Antioxidant status and biomarkers of oxidative stress in dogs with lymphoma.

BACKGROUND: Oxidative stress might play a role in carcinogenesis, as well as impacting morbidity and mortality of veterinary cancer patients. The purpose of this study was to evaluate antioxidant concentrations and biomarkers of oxidative stress in dogs with newly diagnosed lymphoma before treatment and once in remission, with comparison with healthy controls. HYPOTHESIS: Dogs with lymphoma have increased oxidant and reduced antioxidant concentrations compared with healthy controls, and that these abnormalities normalize once remission is achieved. ANIMALS: Seventeen dogs with lymphoma and 10 healthy controls. METHODS: Prospective, observational study. Measures of oxidative stress [malondialdehyde and total isoprostanes (isoP)] and antioxidants [alpha-tocopherol, gamma-tocopherol, oxygen radical absorbance capacity (ORAC), and glutathione peroxidase (GSHPx)] were assessed in dogs with newly diagnosed lymphoma before treatment compared with healthy control dogs. The same parameters were measured in the dogs with lymphoma on week 7 of the chemotherapy protocol when all dogs were in remission. RESULTS: At baseline, dogs with lymphoma had significantly lower alpha-tocopherol (P <.001) and gamma-tocopherol (P= .003) but higher GSHPx (P= .05), ORAC (P= .001), and isoP (P < .001) compared with healthy controls. In the dogs with lymphoma, alpha-tocopherol concentrations were higher (P= .005) and ascorbic acid were lower (P= .04) after treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: Results suggest that dogs with lymphoma have alterations in oxidant and antioxidant concentrations and that the status of some of these biomarkers normalize after remission. Further studies are warranted to determine whether antioxidant interventions to correct these are beneficial in the treatment of canine lymphoma.

Direct adrenergic stimulation of the release of thyrotropin and its subunits from the thyrotrope in vitro.

Adrenergic effects on TSH and subunit secretion were investigated in bovine anterior pituitary monolayer cultures. Epinephrine (E) (10(-6) M) caused a significant increase in TSH, alpha-subunit, and TSH beta release into the medium (P less than 0.001, P less than 0.001, and P less than 0.01, respectively). E, norepinephrine, and phenylephrine, all alpha-adrenergic agonists, caused significant increases in TSH release, with half-maximal effects at 4.3 X 10(-7), 6.8 X 10(-7), and 8.2 X 10(-7) M, respectively. However, isoproterenol, a beta-adrenergic agonist (10(-7)-10(-4) M), did not alter TSH or subunit release. Clonidine, an alpha 2-adrenergic agonist (10(-7)-10(-4) M), had no effect on TSH or subunit secretion; however, coincubation of clonidine (5 X 10(-7) M) with a submaximal concentration of phenylephrine (5 X 10(-7) M) caused a rise in TSH release greater (P less than 0.02) than that seen with P alone. The alpha-adrenergic antagonists phentolamine and fluphenazine completely inhibited (P less than 0.001) the E-induced rise in TSH and subunits. In contrast, the beta-adrenergic antagonists propranolol and metoprolol did not significantly inhibit the stimulation of TSH by E. TSH and subunit secretion is stimulated by adrenergic agonists acting directly on the pituitary, and this is probably mediated via an alpha-adrenergic receptor.

The effect of somatostatin on the release of thyrotropin and its subunits from bovine anterior pituitary cells in vitro.

The effects of somatostatin (SRIF) on the production and release of TSH and its subunits have been investigated in bovine anterior pituitary monolayer cultures. SRIF caused a dose-dependent inhibition of TSH and subunit release by TRH, with a half-maximal effective dose of 3 X 10(-8) M. This effect was time dependent and was greater for TSH than for its subunits. However, the basal release and total production of TSH and its subunits over a 24-h period were not affected by SRIF. The effect of SRIF was additive to that of thyroid hormones in suppressing the release of TSH and its subunits by TRH. A combination of SRIF and thyroid hormone completely suppressed the release of TSH and its subunits by TRH. In contrast, thyroid hormones caused a dose-dependent inhibition of the total production, as well as the release, of TSH and its subunits induced by TRH. Furthermore, thyroid hormones produced a dose-dependent increase (r = 0.81; P less than 0.001) in the effectiveness of a single dose of SRIF in suppressing TSH release by TRH. Analysis of these data revealed that thyroid hormones interacted synergistically with the SRIF effect to suppress TRH-mediated TSH and subunit release.

Kynurenine pathway abnormalities in Parkinson's disease.

We measured metabolites of tyrosine and tryptophan (TRP) in the frontal cortex, putamen (PT), and pars compacta of the substantia nigra (SN) of control and Parkinson's disease (PD) brain tissues. Dopamine concentrations were significantly decreased in the PT and SN of PD tissue, regardless of L-dopa therapy. However, 3-O-methyldopa (3OMD) concentration showed a significant increase in each region of the PD group treated with L-dopa (PD[+]) as compared with both the control group and the PD group without L-dopa therapy (PD[-]). Therefore, 3OMD concentration appears to be a reliable marker of L-dopa therapy. Serotonin concentration was lower in each region of the PD groups than in the control group. Although the magnitude of decrease was greater in the PD(+) group, there was no statistical significance between the two PD groups. The same patterns of decrease were present in kynurenine (KYN) and kynurenic acid (KYA) concentrations, but the molar ratios of TRP to KYN and KYN to KYA were unchanged among three groups. In contrast, 3-hydroxykynurenine (3OHKY) concentration was increased in the PT PD(-) group and in three regions of the PD(+) group. Since the KYN pathway leads to formation of nicotinamide-adenine dinucleotide (NADH), the present results may be a further indication of a defect in NADH:ubiquinone oxidoreductase (complex I) in mitochondria in PD.

Markers of dopamine metabolism in Parkinson's disease. The Parkinson Study Group.

We used two analytic methods (a multichannel coulometric electrode array with high-performance liquid chromatography, and gas chromatography-mass spectrophotometry) to measure CSF dopamine (DA) and its metabolites in mildly affected, unmedicated subjects with Parkinson's disease (PD). The mean (+/- SD) concentration of homovanillic acid (HVA), the most abundant product of DA turnover, was 164.57 +/- 95.05 nM. As sequential aliquots of CSF were collected from the first to 23rd ml, CSF HVA concentration almost doubled. After HVA, 3-O-methyldopa (3-O-MD) was the next most abundant compound. The summed concentrations of 3-O-MD, 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, DA, DA-3-sulfate, homovanillol, and levodopa (LD) amounted to 12.6% of HVA. Concentrations of the DA metabolites did not correlate to a variety of indices of PD severity. The presence of LD and 3-O-MD may be indicators of DA synthesis and possibly could reflect compensatory processes among surviving dopaminergic neurons of the PD brain.

A comparison of changes in plasma thyrotropin beta- and alpha-subunits, and mouse thyrotropic tumor thyrotropin beta- and alpha-subunit mRNA concentrations after in vivo dexamethasone or T3 administration.

Dexamethasone, like T3, inhibits the production of TSH. T3 inhibits TSH synthesis by reducing transcription of the genes encoding TSH-beta and alpha-subunits. Little information is available concerning the effects of dexamethasone on the individual subunits, or the combined effects of dexamethasone with T3. In a preliminary study, hypothyroid mice bearing the thyrotropic tumor TtT 97 were treated with 25, 250, or 500 micrograms dexamethasone ip daily for ten days. Plasma levels of TSH and its subunits were unchanged after 25 micrograms of dexamethasone and maximally suppressed after 250 micrograms. Plasma TSH was reduced to 51% (P less than .02), free TSH-beta to 54% (P less than .01), and alpha-subunit to 62% (P less than .001) of control values. In two similar experiments hypothyroid mice bearing TtT 97 were treated with dexamethasone (250 micrograms), T3 (0.5 or 1 microgram), or both dexamethasone with T3 for 10 days. Total alpha-subunit and TSH-beta were calculated by adding 1/2 TSH + free subunit concentrations. In the experiment using 1 microgram of T3, total plasma alpha-subunit was reduced by dexamethasone to 72%, by T3 to 45% (P less than .02), and by combined treatment to 23% (P less than .01) of control values. In the experiment using 0.5 microgram of T3, total plasma alpha-subunit was reduced by dexamethasone to 66% (P less than .05), by T3 to 67% (P less than .05), and by combined treatment to 46% (P less than .02) of control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Kynurenic acid concentrations are reduced in Huntington's disease cerebral cortex.

Huntington's disease (HD) is characterized by gradually evolving selective neuronal death. Several lines of evidence suggest that an excitotoxic mechanism may play a role. Tryptophan metabolism leads to production of quinolinic acid, an N-methyl-D-aspartate (NMDA) receptor agonist, and to kynurenic acid, an antagonist at these same receptors. We recently found increased kynurenine to kynurenic acid ratios in HD postmortem putamen and decreased kynurenic acid concentrations in cerebrospinal fluid, consistent with decreased formation of kynurenic acid in HD brain. In the present study we used HPLC with 16 sensor coulometric electrochemical detection to measure kynurenic acid and 18 other electrochemically active compounds in 6 cortical regions, caudate and cerebellum from controls, HD, Alzheimer's disease (AD), and Parkinson's disease (PD) patients. Significant reductions in kynurenic acid concentrations were found in 5 of 6 cortical regions examined. Smaller reductions of kynurenic acid in the caudate, cerebellum and frontal pole were not significant. No significant reductions were found in the AD and PD patients. Both uric acid and glutathionine were significantly reduced in several regions of HD cerebral cortex, which could signify abnormal energy metabolism in HD. Since kynurenic acid is an antagonist of excitatory amino acid receptors, a deficiency could contribute to the pathogenesis of neuronal degeneration in HD.

Altered guanosine and guanine concentrations in rabbit striatum following increased dopamine turnover.

The significance of guanine nucleotides and nucleosides in neurodegenerative disorders is suggested by recent reports that these molecules enhance neurite branching and astrocyte proliferation. The objective of this study was to investigate the influence of increased dopamine metabolism, produced by 5-day treatment of rabbits with reserpine (2 mg/kg) or levodopa (LD) (50 mg/kg), on striatal concentrations of guanosine, guanine, and their metabolites. Reserpine treatment decreased striatal guanosine by 41% and increased guanine by 50%, while LD decreased guanosine by 48% (all p < 0.01 vs. vehicle-treated controls). LD also increased guanine by 22% (not statistically significant). Xanthine and uric acid concentrations were unchanged. Because of the neurotrophic properties of guanosine and guanine, changes in striatal concentrations of these purines secondary to increased dopamine (DA) turnover may have relevance for survival of remaining dopaminergic neurons in Parkinson's disease (PD).

Studies on lysyl oxidase of bovine ligamentum nuchae and bovine aorta.

Lysyl oxidase had been purified to near homogeneity from bovine aorta and bovine ligamentum nuchae employing a modification of methods described by Harris et al., and Stassen and his colleagues. The aortic enzyme gives rise to at least three peaks and the ligament enzyme resolves into at least four peaks upon chromatography on DEAE cellulose. The molecular weight of each peak of both enzymes is approximately 30,000 daltons in sodium dodecyl sulfate. The aortic enzyme aggregates to species with molecular weights varying from approximately 60,000 to 1,000,000 daltons upon dialysis out of urea into phosphate-buffered saline. Temperature studies reveal that lysyl oxidase is stable to temperatures as high as 80 degrees C, although the assay optimum is 52 degrees C. Studies in progress suggest the temperature dependency of assay may reflect conformational changes in the elastin substrate.

A comparative bear model for immobility-induced osteopenia.

The National Institutes of Health (NIH) and the National Aeronautics and Space Administration (NASA) are seeking solutions to the human problem of osteopenia, or immobility-induced bone loss. Bears, during winter dormancy, appear uniquely exempted from the debilitating effects of immobility osteopenia. NIH and ESA, Inc. are creating a large database of metabolic information on human ambulatory and bedrest plasma samples for comparison with metabolic data obtained from bear plasma samples collected in different seasons. The database generated from NASA's HR113 human bedrest study showed a clear difference between plasma samples of ambulatory and immobile subjects through cluster analysis using compounds determined by high performance liquid chromatography with coulometric electrochemical array detection (HPLC-EC). We collected plasma samples from black bears (Ursus americanus) across 4 seasons and from 3 areas and subjected them to similar analysis, with particular attention to compounds that changed significantly in the NASA human study. We found seasonal differences in 28 known compounds and 33 unknown compounds. A final database contained 40 known and 120 unknown peaks that were reliably assayed in all bear and human samples; these were the primary data set for interspecies comparison. Six unidentified compounds changed significantly but differentially in wintering bears and immobile humans. The data are discussed in light of current theories regarding dormancy, starvation, and anabolic metabolism. Work is in progress by ESA Laboratories on a larger database to confirm these findings prior to a chemical isolation and identification effort. This research could lead to new pharmaceuticals or dietary interventions for the treatment of immobility osteopenia.

A possible role for elastin ligands in the proteolytic degradation of arterial elastic lamellae in the rabbit.

Thoracic aortae of normal rabbits were perfused with pancreatic elastase in vitro at 37 degrees C and 70 mm Hg pressure in the presence or absence of elastin ligands previously shown to stimulate or inhibit the enzymatic degradation of elastin. Perfusion with elastase results in an average of 3.6 lamellae degraded, whereas addition of sodium linoleate before and during the perfusion with elastase increases this value to 7.9 (P less than 0.001). Conversely, perfusion with the cationic detergent, dodecyltrimethylammonium chloride, completely prevents the degradation of elastic lamellae by elastase. These effects do not reflect alterations of the intrinsic catalytic activity of elastase, but apparently indicate the formation of complexes between the elastin ligands and arterial elastic lamellae, as is consistent with prior studies indicating such interactions between fatty acids or detergents and purified elastin. These studies suggest that agents such as fatty acids may significantly alter the metabolic susceptibility of elastin in vivo and possibly contribute to the degradation of elastic lamellae seen in arteries with advanced atherosclerosis.

Markers of dopamine depletion and compensatory response in striatum and cerebrospinal fluid.

Though depletion of CSF homovanillic acid (HVA) concentration has often been regarded as a direct indicator of dopamine (DA) deficiency in Parkinson's Disease (PD), CSF HVA is normal in mildly affected patients. To explore why, we measured DA and its metabolites in striatum and CSF in rabbits receiving reserpine for 5 days. Reserpine, which depletes striatal DA by disrupting vesicular storage of the neurotransmitter, results in a compensatory increase of DA turnover. In response to a 96% depletion of striatal DA, its catabolic intermediates 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT) decreased 64% and 92% in striatum, although the endproduct, HVA, was unchanged. In contrast, CSF concentrations of HVA and DOPAC increased significantly, though 3-MT and levodopa (LD) were unaltered. A 5-fold rise in striatal LD concentration after reserpine-induced DA depletion provided evidence for enhanced DA synthesis. As in PD, the compensatory increase of DA synthesis after reserpine administration confounds the ability of CSF HVA to reflect DA depletion.

Acute decreases in cerebrospinal fluid glutathione levels after intracerebroventricular morphine for cancer pain.

UNLABELLED: Intracerebroventricular (ICV) morphine administration is effective for the management of refractory cancer pain. Recent preclinical observations of acute depletion of the major endogenous intracellular antioxidant glutathione (GSH) in brain and peripheral organs after ICV morphine in rodents led us to apply microchemical methods to profile the neurochemical effects of ICV morphine in three patients treated for intractable cancer pain. Assessment of morphine, morphine-6-glucuronide, and a panel of endogenous compounds and metabolites in ventricular and cisternal cerebrospinal fluid (CSF) demonstrated transient, postdose increases in morphine and morphine-6-glucuronide in ventricular and cistemal CSF, accompanied by acute decreases in CSF GSH levels. Significant changes were also observed in the CSF levels of 4-hydroxybenzoic acid, homovanillic acid, 5-hydroxyphenyllactic acid, and uric acid. These pilot clinical observations of acute central GSH depletion after ICV morphine suggest a novel mechanism for neuropsychiatric toxicity or preclinical findings, such as hyperalgesia or increased motoric activity observed in nonhuman species after central morphine administration. Because ICV morphine is a mainstay of treatment for refractory cancer pain, elucidation of a mechanism's (or mechanisms') mediating a potential pro-oxidant state in the central nervous system induced by ICV morphine is important. IMPLICATIONS: We observed acute decreases in glutathione levels in cerebrospinal fluid sampled from patients after intracerebroventricular doses of morphine for intractable cancer pain. Such doses may, by depleting the antioxidant glutathione, render the central nervous system vulnerable to damage from oxidative stress.

Characterization of diet-dependent metabolic serotypes: analytical and biological variability issues in rats.

This report, the first in a series on diet-dependent changes in the serum metabolome (metabolic serotype), describes validation of the use of high performance liquid chromatography (HPLC) separations coupled with Coulometric array detectors to characterize changes in the metabolome. The long-term aim of these studies is to improve understanding of the effects of significant variation in nutritive status on physiology and on disease processes. Initial studies focus on identifying the effects of dietary (or caloric) restriction on the redox-active components of rat serum. Identification of compounds of interest is being carried out using HPLC separations coupled with coulometric array analysis, an approach allowing simultaneous examination of nearly 1200 serum compounds. The technical and practical issues discussed in this report are related to both analytical validity (HPLC running conditions, computer-automated peak identification, mathematical compensation for chromatographic drift, etc.) and biological variability (individual variability, cohort-cohort variability, outliers). Attention to these issues suggests approximately 250 compounds in serum are sufficiently reliable, both analytically and biologically, for potential use in building mathematical models of serotype.

Influence of repeated levodopa administration on rabbit striatal serotonin metabolism, and comparison between striatal and CSF alterations.

Parkinson's disease (PD) is characterized by decreased striatal dopamine, but serotonin (5-HT) is also reduced. Because 5-HT decreases following a single levodopa injection, levodopa has been suggested to contribute to PD's serotonergic deficits. However, in a recent study, rat striatal serotonin levels were reported to increase following 15-day levodopa administration. To address this issue, we administered levodopa (50 mg/kg) to rabbits for 5 days, then measured serotonin, its precursors tryptophan and 5-hydroxytryptophan (5-HTP), and its major metabolite 5-hydroxyindole-acetic acid (5-HIAA) in striatum and CSF. Striatal serotonin and tryptophan were unchanged, while 5-HTP and 5-HIAA increased 4- and 7-fold, respectively. CSF 5-HTP and 5-HIAA were also significantly increased. In levodopa-treated animals, 5-HTP concentrations were moderately correlated (r = 0.679) between striatum and CSF, while weak correlations were present between striatal and CSF concentrations of both serotonin and 5-HIAA. These results suggest that repeated levodopa treatment increases striatal serotonin turnover without changing serotonin content. However, levodopa-induced alterations in striatal serotonin metabolism may not be accurately reflected by measurement of serotonin and 5-HIAA in CSF.