Free fatty acids in an animal model of Reye's syndrome.

Recent studies have indicated that viral infections, aspirin treatment and hyperammonemia are associated with Reye's syndrome. It has also been reported that free fatty acids in serum and total lipids in the liver of Reye's syndrome patients are elevated during illness. The role of the lipid changes in the development of the disorder cannot be optimally studied in human patients, because infection and aspirin ingestion occur prior to the earliest symptoms of Reye's syndrome. Effects of influenza B infection, aspirin treatment and hyperammonemia on the level of free fatty acids, total lipids and triacylglycerols in serum and liver of an animal model of Reye's syndrome are reported here. Hyperammonemia was produced in young, male ferrets either by feeding them small amounts of an arginine-deficient diet after overnight fasting or by an intraperitoneal injection of jackbean urease. The ferret model resembled Reye's syndrome in developing increased levels of individual and total serum free fatty acids, liver triacylglycerol and total lipids. The results also indicate that influenza infection or aspirin treatment, or both, while increasing the severity of encephalopathy in the deficient ferrets, did not cause a significant change in the level of serum free fatty acids. Other results suggest that elevation of serum ammonia, serum free fatty acid or liver lipids, either singly or in various combinations, does not provide conditions that can explain the rapidly developing encephalopathy in the arginine-deficient ferrets.

Age-dependent changes in pyruvate uptake by nonsynaptic and synaptic mitochondria from rat brain.

Changes in the uptake of pyruvate by nonsynaptic and synaptic mitochondria from brains of young adult and old rats were investigated. An age-dependent decrease in State 3 respiration in the presence of pyruvate plus malate as substrate was observed in cerebral mitochondrial populations but not in liver mitochondria. Addition of exogenous cytochrome c to nonsynaptic and synaptic mitochondria enhanced the rate of State 3 respiration but the age-dependent decrease in State 3 respiration persisted in both types of mitochondria. A decrease in the uptake of pyruvate as measured by the inhibitor-stop and rapid centrifugation techniques was observed in both nonsynaptic and synaptic mitochondria from 24-month-old rats compared to 3-month-old rats. The results suggest that the decrease in the uptake of pyruvate may be one of the factors responsible for the observed reduction in State 3 respiration in the presence of pyruvate plus malate by both nonsynaptic and synaptic mitochondria from brains of senescent rats compared to young adults.

Age-dependent changes in glutamate oxidation by non-synaptic and synaptic mitochondria from rat brain.

The oxidation of glutamate by non-synaptic and synaptic mitochondria from brains of 3-, 12- and 24-month-old rats was studied. With glutamate plus malate as substrates, non-synaptic mitochondria showed higher respiration rates than synaptic mitochondria in all the three age groups studied. The rate of oxidation of L-[1-14C]glutamate and the activities of NAD-glutamate dehydrogenase and aspartate aminotransferase were also higher in non-synaptic mitochondria compared with synaptic mitochondria in three age groups. With glutamate plus malate as substrates, a significant reduction in state 3 respiration was observed in both mitochondrial populations from 12- and 24-month-old rats compared with 3-month-old animals. Although an age-dependent decrease in the oxidation of L-[1-14C]glutamate was observed in both non-synaptic and synaptic mitochondria from aging rats, the oxidation of [1-14C]-2-oxoglutarate was unaltered in non-synaptic and synaptic mitochondria from senescent rats. The activity of NAD-glutamate dehydrogenase was decreased with age in both mitochondrial populations, whereas aspartate aminotransferase was not altered with age. The results indicate that the oxidation rate of glutamate in rat brain mitochondria is decreased during aging.

Effect of aging on intestinal ischemia and reperfusion injury.

Intestinal ischemia/reperfusion (I/R) is a serious disorder that is prevalent in elderly patients. Reactive oxygen species are implicated in the pathogenesis of intestinal I/R injury. Reactive oxygen species are also implicated in cellular senescence and aging. To test the hypothesis that aging exacerbates intestinal I/R injury, the effects of intestinal I/R on tissue injury were compared between young (3 month old) and aged (12 month old) mice. Intestinal ischemia was induced by occluding the superior mesenteric artery with a microbulldog clamp. Reperfusion was initiated by removing the clamp. Mortality due to intestinal ischemia followed by reperfusion was significantly higher in aged mice. There were no differences in the baseline levels of malondialdehyde or myeloperoxidase activity (indicators of lipid peroxidation and neutrophil infiltration, respectively) between young and aged mice. Although intestinal I/R caused a significant increase in malondialdehyde levels and myeloperoxidase activity in aged mice, similar increases were also observed in young mice. There were no significant differences in the activities of antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase between young and aged mice that underwent sham operation. Intestinal I/R caused a significant decrease in catalase activity only in aged mice. In conclusion, our results indicate that aged mice are more susceptible to mortality due to intestinal I/R and that an age-dependent decrease in catalase activity may contribute to the observed mortality.

Changes in plasma fibronectin in children after elective repair of congenital heart defects.

Plasma fibronectin is an attachment protein important for maintaining capillary integrity and host defense mechanisms. Depletion of plasma fibronectin has been shown to occur in adults after septic shock, major trauma, and burns. Limited laboratory and clinical studies suggest a correlation between decreased plasma fibronectin levels and increased pulmonary capillary permeability and tissue perfusion. Mild and transient plasma fibronectin depletion has been observed in adults after cardiovascular operations. We measured plasma fibronectin by immunoturbidometric assay in 20 children (age 6 months to 12 years) undergoing repair of congenital heart defects. Plasma fibronectin levels immediately after operations and daily thereafter were compared with the preoperative values. Plasma fibronectin declined on postoperative days 1, 2, 3, 4, and 5 (p < 0.05). A nadir was reached on day 3 with a tendency toward recovery thereafter. Patients with a therapeutic intervention score of more than 35 had greater magnitude of plasma fibronectin decline than those with a score of less than 35 at 24 hours after the operation (p < 0.005). We conclude that (1) significant and prolonged plasma fibronectin depletion occurs after cardiovascular operations in children; and (2) postoperative plasma fibronectin depletion is associated with increasingly complex surgical intervention. Reduced plasma fibronectin synthesis and more extensive operations for congenital heart defects are likely reasons for children being more susceptible than adults to plasma fibronectin depletion after cardiovascular operations.

Guanidino compounds in serum, urine, liver, kidney, and brain of man and some ureotelic animals.

Guanidino compound levels were quantitatively determined in serum, urine, liver, kidney, and brain of man and of some ureotelic animals. The guanidino compounds were separated over a cation exchange resin, using sodium citrate buffers, and detected with the fluorescence ninhydrin method. Species-specific differences in the levels of some guanidino compounds in the studied ureotelic animals are shown. alpha-Keto-delta-guanidinovaleric acid is a naturally occurring guanidino compound in ureotelic animals, and is not restricted to the pathobiochemistry of hyperargininemic patients. The fasting serum levels observed in beagles are the same as those found in hyperargininemic patients. In serum, liver, and kidney, the homoarginine, beta-guanidinopropionic acid, and gamma-guanidinobutyric acid levels are the highest in rats. The last two compounds have the highest levels of the studied guanidino compounds, with the exception of creatinine, in kidney. Specific high levels of gamma-guanidinobutyric acid and argininic acid are found in brain of rabbits.

Aggrecan degradation in chondrocytes is mediated by reactive oxygen species and protected by antioxidants.

Reactive oxygen species (ROS) are implicated in aging of cartilage and in the pathogenesis of osteoarthritis. However, the biological role of chondrocytes-derived ROS has not been elucidated. An in-vitro model was developed to study the role of chondrocyte-derived ROS in cartilage matrix degradation. The primary articular chondrocytes were cultured and the aggrecan matrix was radiolabeled with 35-sulfate. The labeled aggrecan matrix was washed to remove unincorporated label and chondrocytes were returned to serum free balanced salt solution. The cell-monolayer-matrix sensitivity to oxidative damage due to either hydrogen peroxide or glucose oxidase was established by monitoring the release of labeled aggrecan into the medium. Lipopolysaccharide (LPS) treatment of chondrocyte-monolayer enhanced the release of labeled aggrecan. Catalase significantly prevented the release of labeled aggrecan in LPS-chondrocyte cultures, suggesting a role for chondrocyte-derived hydrogen peroxide in aggrecan degradation. Superoxide dismutase or boiled catalase had no such inhibitory effect. The effect of several antioxidants on LPS-chondrocyte-dependent aggrecan degradation was examined. Hydroxyl radical scavengers (mannitol and thiourea) significantly decreased aggrecan degradation. A spin trapping agent N-tert-butyl-phenylnitrone (but not its inactive analog tert-butyl-phenylcarbonate) significantly decreased aggrecan degradation. Butylated hydroxytoluene also inhibited aggrecan degradation, whereas the other lipophilic antioxidant tested, propyl gallate, had a marked dose-dependent inhibitory effect. These data indicate that general antioxidants, hydroxyl radical scavengers, antioxidant vitamins, iron chelating agents, lipophilic antioxidants, and spin trapping agents can influence chondrocyte-dependent aggrecan degradation. These studies support the role of a chondrocyte-dependent oxidative mechanism in aggrecan degradation and indicate that antioxidants can prevent matrix degradation and therefore may have a preventive or therapeutic value in arthritis. The enhancement of oxidative activity in chondrocytes and its damaging effect on matrix may be an important mechanism of matrix degradation in osteoarthritis.

Histopathology of fasting and bluecomb disease in turkey poults and embryos experimentally infected with bluecomb disease coronavirus.

The histopathology of fasting and bluecomb disease in one-day-old turkey poults inoculated with bluecomb disease coronavirus (BCDCV) was studied. Uninoculated fasting poults produced clinical signs similar to those observed in BCDCV-inoculated poults. No histological changes in the intestines were observed in the fasted poults whereas definite lesions were observed in the BCDCV-inoculated poults. The lesions did not differ significantly with whether they were fed or fasted. The severity of the lesions in the intestinal epithelium was in decreasing order in the jejunum, ileum, and cecum. The lesions first appeared 24 hours postinoculation (PI) and progressed through 96 hours PI, as marked shortening of the villi, loss of microvilli, granular appearance of the cytoplasm of epithelial cells with nuclear margination of chromatin, and accentuation of the nucleolus. Similar lesions were observed in the jejunum, ileum, and cecum of turkey embryos inoculated at 24 days old as well as poults from these embryos. Signs of healing were first seen at 120 hours PI. No histopathological changes were observed in the pancreas, brain, kidneys, liver, adrenal, and bursa of Fabricius. The intestinal lesions observed should be a useful histological technique for differentiating fasting from bluecomb disease in turkey poults.

Effects of influenza infection, aspirin, and an arginine-deficient diet on the inner ear in Reye's syndrome.

The individual effects of an influenza B viral infection, aspirin, and an arginine-deficient diet on the inner ear were assessed in the ferret model for Reye's syndrome using both functional and morphological parameters. Auditory brainstem evoked responses recorded from inoculated ferrets revealed threshold elevations and increased latencies during the first 72 hours, but approximated those of control animals by 96 hours. Although there was a mild distention of Reissner's membrane, no pronounced structural alterations in sensory or supporting cells were observed in cochleas from inoculated ferrets. The administration of aspirin appeared to alter neither the functional nor the structural integrity of the cochlea. The presentation of an arginine-deficient diet, creating a hyperammonemic condition, led to both altered auditory evoked responses and vacuolization of cochlear tissues after treated animals had undergone seizures and coma. These data demonstrated that both influenza B and the arginine-deficient diet individually affected the hearing of treated animals. The individual agents did not alter the cochlea as severely as when they were presented in combination. These results suggest that hearing impairment in patients with Reye's syndrome may be a result of potentiation of certain metabolic-altering agents.

Altered auditory brainstem evoked responses in the ferret model for Reye's syndrome.

The effects of influenza B, aspirin and hyperammonemia on the functional integrity of the cochlea were examined using the ferret model for Reye's syndrome. Auditory brainstem evoked responses (ABR) were recorded from treated ferrets and compared to those recorded from controls. Delayed latencies of all waves, as well as increased I-IV Interwave latencies were observed in recordings from treated ferrets. Importantly, the latencies of Wave I, nerve generator potential from the eighth nerve, and Wave IV, response potential from the brainstem nuclei, increased on Days 3 and 5 of the study. However, recorded ABR from treated ferrets on Day 10 showed that while the latencies of Wave IV were approximate to those of controls, Wave I remained delayed. These results suggest that systemic effects of influenza B, aspirin and hyperammonemia may lead acutely to both peripheral and central auditory dysfunction, and that the functional integrity of the central auditory system may recover more quickly than that of the peripheral system. Furthermore, these results suggest that such effects may lead to acute transient hearing impairment in patients with Reye's syndrome.

Fluorescent antibody test for rapid diagnosis of coronaviral enteritis of turkeys (bluecomb).

Intestinal tissues obtained from coronavirus-infected embryos and turkeys were examined by fluorescent antibody tissue section technique (FAT). Evidence of viral antigen was demonstrated in the cytoplasm of the intestinal epithelial cells covering the villi. Embryo intestines that were examined from 24 to 96 hours after inoculation were positive for immunofluorescence (IF), whereas bursa of Fabricius was negative. Poults hatched from infected embryos were examined at 2 days of age and were positive for IF. Coronaviral antigen was detected by FAT in the cytoplasm of intestinal epithelial cells of the jejunum, ileum, duodenum, and cecum in all turkeys that were examined from 24 hours to 28 days.

Immunity to transmissible (coronaviral) enteritis of turkeys (bluecomb).

Turkey flocks recovering from natural and laboratory induced coronaviral enteritis developed lifelong immunity. Virus neutralization tests indicated that neutralization capacity of serums from recovered turkeys was low. One-way cross challenge test using turkeys inoculated with various bluecomb isolates revealed close antigenic relationship or antigenic identity among bluecomb isolates. The controlled application of intestinal preparations containing coronavirus as a vaccination procedure had value in developing active lasting immunity but it has limitations in areas that are attempting to eliminate the disease by a depopulation program. Killed vaccines injected parenterally did not produce a detectable immunity.

Effect of endotoxemia on plasma and tissue levels of nitric oxide metabolites and guanidino compounds.

The effect of endotoxemia on the levels of amino acids, nitrates, nitrites and guanidino compounds was investigated. Plasma levels of nitrate and nitrite were significantly increased indicating increased production of nitric oxide during endotoxemia. Plasma concentrations of alanine, glutamine, leucine, methionine, phenylalanine, proline and taurine were also significantly elevated. These results indicate that endotoxin produces a hypercatabolic state. The plasma concentration of arginine was significantly decreased whereas the concentrations of ornithine and urea, the catabolites of arginine were increased. Decreased plasma arginine coupled with increased plasma ornithine and urea indicate that arginine catabolism is increased and arginine synthesis is decreased during endotoxemia. Plasma levels of creatine, creatinine, guanidine and guanidinosuccinic acid were significantly elevated whereas homoarginine levels were significantly decreased. Nitric oxide synthase utilizes arginine as well as homoarginine as substrates. The decreased concentration of both substrates may be related to alterations in nitric oxide synthase activity during endotoxemia. These results suggest that in addition to nitric oxide, other catabolites of arginine such as guanidino compounds may be important in the pathophysiology of endotoxemia. Because of the marked increase in guanidinosuccinic acid, a known uremic toxin, we speculate that guanidinosuccinic acid may be important in the pathophysiology of endotoxemia.