in preventing oxidative LDL modification by macrophages.

The purification of ovine pancreatic lipase that is free of colipase using an improved delipidation method.

A modified procedure for the purification of ovine pancreatic lipase (triacylglycerol acyl-hydrolase, EC3.1.1.3) is described. The method is more rapid and more reproducible than that reported previously and results in a pure lipase preparation, that gives a better yield at the same specific activity, free of colipase and uncontaminated by lipid. The procedure involves the preparation of a lipid-free acetone powder from fresh pancreas without the use of chloroform or butanol as was used in the procedure described earlier. The aqueous purification of the lipase from the delipidated powder is similar to that described earlier, but includes the use of beta-mercaptoethanol and uses salt gradient elution from CM-Sepharose. An assay procedure for lipase is reported involving the extraction of released free fatty acids with chloroform/methanol before titrating with sodium hydroxide. A modification of this assay is used for the determination of colipase. The above assay procedure is compared to the potentiometric method reported previously. Polyacrylamide gel, amino acid composition analysis and N-terminal sequence data for the purified ovine lipase are presented.

Sequence and immunological characterisation of ovine pancreatic lipase.

Purified ovine pancreatic lipase has been subjected to a limited protein sequence analysis. Cyanogen bromide fragments from the molecule were isolated and characterised to enable the structure of the molecule to be mapped. Some tryptic peptides were also isolated, sequenced, and aligned by homology to lipase sequences from other species. A total of 172 residues out of a possible 456 have been assigned, including 45 residues at the N-terminus and 10 residues at the C-terminus of the protein. A polyclonal antibody has been prepared to ovine lipase which has been characterised by Ouchterlony immunodiffusion and by Western blotting experiments. These experiments showed that the ovine pancreatic lipase was immunologically different from the ovine hepatic and lingual lipase, whereas there was considerable immunological similarity amongst ovine, bovine and rabbit pancreatic lipase, but less with porcine pancreatic lipase.