Reye's Syndrome Patients Research Articles

From Australia to Chinchilla to Jamaica

A general impairment of liver mitochondrial enzymes is central to Reye's syndrome (RS). The respiration of isolated liver mitochondria was measured after the addition of concentrated normal serum or RS serum derived from 12 patients. RS serum stimulates oxygen consumption in isolated rat liver mitochondria. This effect is due to (a) the oxidation of uric acid by peroxisomes contaminating the preparation and (b) a stimulation of mitochondrial respiration (1.05 ± 0.14 nmole of O2/ min · mg of protein; control 0.30 ± 0.08 nmole O2/min · mg). The stimulation of respiration occurs in the presence of all respiratory substrates, is dependent on the amount of serum added, and represents an uncoupling of oxidativephosphorylation. RS serum reduces ATP formation by 15 to 76%. The uncoupling effect correlates with the amount of free fatty acid in the serum sample and resembles theeffect induced by the addition of a dicarboxylic fatty acid. Dicarboxylic fatty acids, especially long–chain dicarboxylic acids, impair ATP formation. Dicarboxylic acids were found in the serum of all RS patients and comprised as much as 54% of the total serum free fatty acids. Ninety percent of the serum dicarboxylic acids were of 16 to 18 carbon lengths. The amount of dicarboxylic acids in the RS serum corresponded directly with the reduction in ATP formation by the RS serum. This demonstrates that dicarboxylic acids occur in RS and may be important in the general impairment of mitochondrial function in RS and other disorders where they are present.

Elevated Serum γ‐Aminobutyric Acid Levels in Children with Reye's Syndrome

SummaryElevated serum levels of γ‐aminobutyric acid (GABA), a potent inhibitory neurotransmitter, have recently been implicated in the pathogenesis of hepatic encephalopathy. In this study, serum GABA levels were measured in five children with severe Reye's syndrome, 10 children with acute viral hepatitis, and seven healthy volunteers. Serum GABA levels were highest in the five Reye's syndrome patients. The mean serum GABA level for the Reye's syndrome group (3.0 ± 1.3 üM, mean ± SEM) was significantly elevated as compared to the mean of the viral hepatitis group (0.72 ± 0.07 μM, p < 0.05) and the healthy volunteers (0.38 ± 0.04 μM, p < 0.05). These results provide preliminary evidence to suggest that elevated serum GABA levels may in part be responsible for the encephalopathic state observed in children with Reye's syndrome.

Cerebral Tubuloreticular Inclusions In Reye's Syndrome

IntroductionTubuloreticular inclusions (TRI) were first reported in patients with herpesvirus encephalitis or systemic lupus erythematosus (SLE). TRI are abnormal subcellular structures which consist of fine irregularly branched, anastomosing tubules within cisternae of endoplasmic reticulum. TRI have also been reported in blood lymphoreticular cells, vascular endothelial cells, mesenchymal cells, and epithelial cells in a variety of clinical conditions which include SLE and other autoimmune diseases, congenital immunodeficiency, lymphoproliferative disorders, neoplasms, neurodegenerative disorders, T-cell leukemia, and acquired immune deficiency syndrome (AIDS). We have recently reported an association of TRI with Reye's syndrome (RS). The inclusions were found in peripheral leukocytes of two unrelated patients with RS following a prodromal influenza respiratory infection. Subsequent to those studies we have retrospectively examined biopsy material from earlier cases of RS, and have identified TRI in the capillary endothelial cells from brain biopsies of three RS patients. The purpose of this report is to present these observations and to discuss the association of TRI with RS.

Effect of Reye's syndrome serum on isolated chinchilla liver mitochondria.

A general impairment of liver mitochondrial enzymes is central to Reye's syndrome (RS). The respiration of isolated liver mitochondria was measured after the addition of concentrated normal serum or RS serum derived from 12 patients. RS serum stimulates oxygen consumption in isolated rat liver mitochondria. This effect is due to the oxidation of uric acid by peroxisomes contaminating the preparation and a stimulation of mitochondrial respiration (1.05 +/- 0.14 nmol of O2/min X mg of protein; control 0.30 +/- 0.08 nmol O2/min X mg). The stimulation of respiration occurs in the presence of all respiratory substrates, is dependent on the amount of serum added, and represents an uncoupling of oxidative phosphorylation. RS serum reduces ATP formation by 15-76%. The uncoupling effect correlates with the amount of free fatty acid in the serum sample and resembles the effect induced by the addition of a dicarboxylic fatty acid. Dicarboxylic fatty acids, especially long-chain dicarboxylic acids, impair ATP formation. Dicarboxylic acids were found in the serum of all RS patients and comprised as much as 54% of the total serum free fatty acids. 90% of the serum dicarboxylic acids were of 16-18 carbon lengths. The amount of dicarboxylic acids in the RS serum corresponded directly with the reduction in ATP formation by the RS serum. This demonstrates that dicarboxylic acids occur in RS and may be important in the general impairment of mitochondrial function in RS and other disorders where they are present.

Absence of diffusible inhibitor of glutamate dehydrogenase in the hepatocytes of Reye syndrome patients.

Hepatic glutamate dehydrogenase (GDH) activity was measured in postmortem specimens obtained from two stage V Reye syndrome patients and in three postmortem specimens of normal human liver. The Reye syndrome specimens showed the hepatic mitochondrial enzyme deficits in GDH and monoamine oxidase activities that are characteristic of Reye syndrome. GDH activity was linear with the amount of supernatant fraction added, both for Reye and normal liver preparations: moreover, the activities of mixtures of Reye and control supernatant fractions were the sums of the activities of the individual components. This means that the activity difference between Reye and normal GDH activity is not due to a diffusible inhibitor in the Reye hepatocytes or to an activator of GDH in the normal control hepatocytes. Serum obtained from six Reye cases during neurologic deterioration was added to normal hepatic GDH preparations to test for a serum inhibitor of FDH. Highly variable effects were found, with two serum samples producing marked inhibition and others showing weak inhibition, no effect, or stimulation of GDH activity. The inhibitor was not removed by charcoal treatment and most of the activity was retained by a 10,000 dalton Diaflo membrane, signifying either that the compound had a high molecular weight or that it was bound to serum protein. We conclude that the decreased activity of GDH in Reye hepatocytes is not due to an intracellular diffusible inhibitor, and that serum effects are quite variable and are not directly related to intracellular changes in GDH activity.

Morphologic grading of hepatic mitochondrial alteration in Reye's syndrome: potential prognostic implication.

Mitochondrial alterations are known to be constantly associated with Reye's syndrome. Fifty-eight liver specimens from 50 patients who fulfilled the clinical and pathologic criteria were examined by electron microscopy. The degree of mitochondrial injury was graded I, II, and III according to the severity of the alteration of mitochondrial matrix. The results correlated well with clinical parameters--specifically initial clinical stage, peak clinical stage, and clinical outcome. Electron microscopic examination of hepatic mitochondria is therefore potentially useful in evaluating Reye's syndrome patients.

Serum glutamate dehydrogenase and ornithine carbamyl transferase in Reye's syndrome.

Parallel increases in the ornithine carbamyl transferase and glutamate dehydrogenase activities were observed in the serum of 22 Reye's syndrome patients. The increase in the activity of glutamate dehydrogenase was masked by a dialyzable inhibitor. It is proposed that the measurement of serum ornithine carbamyl transferase and glutamate dehydrogenase activities may be useful as an indicator of liver mitochondrial damage in Reye's syndrome.

Ornithine carbamyl transferase in Reye's syndrome

Serum levels of ornithine carbamyl transferase activities were determined in the acutely ill and convalescent Reye's syndrome patients and in their parents. Acutely ill Reye's syndrome patients had elevated levels of serum ornithine carbamyl transferase activities as compared to those in controls. The convalescent Reye's syndrome patients and their parents had normal levels of serum ornithine carbamyl transferase activities. These results suggest that an inborn metabolic defect was not responsible for the increase in serum ornithine carbamyl transferase activities in Reye's syndrome.

Short-chain fatty acids and encephalopathy of Reye's syndrome.

Plasma levels of six short-chain fatty acids (SCFA) were measured in 23 Reye's syndrome patients. In sequential measurements, only propionic acid correlated closely with neurologic severity. Although admission SCFA levels were slightly elevated, there were no significant differences between patients grouped by severity of encephalopathy. Admission SCFA did not predict neurologic outcome; also, they correlated poorly with admission blood ammonia, amino acid nitrogen, and lactate.

DICARBOXYLIC ACIDS AS UNCOUPLERS IN REYE'S SYNDROME

A general impairment of liver mitochondrial enzymes is central to Reye's Syndrome(RS). The effect of RS serum from 8 patients in profound coma on respiration of isolated liver mitochondria was measured with 2.5mg of mitochondrial protein, 3mM substrates, and 5x concentrated serum. RS serum stimulates respiration(1.05± .14 nmol O2/min/mg protein, control .30± .08). This effect is due to: 1.)oxidation of uric acid by microsomes contaminating the preparation, 2.)fatty acids providing substrate, 3.)uncoupling of oxidative phosphorylation by 30-40%. The amount of uncoupling is additive, correlates with the amount of free fatty acids in the serum but is not inhibited by 1% defatted albumin as is oleate added to normal serum. The degree of stimulation/mcg of fatty acid is distinct from that of oleate or octanoate added to normal serum. RS serum stimulates respiration in the presence of antimycin and 3mM ascorbate by 80-149%: oleate+ normal serum causes a 0-10% increase. 3mM medium chain dicarboxylic acids are not readily bound to 1% albumin and induce a 90-105% increase in antimycin/ascorbate respiration; they require intact mitochondria and exogenous substrate. Dicarboxylic acids are markedly elevated (1-3mg/mg creatinine, N=12; controls<.10, N=40) in urine from our RS patients and unusual long and unsaturated dicarboxylic acids were reported in the serum of 2 RS patients (Amer. Soc. Mass. Spec. 31:98, 1983). Thus, dicarboxylic acids act as uncouplers and may be important in the general impairment of mitochondrial function in RS and other disorders where large amounts of dicarboxylic acids are present.

BIOCHEMICAL PROFILES FOLLOWING ASPIRIN ADMINISTRATION IN REYE'S SYNDROME PATIENTS AND CONTROLS

Is aspirin an important factor in the pathogenesis of Reye's syndrome (RS)? We have challenged 11 control individuals (C), 5 first degree relatives of RS patients (FR) and two patients who have recovered from RS (RS-1 & RS-2) with standard doses of aspirin for 12 hrs. (600 mg. q 4 hrs.) while fasting. There were no differences in the preaspirin and postaspirin concentrations of ammonia, glucose, AST, or LDH in the RS patients compared to the controls. However, both RS patients showed increased lactate, decreased pyruvate, and decreased β-hydroxybutyrate concentrations following the aspirin challenge while controls showed an opposite trend as illustrated below:These biochemical differences in the RS patients compared to controls suggest an altered cytosolic redox state which is also an indirect index of mitochondrial function. One interpretation of these preliminary observations is that individuals who develop RS may be susceptible to mitochondrial injury when exposed to aspirin during fasting.

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.

Measurement of true salicylate concentrations in serum from patients with Reye's syndrome.

Patients with Reye's syndrome who have been given aspirin are said to maintain higher-than-anticipated salicylate concentrations in blood, for longer than expected. We explored whether this could be attributed to spurious results from nonsalicylate compounds in the Trinder reaction for salicylates. All of 63 organic acids and amines examined that form colored complexes with Trinder's reagent had detectable absorbance at 540 nm at 0.2 g/L, including some endogenous compounds known to be increased in Reye's syndrome patients and many others endogenous in humans. By subjecting deproteinized sera to thin-layer chromatography and eluting the salicylate fraction before complexing it with ferric ion, true salicylate can be measured quantitatively and differentiated from interfering compounds. In addition, when we examined the effect of salicylate on palmitate binding to serum proteins, we found that salicylate concentrations of 0.2 g/L displaced [16-14C]palmitate binding to protein more in Reye's syndrome patients than in Reye's syndrome survivors or children with influenza. This suggests the presence of atypical binding characteristics for salicylate and palmitate in the acute disorder but not in survivors or children with influenza.

Somatic evoked potential evaluation of cerebral status in Reye syndrome

Median nerve somatic evoked potentials (SEPs) were serially recorded in 12 Reye syndrome patients from shortly after admission to discharge. Recovery was clinically satisfactory in nine, unsatisfactory in two, and one died. All SEP components were absent or markedly depressed in initial recordings. Early progressive recovery of primary cortical components was associated with patient survival; lack of it was associated with death. Progressive recovery of SEP components later than 100 msec was associated with satisfactory clinical recovery; failure of recovery of these components was associated with residual neuropsychological deficit. Evaluation of late component recovery required comparison with age-dependent SEP configurations in normal children which differ from adults. We conclude that serial SEP recording is of significant value and superior to the EEG in early prognosis for survival, prognosis for clinically satisfactory or unsatisfactory recovery, and general evaluation of neurologic status in Reye syndrome.

Liver coenzyme A ester content: comparison between Reye's syndrome and control subjects.

1. The concentrations of the acid soluble and insoluble coenzyme A (CoA) esters were measured in samples of liver obtained at autopsy from Reye's syndrome and control subjects because the long chain fatty CoA compounds which make up the bulk of the acid insoluble CoA esters are known to inhibit a number of mitochondrially located enzymes, several of which may be affected in Reye's syndrome. 2. Concentrations of the acid insoluble esters varied widely in both control and Reye's liver samples. The difference between the mean values was not statistically significant (1·06 ± sem 0·33 nmol/g wet weight in Reye's samples vs 0·88 ± 0·21 in control samples). 3. Concentrations of the acid soluble CoA esters, which include the short chain fatty CoA compounds, were higher in Reye's liver samples than in samples from controls. The mean value for Reye's samples was 104·8 ± sem 29·4 nmol/g of liver compared with 26·4 ± 10·1 nmol/g for control samples (P &amp;lt; 0·05). 4. Studies with rats designed to assess postmortem change indicate that the liver concentration of the acid insoluble CoA compounds does not change during a 4 h period at 4°C. This finding suggests that the observations made in Reye's liver was probably due to a premorbid abnormality. 5. These findings implicate a block in the β-oxidation of fatty acids and could account for the reported relative increase in the concentrations of the short to medium chain fatty acids in the plasma of Reye's syndrome patients.

Reye's syndrome: Plasma-induced alterations in mitochondrial structure and function

Preincubation of rat liver mitochondria with plasma from Reye's syndrome (RS) patients induces a transient stimulation of the State 4 respiratory rate of the oxidation of NAD-linked substrates which is followed by inhibition. A loss of nearly 90% of the intramitochondrial NAD + and NADP + is also seen. The respiratory rate cannot be stimulated upon subsequent addition of ADP, but can be fully restored upon the addition of either NAD + or succinate (plus rotenone). The degree of effectiveness depends on the incubation time and the ratio of RS-plasma/mitochondrial protein. The RS-plasma effects can be eliminated by an inhibitor of mitochondrial Ca 2+ transport (ruthenium red) or by a Ca 2+ chelator (ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid). Control plasma at a concentration of 2 mg dry wt per milligram of mitochondrial protein, or 30 μ m Ca 2+ gives no effect, but can reproduce the RS-plasma effects completely when a minute amount of allantoin (10 −11 mol/mg mitochondrial protein) is also present. We conclude that allantoin and Ca 2+ can increase the permeability of mitochondrial membrane, and may be the key components responsible for the mitochondrial injuries produced by RS-plasma.

Hepatic Monoamine Oxidase Deficiency in Reye Syndrome

Faraj et al1 recently reported a positive correlation between plasma tyramine levels and the number of days in coma, for patients with biopsy-proven Reye's syndrome. They suggested that the mitochondrial injury might have a disruptive effect upon hepatic monoamine oxidase, with a subsequent decrease in clearance of tyramine. We have reached a similar conclusion on the possible role of tyramine in the development of the encephalopathy, based on the finding that the hepatic monoamine oxidase activity was reduced about 60% in stage V Reye's syndrome patients.

Letters to the Editor

We read with great interest the letter by Mitchell and Arcinue concerning our finding of hypertyraminemia in Reye's syndrome patients. Since the acceptance of our manuscript we have confirmed this finding in an additional 16 patients with biopsy-proven Reye's syndrome. The discovery of depressed hepatic monoamine oxidase activity described by the authors confirms our preliminary findings in four patients whose hepatic monoamine oxidase levels were measured in our laboratories. We feel that the work of Mitchell and Arcinue supports our hypothesis that derangement in biogenic amine metabolism may contribute to the encephalopathy of Reye's syndrome.

Reye??s Syndrome: Preservation of Mitochondrial Enzymes in Brain and Muscle Compared with Liver

The activities of five mitochondrial enzymes tested in liver from patients with Reye's syndrome were measured. Citrate synthase, glutamic dehydrogenase, succinic dehydrogenase, pyruvate carboxylase, and pyruvate dehydrogenase were all outside of the range shown by control samples and well below them in activity. The activity of two extramitochondrial enzymes, glucose-6-phosphatase, which is a microsomal enzyme, and fructose-1,6-diphosphatase, which is a soluble enzyme, were in the normal range in samples from Reye's syndrome patients. In both muscle and brain the activities of the mitochondrial enzyme, citrate synthase, glutamic dehydrogenase, and succinic dehydrogenase were all within the control range. Pyruvate dehydrogenase was found to be normal in muscle from these patients.

392 THE EFFECT OF REYE'S SYNDROME SERUM ON MITOCHONDRIAL RESPIRATION IN VITRO

Recently we showed that serum from Reye's Syndrome (RS) patients has an effect on the respiratory function and morphology of isolated rat liver mitochondria (mito.) suggesting the existence of a pathogenic serum factor. We now report further investigation into the biochemical mechanism of action of the serum factor. In vitro respiratory rates of isolated rat liver mito. were assessed polarographically as described previously (Science 197:908, 1977). State 4 respiration was markedly increased in the presence of RS serum as compared to control serum. To distinguish among several mechanisms for the increase in state 4 respiration, inhibitors of specific mito. functions were tested as possible antagonists of the RS effect. The inhibitors used were: eligomycin, an inhibitor of mito. ATPase, ruthenium red, which blocks mito. Ca++ transport; and three site-specific e− transport chain inhibitors: rotenone (site I), antimycin A (site II), KCN (site III). In each case RS serum was added to the assay in the presence of inhibitor to see if the usual stimulation of respiratory rate could be blocked. Of the inhibitors thus tested only KCN abolished the effect of RS serum. We concluded that the putative serum factor stimulated respiration by directly or indirectly reducing components of the e− transport chain at a point beyond phosphorylation site II. (Sup. by Chas. H. Hood Foundation)