The effect of dietary n-3 and n-6 fatty acids on tumor necrosis factor-alpha production and leucine aminopeptidase levels in rat peritoneal macrophages.

The purpose of this study was to determine the effect of dietary n-3 and n-6 fatty acids on tumor necrosis factor-alpha (TNF-alpha) production and macrophage (MO) activation state. Rats were fed diets containing 12.5% linseed oil (LO) or corn oil (CO) that are high in n-3 and n-6 fatty acids respectively. The LO diet resulted in a significant increase in basal and endotoxin (LPS)-induced levels of TNF-alpha from resident MO cultured in vitro. There was no difference between the diets in LPS-induced TNF-alpha production by complete Freund's adjuvant (CFA) elicited macrophages. Variable responses were also observed between LO and CO MO in response to prostaglandin E2, indomethacin (INDO), and the prostaglandin E receptor antagonist SC-19220. This may indicate differences in signal transducing secondary messengers due to different activation states, receptor expression or ligand binding. Fluorescence due to leucine aminopeptidase (LAP) staining was determined by flow cytometry. Resident LO MO had a 15% increase in LAP fluorescence compared to CO MO. In CFA-elicited MO, the CO MO had a 43% increase in fluorescence compared to LO MO. Resident LO MO increased in LAP fluorescence by 35% to the activated state whereas resident CO MO increased in LAP fluorescence by 93%. The smaller window of activation for the LO MO may explain some of the antiinflammatory properties of dietary n-3 fatty acids.

Phase I trial of piroxicam in 62 dogs bearing naturally occurring tumors.

Piroxicam, a nonsteroidal antiinflammatory drug, was given to 62 dogs bearing naturally occurring tumors in a phase I clinical trial. Dose escalation was performed, with oral doses ranging from 0.5 mg/kg every 48 h (q48h) to 1.5 mg/kg q48h being tested. Dose-limiting gastrointestinal irritation/ulceration occurred in all four animals that received 1.5 mg/kg q48h. The maximum tolerated dose was 1 mg/kg q48h. Subclinical renal papillary necrosis occurred in two dogs (initial dosages, 1 and 1.5 mg/kg q48h, respectively). Following dose escalation, an additional group of dogs was treated with 0.3 mg/kg piroxicam q24h per os, the accepted canine dosage prior to this trial. Inclusion of this treatment group enabled evaluation of the toxicity of and tumor response to a daily dosage regimen. No complete remissions occurred in this trial. Partial remission was documented in three of ten dogs exhibiting transitional-cell carcinoma, in three of five animals bearing squamous-cell carcinoma, in one of three dogs displaying mammary adenocarcinoma, and in the one dog that exhibited a transmissible venereal tumor. The results of this study support the additional evaluation of piroxicam in a phase II clinical trial in dogs bearing naturally occurring tumors.

Models for the pharmacokinetics and pharmacodynamics of insulin in alloxan-induced diabetic dogs.

A combined pharmacokinetic/pharmacodynamic model was proposed to describe the pharmacokinetics of intravenously administered regular insulin (0.55 units/kg) in alloxan-induced diabetic dogs. Serum insulin concentrations were described by either a one- or two-compartment open model, in which a hypothetical effect compartment was linked to the central pharmacokinetic compartment, or in which the effect compartment was linked to the peripheral compartment. Response, as measured by percent change in glucose concentration from adjusted basal plasma concentrations, was modeled using the sigmoidal Emax effect model, a linear effects model, a log-linear effects model, and a gamma-linear effects model, using the insulin pharmacokinetic parameters to describe the amount in the hypothetical effect compartment. The results indicated that insulin pharmacokinetics are usually described by a two-compartment open model. Response to insulin was predicted more accurately in half of the dogs using the gamma-linear effects model in which the effect compartment was linked to the central compartment. In the other half of the dogs the best model was the sigmoidal Emax model in which the effect compartment was linked to the central pharmacokinetic compartment. The parameters in the latter model were correlated with each other and the confidence limits of the parameter estimates were larger than the parameters of the gamma-linear effects model. These models should be further investigated, but may offer an alternative method for distinguishing rapid insulin metabolism from insulin resistance.

Effects of multiple dose infections with Ascaris suum on blood gastrointestinal hormone levels in pigs.

Ten consecutive daily doses of infective Ascaris suum eggs were administered to pigs in two experiments and the levels of gastrointestinal hormones in their blood were measured. The piglets in each experiment were divided into low-dose (LDI) and high-dose (HDI) infections and control groups. Infected pigs had lower feed consumption, lower weight gains, and lower feed efficiency than control pigs. Serum gastrin levels in infected pigs were significantly lower than the controls from Days 7 to 17 post first inoculation (PFI), and so were their serum glucagon levels from Days 12 to 24 PFI. Serum insulin levels in infected animals were sometimes lower than those in controls. These differences were usually more intense in the LDI pigs than in HDI pigs. The plasma cholecystokinin (CCK) levels in the LDI group were significantly higher than those in controls from Day 10 PFI to the end of the experiment, while the CCK levels in the HDI group did not differ significantly from the controls. Increased plasma CCK levels could be a satiety factor in A. suum infection since the time of occurrence of high levels of CCK matched the period of reduced feed consumption.

Tumor necrosis factor-like cytotoxicity and anorexia in Ascaris suum infected pigs.

Tumor Necrosis Factor-Like Cytotoxicity (TNF-LC) was examined in sera from 12 pigs experimentally infected with Ascaris suum. The difference of TNF-LC levels between eight infected and four uninfected controls was not significant. When an endotoxin challenge was intravenously administered 1 month after the first dose of A. suum, the levels of TNF-LC in the sera of infected pigs were one-third that of the controls 125 min post-challenge (PC). In a more detailed study on four infected and two uninfected control pigs, TNF-LC was monitored every 10-15 min until 125 min after endotoxin challenge. The TNF-LC levels in these pigs increased at 40 min PC, reached maximum in another 10-25 min and then decreased. This pattern was seen in all except one infected pig. The infected pigs showed milder shock symptoms and their serum TNF-LC levels returned to pre-challenge levels 30 min earlier than controls.

Serum levels of gastrin, insulin and glucagon as possible factors of anorexia in pigs infected once with Ascaris suum.

In order to determine possible mediators for development of anorexia in pigs infected with Ascaris suum, serum levels of gastrin, insulin and glucagon were measured. After a single high oral dose of 100,000-200,000 embryonated eggs the serum levels of gastrin and insulin in the infected pigs did not significantly differ from those in controls. Serum glucagon levels in the infected groups, however, were lower than those in controls and the difference was more evident 24 days postinoculation and later.

The effect of starting time on dexamethasone suppression test results in horses.

This study was conducted to investigate the effect of starting time on dexamethasone suppression test results in horses. Eight adult horses were used throughout the trial. Baseline cortisol levels were established by collecting cortisol levels twice daily, at 8:00 A.M. and 8:00 P.M. for 4 consecutive days. Morning baseline cortisol levels were 46.3 +/- 5.94 ng/ml, and evening baseline cortisol levels were 32.8 +/- 5.59 ng/ml. Although lower, the evening cortisol levels were not statistically different (P = 0.154) from the morning levels. Dexamethasone suppression tests initiated at either 9:00 A.M. or 9:00 P.M. were performed by collected a control blood sample, administering either 0.044 mg/kg dexamethasone or its vehicle intravenously and then collecting additional blood samples at 6, 12, 24, 36, and 48 hr after treatment. Mean cortisol levels at hr 0, 6, 12, 24, 36, and 48 after a dexamethasone injection given at 9:00 A.M. were 55.6 +/- 3.08, 6.4 +/- 2.05, 0.73 +/- 0.48, 11.0 +/- 5.82, 12.6 +/- 4.30, and 40.5 +/- 5.38 ng/ml respectively. Mean cortisol levels at hr 0, 6, 12, 24, 36, and 48 hr after a dexamethasone injection given at 9:00 P.M. were 45.0 +/- 6.03, 4.5 +/- 1.28, 0.20 +/- 0.12, 4.5 +/- 2.49, 23.4 +/- 5.88, and 29.5 +/- 6.61 ng/ml respectively. There was no statistical difference in cortisol values between A.M. and P.M. initiated tests at any hour post dexamethasone administration. There was no decrease in cortisol level after administration of dexamethasone vehicle.

Effect of thyrotropin storage on thyroid-stimulating hormone response testing in normal dogs.

The stability of reconstituted, refrigerated thyrotropin was evaluated. Thyrotropin (TSH) was reconstituted at the start of the study and stored at 4 degrees C. A TSH stimulation test was performed in eight healthy, euthyroid dogs at weekly intervals for 1 month. In seven of eight dogs, there was no significant difference (P less than 0.05) between the post-TSH T3 concentrations and the post-TSH T4 concentrations for the duration of the study. For one dog, the post-TSH T4 concentration was below the normal post-TSH T4 range following the administration of reconstituted TSH that had been stored 4 weeks. The T3 response to the TSH, however, was normal. This dog responded normally to freshly reconstituted TSH. The results of this study suggest that reconstituted bovine TSH can be stored at 4 degrees C for at least 3 weeks without loss of biologic activity in the dog.

Ultracentrifugal and electrophoretic characteristics of the plasma lipoproteins of miniature schnauzer dogs with idiopathic hyperlipoproteinemia.

To better characterize the idiopathic hyperlipoproteinemia of Miniature Schnauzer dogs, the plasma lipoproteins of 20 Miniature Schnauzers (MS) and 11 dogs of other breeds (DOB) were evaluated by ultracentrifugation, electrophoresis, and biochemical tests. Seventeen MS were healthy; 3 had diabetes mellitus. Plasma from 6 of 17 healthy and all 3 diabetic MS was visibly lipemic. Lipemia was slight to marked in healthy lipemic MS, and marked in diabetic ones. All DOB had clear plasma; 8 were healthy and 3 had diabetes. All healthy lipemic MS and diabetic lipemic MS had hypertriglyceridemia associated with excess very low density lipoproteins. Chylomicronemia was present in 4 of 6 healthy lipemic MS and all 3 diabetic lipemic MS. Lipoproteins with ultracentrifugal and electrophoretic characteristics of normal low density lipoprotein were lacking in 4 of 6 healthy lipemic MS. The lipoprotein patterns of 4 of 11 healthy nonlipemic MS were characterized by mild hypertriglyceridemia associated with increased very low density lipoproteins and a lack of lipoproteins with characteristics of normal low density lipoproteins. Lipoprotein patterns of diabetic DOB closely resembled those of healthy DOB; those of diabetic lipemic MS resembled those of markedly lipemic healthy lipemic MS. In conclusion, the hyperlipoproteinemia of Miniature Schnauzers is characterized by increased very low density lipoproteins with or without accompanying chylomicronemia; some affected dogs may have decreased low density lipoproteins.

Piroxicam therapy in 34 dogs with transitional cell carcinoma of the urinary bladder.

Thirty-four dogs with histopathologically confirmed, measurable, nonresectable transitional cell carcinoma of the urinary bladder were treated with piroxicam (0.3 mg/kg PO sid) and were evaluated for tumor response and drug toxicity. Dogs were evaluated at the Purdue University Veterinary Teaching Hospital by means of physical examination, thoracic and abdominal radiography, cystography, complete blood count, serum biochemistry profile, and urinalysis. In selected cases, prostaglandin E2 (PGE2) concentrations in plasma and in supernatants of stimulated monocytes, and natural killer cell activity were quantified. Dogs were evaluated before therapy and at 28 and 56 days after initiation of therapy. Dogs with stable disease or remission at 56 days remained on the study and were evaluated at 1 to 2 months intervals. Tumor responses were 2 complete remissions, 4 partial remissions, 18 stable diseases, and 10 progressive diseases. The median survival of all dogs was 181 days (range, 28 to 720+ days), with 2 dogs still alive. Piroxicam toxicity consisted of gastrointestinal irritation in 6 dogs and renal papillary necrosis (detected at necropsy) in 2 dogs. Monocyte production of PGE2 appeared to decrease with therapy in dogs whose tumors were decreasing in size, and increased in dogs with tumor progression. A consistent pattern in natural killer cell activity was not observed. In vitro cytotoxicity assays against 4 canine tumor cell lines revealed no direct antitumor effects of piroxicam. In summary, antitumor activity, which was not likely the result of a direct cytotoxic effect, was observed in dogs with transitional cell carcinoma of the bladder treated with piroxicam.

Metabolism of estrogens in the gastrointestinal tract of swine. I. Instilled estradiol.

One minute after instillation of 14C-estradiol-17 beta (14C-E2 17 beta) into selected sections of the gastrointestinal tract of swine, radioactive estradiol metabolites were present in blood collected from the portal and jugular veins. Ether was used to extract free but not conjugated estrogens. The percentage of plasma radioactivity that was ether extractable (EE) was low in portal plasma and even lower in jugular plasma following instillation of 14C-E2 17 beta into the stomach, ileum and colon. EE radioactivity was not detectable in either portal or jugular plasma when estradiol was instilled into the duodenum or jejunum. Therefore, estrogens were conjugated either in the lumen of the gastrointestinal tract or as they crossed the intestinal mucosa. The liver played only a minor role in conjugation of these steroids, since the estrogen metabolites present in portal plasma were very similar to those in jugular plasma, and metabolites in the urine were similar to those in plasma. The principal estrogen conjugate found in both portal and jugular plasma, regardless of the gastrointestinal section into which 14C-E2 17 beta was instilled, was estrone glucuronide. There was no uniform metabolic pattern observed in the metabolites of estradiol that remained in the lumen of each gastrointestinal section; however, many metabolic transformations occurred. We concluded that almost all estrogens absorbed were metabolized during the absorption process. The liver was active only in the metabolism of estrogens that escaped conjugation in the intestinal mucosa.

Metabolism of estrogens in the gastrointestinal tract of swine. III. Estradiol-17 beta-D-glucuronide instilled into sections of intestine.

Studies were conducted to determine the absorption and metabolic fate of 3H-estradiol-17 beta-glucuronide (3H-E2-G) in swine. The conjugate, 3H-E2-G (48.7 x 10(6) DPM, 45.5 Ci/mmol), was injected into ligated 15-cm sections of duodenum, proximal jejunum, distal jejunum, ileum and spiral colon of 10 kg female pigs. Blood from the jugular and portal veins and urine were collected at .5-h intervals for 5 h. Absorption from the colon was rapid and radioactivity peaked in both portal and jugular plasma by .5 h postinjection. In contrast, the highest plasma estrogen concentration from most other sections was reached at 5 h, the last sampling time. The urinary excretion patterns were nearly identical to those seen in plasma, with the radioactivity peaking early (1.5 h) after instillation of 3H-E2-G into the colon, but still rising at the end of the experiment after instillation into the duodenum, distal jejunum and ileum. The proximal jejunum, which produced low plasma estrogen concentrations, also produced low urine concentrations. The slower absorption of 3H-E2-G compared to 14C-estradiol-17 beta is consistent with the view that the limiting factor for the absorption of the conjugate is hydrolysis to a free estrogen. The predominant metabolites in portal venous plasma from all sections of the intestine at the end of the experiments were the monoglucuronides of estrone and estradiol. Because the administered 3H-E2-G was conjugated at C-17, the presence of estrone glucuronide in portal plasma indicates that, at least in the duodenum, ileum and colon, 3H-E2-G undergoes cleavage, followed by the oxidation of estradiol to estrone, which is subsequently reconjugated by the intestinal mucosa.

Metabolism of estrogens in the gastrointestinal tract of swine. II. Orally administered estradiol-17 beta-D-glucuronide.

Studies were conducted to determine the absorption and metabolic fate of orally administered 3H-estradiol-17 beta-glucuronide (3H-E2-G) in swine. Xylazine-tranquilized female pigs (5 to 6 wk old) were given .04, .4 or 4 mumol 3H-E2-G via stomach tube, and blood samples were collected from previously implanted jugular cannulas for 12 or 72 h. The entire gastrointestinal tract was removed from gilts euthanatized 12 h post-treatment, and free and conjugated estrogens were isolated from plasma and intestinal chyme by diethyl ether extraction and adsorption to Amberlite XAD-2 resin columns. After preparative thin layer chromatography of the conjugate fractions, the conjugates were cleaved by enzyme hydrolysis, solvolysis or acid hydrolysis. The freed estrogens were identified by thin layer chromatography. Plasma radioactivity peaked between 6 and 8 h after administration of the conjugate. None of the radioactivity in plasma was ether extractable. There was evidence for a decrease in absorption rate of radioactive estrogen in the high dosage group. The pattern of metabolites and urinary excretion or orally administered 3H-E2-G was similar to that reported for 14C-E2, except for the greater proportion of polar metabolites and delayed absorption, probably reflecting the need for the conjugate to be hydrolyzed first. The greater proportion of polar metabolites found in this study may have been due to the longer treatment period rather than the administration of the conjugated form of estradiol.

Dexamethasone treatment during hemorrhagic shock: changes in extracellular fluid volume and cell membrane transport.

Changes in extracellular fluid volume and cell membrane transport during hemorrhagic shock and the effects of dexamethasone treatment on these changes were measured. It is well known that prolonged hemorrhagic shock leads to irreversible changes and a progressive decrease in blood pressure despite reinfusion for lost blood. Pharmacologic doses of glucocorticoids provide some protection against these changes. Therefore, one purpose in the present study was to identify possible sites of glucocorticoid action whicy may prevent the irreversible changes from occurring. The extracellular fluid volume in normal control, nontreated dogs in shock, and dexamethasone-treated dogs in shock were measured by a dilution technique, using [35S] sodium sulfate. Cell membrane cation transport capabilities were measured in liver slices, diaphragm slices, and red blood cells taken from normal control, nontreated rats in shock, and dexamethasone-treated rats in shock. The accumulation of radioactivity by the tissues incubated with 22Na served as an indicator of cell membrane ion transport capabilities. The results indicate that in animals subjected to prolonged hemorrhagic shock, there is a fluid shift from the extracellular space into intracellular spaces, reducing blood volume. Cell membranes are damaged and transport mechanisms are altered; therefore, the cells are unable to extrude ions along with water. Dexamethasone treatment was shown to prevent extracellular fluid volumes from decreasing below that amount due to the plasma lost during hemorrhage. Also, it prevented some cell membrane damage and maintained membrane transport mechanisms near normal. In addition, at the onset of dexamethasone injection, blood pressure increased, and urine output was restored.

Characterization of release of tumor necrosis factor, interleukin-1, and superoxide anion from equine white blood cells in response to endotoxin.

Direct effects of endotoxin (lipopolysaccharide [LPS]) on equine WBC are known to stimulate the release of a variety of mediators including thromboxane, prostacyclin, and leukotrienes. In this study, 0.1 microgram of LPS/ml stimulated an early increase in tumor necrosis factor, succeeded by an increase in interleukin-1, but concentrations of LPS up to 5.0 micrograms/ml caused no significant increase in superoxide anion release. The concentration of LPS (0.1 microgram/ml) used in this experiment was in the range of concentrations measured in plasma of some horses with gastrointestinal problems. These results indicate that mediators released in response to low concentrations of LPS may be responsible for many of the LPS-induced pathophysiologic effects. This is indicated because concentrations of LPS detected in plasma of some horses with severe gastrointestinal problems are approximately 0.1 microgram/ml, a concentration that will stimulate cells to produce tumor necrosis factor, but will not stimulate any other measurable cytotoxic effect.

Serum triiodothyronine, total thyroxine, and free thyroxine concentrations in horses.

The objectives of this experiment were to determine serum concentrations of triiodothyronine (T3), thyroxine (T4), and free thyroxine (fT4) at rest, following thyroid-stimulating hormone (TSH) administration, and following phenylbutazone administration in healthy horses. This was done to determine which available laboratory test can best be used for diagnosis of hypothyroid conditions in horses. Serum T3, T4, and fT4 concentrations in serum samples obtained before and after TSH stimulation and following phenylbutazone administration for 7 days were determined. Baseline values ranged from 0.21 to 0.80 ng of T3/ml, 6.2 to 25.1 ng of T4/ml, and 0.07 to 0.47 ng of fT3/dl. After 5 IU of TSH was administered IV, serum T3 values increased to 6 times baseline values in 2 hours. Thyroxine values increased to 3 times baseline values at 4 hours and remained high at 6 hours. Free T4 values increased to 4 times baseline values at 4 hours and remained high at 6 hours. Administration of 4.4 mg of phenylbutazone/kg, every 12 hours for 7 days significantly decreased T4 and fT4 values, but did not significantly affect serum T3 concentrations. It was concluded that a TSH stimulation test should be performed when hypothyroidism is suspected. Measurement of serum fT4 concentrations, by the single-stage radioimmunoassay, does not provide any additional information about thyroid gland function over that gained by measuring T4 concentrations. Phenylbutazone given at a dosage of 4.4 mg/kg every 24 hours, for 7 days did significantly decrease resting T4 and fT4 concentrations, but did not significantly affect T3 concentrations in horses.

Dexamethasone treatment during hemorrhagic shock: blood pressure, tissue perfusion, and plasma enzymes.

In a study to determine beneficial effects of dexamethazone (5 mg/kg) during hemorrhagic shock, perfusion (as measured by radioactive microspheres) and plasma enzymes were measured. Hemorrhagic shock (mean arterial blood pressure, MABP, of 50 mm of Hg) was induced in dogs and then the dogs were isolated from the shed-blood reservoir and were allowed to compensate their MABP. Hemodynamic changes, tissue perfusion, and plasma enzymes were measured at different intervals of time in nontreated and dexamethasone-treated dogs. Beneficial effects were increased MABP, improved blood flow to the lungs, gastrointestinal tract, and kidneys, and less cell damage as indicated by amounts of plasma enzymes released from damaged tissues. These effects favor the maintenance of the homeostatic state and, in turn, a greater chance for survival of the dog.

Dexamethasone treatment during hemorrhagic shock: effects independent of increased blood pressure.

Effects of dexamethasone (5 mg/kg of body weight) on organ blood flow, enzyme release, and hemodynamics were studied in dogs with hemorrhagic shock to determine if the consequences observed were due to increased mean arterial blood pressure (MABP) or intrinsic effects of dexamethasone. Hemorrhage was induced in anesthetized dogs until the MABP was 50 mm of Hg and then the dogs were treated with dexamethasone or an equal volume of saline solution. Dogs remained connected to the blood reservoir during the entire experiment and MABP was maintained at 50 mm of Hg in the treated and control groups of dogs. Beneficial actions of dexamethasone treatment independent of increased MABP were observed. Increased survival rate, differential blood flow to some organs, and less tissue damage occurred as a result of dexamethasone treatment and were independent of increased MABP.

Thyrotropin-releasing hormone stimulation testing in healthy dogs.

Serum triiodothyronine (T3) and thyroxine (T4) concentrations were determined after IV administration of 200 micrograms of thyrotropin-releasing hormone (TRH) to 10 healthy euthyroid dogs. Significant (P less than 0.05) changes were not found in the T3 concentration throughout an 8-hour sampling interval. All dogs had a significant increase (P less than 0.05) in the T4 concentration at 4, 5, 6, 7, and 8 hours after TRH administration. The largest increase in the serum T4 concentration occurred 4 hours after TRH injection. From 4 to 8 hours after TRH administration, the mean increase above basal T4 concentrations was 13.9 +/- 5.4 ng/ml.

Endotoxin-induced hemodynamic changes in dogs: role of thromboxane and prostaglandin I2.

Plasma concentrations of thromboxane and prostaglandin I2 (PGI2) before and after IV injection of endotoxin and resulting hemodynamic changes were evaluated. Effects of flunixin meglumine on plasma concentrations of these prostaglandins and the related hemodynamic changes were also determined. Shock was induced in 2 groups of anesthetized dogs. Four dogs were given endotoxin only and 4 dogs were given endotoxin and then were treated with flunixin meglumine. Arterial blood pressure (BP), cardiac output (CO), and heart rate were measured, and blood samples were collected at postendotoxin hours (PEH) 0, 0.1, 0.25, 0.5, 1, 2, 3, and 4. Plasma thromboxane and PGI2 concentrations were increased in canine endotoxic shock. Thromboxane concentration was highest early in shock, and appeared to be associated with an initial decrease in BP and CO. The increased concentration of PGI2 was associated with systemic hypotension at PEH 1 to 2. Treatment of dogs with flunixin meglumine at PEH 0.07 prevented further increase of thromboxane and blocked the release of PGI2, resulting in an increased CO, BP, and tissue aerobic metabolism.