003 1-3998/85/19 12-1346$02.00/0 PEDIATRIC RESEARCH Vol. 19, No. 12, 1985 Copyright 0 1985 International Pediatric Research Foundation, Inc. Printed in U.S.A.

Investigation of an Animal Model of a Reye-Like Syndrome Caused by Margosa Oil

DAVEN SINNIAH, PHILIP H. SCHWARTZ, ROBERT A. MITCHELL, AND EDGARDO L. ARCINUE Division of Pediatric Intensive Care, Department of , Children's Hospital of Los Angeles, Los Angeles, California [P.H.S., E.L.A.]; Department of Pediatrics, University of Malaya, Kuala Lumpur, Malaysia [D.S.]; Department of Neuroscience, Brain Research Institute, University of California, Los Angeles, California [P.H.S.], and Department of Biochemistry, Wayne State University, Detroit, Michigan [R.A.M.]

ABSTRACX. Following reports of a Reye-like syndrome AlaAT, alanine aminotransferase (E.C. 2.6.1.2) in children resulting from Margosa oil (MO) ingestion, we LADNADH, lipoamide dehydrogenase, nicotinamide ad- administered MO to laboratory rats in an attempt to enine dinucleotide, reduced form (NADH) (E.C. 1.6.4.3., produce an animal model of Reye's syndrome. Male rats NADH diaphorase, NADH tetrazolium reductase) were injected intraperitoneally with either MO or corn BHT, butylated hydroxytoluene oil and observed for clinical signs of a toxic response. After PAS, periodic acid-Schiff 15 h the animals were administered a second dose and the H+E, hematoxylin and eosin MO-treated animals developed florid neurological symp- FFA, free fatty acid toms. The animals were then sacrificed and blood samples were analyzed for glucose, ammonia, aspartate aminotrans- ferase, and alanine aminotransferase. Sections of , kidney, and brain were examined by light microscopy after Sudan black B, hematoxylin and eosin, and periodic acid- RS was first described in 1963 as a childhood disease charac- Schiff staining. Liver was additionally examined by elec- terized by encephlopathy with fatty degeneration of the viscera tron microscopy. Liver samples were analyzed for hepatic (1). Since that time epidemiological studies have revealed that enzyme levels and brain samples were analyzed for water RS is one of this country's top 10 killers of children (2, 3). The content. There were greatly increased levels of ammonia, design of specific therapies and effective preventive measures for aspartate aminotransferase, and alanine aminotransferase RS have been severely hampered by the lack of knowledge and decreased glucose levels in the blood of MO-treated concerning the etiology of the disease and the lack of a suitable animals. Light microscopy of MO-treated revealed animal model for the disease (4). Numerous investigators have fatty infiltration, granularity of the cytoplasm with normal attempted to induce the illness in laboratory animals (5). nuclei, and glycogen depletion; electron microscopy re- Sinniah and Baskaran (6, 7) have reported a syndrome similar vealed mitochondrial pathology in the livers of MO-treated to RS occumng in children after ingestion of MO-an extract animals. There were no significant morphological changes of the seeds of the Neem tree, Azadirachta iizdica A. Juss. They in brain or kidney specimens although the kidneys did described the syndrome as one of ", drowsiness, meta- show some fatty infiltration. Hepatic mitochondrial enzyme bolic acidosis, polymorphonuclear leukocytosis, and encephalo- levels were unchanged and there was no increase in brain pathy" in 13 children who had ingested MO. The mortality rate water content in the MO-treated animals. Thus, many of was greater than 80% and occurred after the children had the abnormalities seen in Reye's syndrome were seen in lapsed into and developed severe . this model; however, there were no hepatic enzyme changes demonstrated pronounced fatty infiltration of the liver. Post- despite altered mitochondrial morphology and no evidence mortem studies in an infant who died of MO poisoning revealed of cerebral edema despite a florid . None- histopathological and electron microscopic features resembling theless, this model may have important implications for those found in RS. The clinical symptomatology and results of the understanding of the pathogenetic mechanisms of this biochemical and pathological studies were consistent with those Reye-like syndrome and, perhaps, Reye's syndrome. (Pe- of a RLS. diatr Res 19: 1346-1355,1985) The present study was performed to ascertain whether or not some of the cardinal manifestations of RS could be elicited in Abbreviations the laboratory rat by the administration of MO. Clinical, patho- logical, and biochemical measures were made. RS, Reye's syndrome RLS, Reye-like syndrome MO, Margosa oil MATERIALS AND METHODS CO, corn oil Fourteen male Sprague-Dawley rats (250 g, Simonsen Labs, AspAT, aspartate aminotransferase (E.C. 2.6.1.1) Gilroy, CA) were separated into two treatment groups of seven Received December 24, 1984: accepted ~uly26, 1985. animals each. The animals were acclimated to a 12-h lightldark Correspondence and reprint requests may be addressed to Philip H. Schwartz, cycle in individual cages and were allowed food and water ad ICU Administration, Children's Hospital of Los Angeles, 4650 Sunset Boulevard, libitum. The control group received an intraperitoneal injection Los Angeles, CA 90027. of 5 ml.kg-' corn oil (CO, Mazola Oil, Best Foods, Englewood This work was supported by the Division of Pediatric Intensive Care, Department of Pediatrics, Children's Hospital of Los Angeles. This work was presented in part Cliffs, NJ) at 0 and 15 h, the test group received a before the Society for Pediatric Research, Washington, D.C., May 5, 1983. dose of MO (obtained in India by DS) at the same time intervals. 1346 REYE-LIKE SYNDROME CAUSED BY MARGOSA OIL

This dosage regimen was chosen to mimic, as close as possible, RESULTS the human situation (i.e. the approximate dose and time se- quence reported by parents of children who had developed the Of the behaved throughout Reye-like syndrome after ingestion of MO) (Sinniah D, personal the experiment. The MO-treated animals, however, exhibited The animals were observed for clinical signs of tach~~neaand lethargy within 2 h after the first dose of MO. toxicity and were sacrificed at 18-20 h by cervical dislocation. Just Prior to the second dose, these animals were lethargic and Blood was collected by cardiac puncture, placed in heparinized h~~emeactiveto a sudden noise. Shortly after the second dose centrifuge tubes cooled to 5° C, and centrifuged at 1~~~ for the animals exhibited extreme prostration and hyporeactivity/ min in a refrigerated centrifuge. ~li~~~~~of plasma were coma and five of the seven animals had just before removed and analyzed for ammonia, glucose, AspA~,and sacrifice. Preliminary studies had shown that seizures foreboded AlaA~by standard clinical laboratoIy techniques. (samples of death in all cases examined and that the intensity of the clinical blood had been removed by the tail vein prior to the beginning symptomotology was dose of the experiment to provide initial blood chemistry data.) R~- The results of the laboratory analysis of initial and final are expressed as IU .dl-' for ASPAT and AlaAT, mg. dl-' ASPAT, AlaAT, glucose, and anmonia levels are shown in Table for glucose, and pg NH3 N. dl-' for ammonia. 1. It can be seen that AspAT and AlaAT levels were significantly samplesof brain, kidney, and liver were removed and speci- increased in the MO-treated animals. AspAT levels were raised mens were placed in buffered formalin (10%) for histopatho~og- 6- to 13-fold (versus final CO values or initial MO values, ical studies. Additional samples of liver were placed in 0.c.~.respectively) while AlaAT levels were raised 1 I-fold. Blood glu- ~~~~~~~d (~~b~~k products, ill^, and frozen for cose showed a significant decrease in the MO-treated animals, histochemical and histopatho~ogica~studies or in cold isotonic to 55% of control levels. Blood mmonia 2% glutaraldehyde in phosphate buffer, p~ 7.4, for electron levels showed no significant increase in the MO-treated animals microscopic evaluation. Liver samples also were frozen at -70" at the end of the experiment as compared to the beginning (due c for enzyme analysis. Brain samples were frozen for later to high variance), but the levels were elevated 3.8-fold compared analysis of water content. hi^ was done by weighing of a 400- to the CO-treated group. Elimination of an outlying data point 500 mg specimen of forebrain, drying it at 10q c for 48 h, and (Q test) for final MO values yielded statistical significance when reweighing it. Brain water content is expressed as the ratio of the compared to initial values. The CO-treated group demonstrated dry to the wet weight. a significant decrease in blood ammonia at the end of the ~h~ formalin samples were prepared by conven- experiment compared to initial values, probably a reflection 01 tional histologic procedures. sections were stained with sudan the higher ammonia concentrations known to occur in venous black B, H+E, or PAS stains, and were mounted for examination blood draining skeletal muscle (i.e. tail vein) versus arterial blood under the light microscope. (i.e. cardiac puncture). -,-he samples in 0.c.~.were cut into 6-pm thick sections on a Sudan black B staining of unfixed frozen and formalin fixed cryostat and either stained with Sudan black B and mounted or liver, kidney, and brain of the MO-treated animals revealed microvesicular fat droplets in the liver and kidney, but not the incubated for LADNADH. The histochemical procedure jin- valved incubating the tissue slices for 30 min at 370 c in a 0. brain (representative sections from unfixed frozen liver are shown M ~ri~buffer solution at p~ 7.4 containing 4 mgml-~N~DH in Fig. 1). The effects seen in the liver were much more dramatic and mg.ml-~nitro blue tetrazolium. After the reaction had than those seen in the kidney. There was no significant reduction taken place the slices were rinsed in distilled water and then of LADNADH staining in the MO-treated livers, but several mounted in jelly for light microscopic examination. developed a coarse, larger grain Pattern than normal (data not The specimens fixed for electron microscopic examination shown). H+E staining revealed a granularity of the cytoplasm were postfixed in osmium tetroxide and worked up by standard and normal nuclear staining of livers of MO-treated animals histological techniques. They were stained with uranyl acetate 'Ompared to but no changes were seen in kidney Or lead citrate and viewed and photographed using a siemens brain (representative sections from liver are shown in Fig. 2). 1 A electron microscope. Additionally, no areas of necrosis or were seen Assays of hepatic enzyme activities were performed according and mitotic figures were rarely seen. PAS staining revealed a to our previously published (8, 9). Briefly, small speci- Of glycogen in the livers of MO treated animals but no mens of liver tissue (20 mg wet weight) were homogenized in cold 0.1 M potassium phosphate buffer, pH 7.4. The homogenate was centrifuged for I h at 100,000 X g and the supernatant Table 1. Glucose, ammonia, ASPAT and AldTlevels in fraction was used for assays of lactate dehydrogenase (E.C. plasma of CO- and MO-treatedrats (means * SEMStudent's 1.1.1.27), glutamate dehydrogenase (EC 1.4.1.3), catalase (EC t test) 1.1 1.1.16), and glucose-6-phosphate dehydrogenase (EC Parameter Group (n = 7) Initial Final 1.1.1.49). The pellet was used for measuring monoamine oxidase (EC 1.4.3.4) activity. Up to 12 liver specimens were worked up CO 136k3 140+3 in a batch procedure and all assays of a particular enzyme were (mg'dl-') MO 128+ 3 73 a 7*3t completed within 2 h to minimize differences due to handling. Ammonia CO 294 k 59 189 a 144 All measurements were made at 25" C and the results expressed (NH3N.dl-') MO 367k89 716~167p11 as IU .mg of soluble or pellet protein-', depending on the location CO 105 k 10 217 + 46 of the activity in the fractionated homogenate. Catalase activity (lu.d'-') MO 104 a I1 1359 + 3751.** was expressed as the first order rate constant for the disappear- *IaAT CO 48 c 3 48 + 3 ance of hydrogen peroxide, divided by the amount of protein (lu'dl-l) MO 48 + 3 538 zk 180**.tt present (i.e. as min-' .mg-I). * p < 0.00 1 vs CO final value. Student's t test was used to test for differences between the tp< 0.01 vs MO initial value. mean values of the treated and control groups. In situations tp< 0.01 vs CO initial value. where the two populations had unequal variances (p < 0.05 as $p< 0.02 vs CO final value. judged by the F test of sample variances), a separate rather than 11 NS, p < 0.01 vs MO initial value after elimination of outlying data pooled variance estimate was used for the t test. All analyses point by the Q test. were performed on an IBM 3078 computer using the SAS np<0.01 vsCO final value. statistical package, release 82.4 (University of Southern Califor- **p < 0.05 vs MO initial value. nia). ttp< 0.05 vs CO final value. 1348 SINNIAH ET AL.

Fig! 1. Sudan black B stained sections of liver tissue from a CO- (A)and a MO- (B)treated animal. The magnification is 600. Note the sparsity of black stained particles in A and the abundance of black stained fat droplets throughout the section in B. changes were seen in kidney or brain (representative sections drawn: 1) the CO-treated animals appear to have normal liver from liver are shown in Fig. 3). ultrastructure and, 2) the MO-treated animals appear to have After electron-microscopic examination of the livers of both abnormal liver ultrastructure. The latter is exemplified by the CO and MO treated animals the following conclusions were following: a) greatly increased lipid as small bodies, b) dilatation REYE-LIKE SYNDROME CAUSED BY MARGOSA OIL 1349

Fig. 2. H+E stained section of liver tissue from a CO- (A)and a MO- (B) treated animal. The magnification is 600. Note the uniformly stained cells and central and normal appearing nuclei in A. Note also the nonuniformity of staining throughout the section in B, the obvious "ground-glass" appearance of the cells in the lower right portion of the section, and the normal appearing nuclei in that same area. 1350 SINNIAH ET AL.

Fig. 3. Periodic acid-Schiff stained section of liver tissue from a CO- (A)and a MO- (B) treated animal. The magnification is 600. Note the uniformly dense staining of glycogen throughout the section in A and the almost complete absence of stainable glycogen throughout the section in B. REYE-LIKE SYNDROME CAUSED BY MARGOSA OIL 1351

Fig. 4. Low power electron micrograph of a hepatocyte from a CO- (A)and a MO- (B)treated animal. The magnification is 5700 and the bar represents 3 pm. Seen in A are abundant rough endoplasmic reticulum (white arrow), electron-dense mitochondria (M)with dense bodies, the centrally located nucleus (N), and abundant glycogen granules (small black spots). Note in B the centrally located nucleus (N), enlarged and electron-lucent mitochondria (black arrow) without dense bodies, the absence of rough endoplasmic reticulum, abundant lipid inclusions (L), and the absence of glycogen granules. 1352 SINNIAH ET AL. and hyperplasia of smooth endoplasmic reticulum, c) greatly degeneration of cytoplasmic integrity. With the degeneration decreased amounts of rough endoplasmic reticulum, d) greatly there is no obvious increase in lysosomes. The changes were not decreased glycogen content, e) mitochondria1 degeneration with entirely uniform throughout the liver with some regions appear- swelling and loss of matrix, cristae, and dense bodies, f) normal ing normal. However, these areas did not appear to be segregated appearing and central nuclei, and g) granularity of cytoplasm/ to any particular anatomical substructure of the liver. Repre-

Fig. 5. High power electron micrograph of a hepatocyte from a CO- (A)and a MO- (B) treated animal. The magnification is 45,000 and the bar represents 0.2 gm. Seen in A are normal mitochondria (M) with dense bodies (D) and cristae (C), rough endoplasmic reticulum (black arrow), and glycogen granules (G). Note in B the lipid inclusions (L), enlarged mitochondria (M) without dense bodies and few cristae, little glycogen, and swollen smooth endoplasmic reticulum (S). REYE-LIKE SYNDROME CZAUSED BY MARGOSA OIL 1353 Table 2. Hepatic enzyme activities in CO- and MO-treated hallmark. Fatty liver, however, is a nonspecific lesion and may animals (means + SEM) occur in response to a number of unrelated toxins including Treatment group ethanol, ethionine, galactosamine, and carbon tetrachloride (21). Thus, 4-pentenoic acid administration (22), octanoic acid infu- Enzyme CO (n = 6) MO (n = 5) sion (23), encephalomyocarditis virus infection with BHT ad- Monoamine oxidase 201+10 222C24 ministration (24), type A virus infection with pesticide (lo5 IU . mg protein-') camer administration (25), aflatoxin B1 exposure (26), influenza Glutamate dehydrogenase 81 +.2 73 + 4 B infection (27), influenza B infection with acetaminophen ad- ( 1O3 IU .mg protein-') ministration (28), and and treatment Catalase 13.2 C 0.7 10.4 + 0.4* with (29) and without (30) influenza infection have been pro- (min-' . mg protein-') posed as animal models of RS. While each of these models has Glucose-6-phosphate dehydrogenase 12 1 k 8 137 k 6 heuristic value, none is entirely satisfactory as an animal model of RS for a number of different reasons. ( 1O4 IU .mg -. protein-') Lactate dehydrogenase 1.95 + 0.07 1.96 + 0.08 In trying to develop an animal model of a human disease as (IU. mg motein-') complex as RS it is essential that as many representative param- eters as possible be measured (3 1). The criteria for the diagnosis * p < 0.02 compared to corn oil treated animals (Student's t test). of a RLS in animals presumably are identical to those required for RS in children and nearly all of the models suffer either some major dissimilarity to RS or some major parameter was not Table 3. Brain water content in CO- and MO-treated animals studied. For example, the 4-pentenoic acid model, in experiments (means + SEM) from the same laboratory, does not demonstrate sufficiently large Treatment Dry-to-wet wt ratio increases in serum transaminases nor is liver ultrastructure sig- nificantly altered (22). The encephalomyocarditis virus and BHT CO (n = 6) 0.2088 0.0002 + model does not demonstrate mitochondria1 lesions similar to MO (n = 6) 0.2 104 C 0.0005 those seen in RS (24) and the octanoic acid model of RS fails to demonstrate changes in a number of parameters of interest in RS (23). The influenza B model, while appearing to be the most sentative electron micrographs of both treatment groups are relevant to the actual syndrome, also does not demonstrate shown in Figures 4 and 5. Both low (5,700X) and high (45,000x) mitochondrial pathology (27). Two of the reported animal level magnifications are shown. models of RS, the aflatoxin B1 (26) and the influenza type A Analysis of hepatic enzyme activities in the MO- and CO- virus infection with insecticide carrier (25), measured only a treated animals indicated that with the exception of catalase, no single parameter: urea cycle enzymes and lethality, respectively. enzyme differed significantly from control levels (Table 2). The None of the models has demonstrated the combination of mi- MO-treated animals showed a small (2 1%) but significant (p < tochondrial biochemical and morphological abnormalities seen 0.02) decrease in catalase. in RS. As this is the pathognomonic feature of RS its absence is Measurements of brain water content by the dehydration an important failing in all of the above models. method revealed no differences between the CO- and MO-treated Investigations of a number of putative animal models of RS animals with both sets of animals having ratios of dry to wet have revealed that viral infection modifies the effects of the weights of approximately 0.210 for specimens taken from the chemical agents used. For instance, Hug et al. (24) showed that forebrain (Table 3). This was repeated in small (65 g) animals while BHT alone or virus alone decreased carbamoyl phosphate and similar results were observed except that the ratios were synthetase activity in the liver, both together decreased the slightly lower (data not shown). activity but not in an additive manner. Conversely, virus alone had no effect on arginase activity while BHT increased it and DISCUSSION the combination of the two decreased the activity. Deshmukh et al. (29) demonstrated synergistic actions of viral infection, hy- RS is characterized by the following clinical, pathological, and perammonemia, and aspirin administration. Thus, it can be seen biochemical criteria: 1) clinical features of vomiting, lethargy, that the interaction of virus and chemical agents is complex and , coma, and seizures (in children less than 1 yr not easily predictable or interpretable. of age) during the recovery phase of a viral illness (1, lo), 2) These preliminary studies in our laboratory of the effects of elevated serum transaminases (at least three times the upper limit MO have demonstrated some striking similarities to RS. The of normal) and ammonia, a characteristic hyperaminoacidemia clinical symptomotology including tachypnea, lethargy, and (1 I), elevated long-chain, polyunsaturated, free fatty acids (12), coma resembles that seen in RS and its development is dose and a decreased prothrombin time with normal bilirubin levels related (pilot studies). Hyperammonemia, elevated aminotrans- and a normal examination (13), 3) light ferases, and light and electron microscopic evidence of fatty microscopic and ultrastructural analysis of the liver demonstrat- infiltration of the liver and mitochondria1 lesions, respectively, ing microvesicular fatty accumulation, glycogen depletion, and including swelling, rarefication, and loss of dense bodies were typical swollen and pleiomorphic mitochondria (14), the latter seen. Additionally, the livers demonstrated no necrosis, glycogen consisting of loss of dense granules, rarefaction, and dissolution depletion was very apparent with both PAS staining and electron of matrix, 4) acute cerebral edema characterized by swollen microscopy, and rough endoplasmic reticulum had virtually astrocytes and normal appearing neurons under the light micro- disappeared while there appeared a relative proliferation of scope (15), and, 5) reduction in the activity of all hepatic mito- smooth ER. The nuclei of the hepatocytes were central and chondrial enzymes measured (8, 9, 16-20) with little or no appeared normal under both the light and electron microscope. change in the activity of most cytosolic and microsomal enzymes These clinical, laboratory, and morphological findings are very (9, 19). similar to those seen in RS and demonstrate that MO can, indeed, Since the etiology of RS is thought to be multifactorial and to give rise to many of the features of RS in the laboratory rat in a include a viral component, individual susceptibility, and expo- similar fashion to its effects on children. sure to environmental toxins, attempts to link RS with a variety We were not able to demonstrate cerebral edema in these of chemical and viral agents have formed the basis for a number encephalopathic animals by determining dry-to-wet weight brain of different proposed animal models of RS (5). Each proposed ratios, however, cerebral edema has been convincingly demon- model has hinged on the development of a fatty liver as its strated in humans who consumed the toxic oil (6, 7). This was 1354 SINNIAH ET AL. obvious at the gross as well as microscopic levels. The method undertaken thus far does not conclusively prove this supposition. of measuring dry-to-wet weight brain ratios has been used pre- While longer chain saturated FFAs such as palmitic or myristic viously to demonstrate cerebral edema in animal models of would distill in the appropriate range, they would not co-chro- cerebral edema of the cytotoxic type such as water intoxication matograph with the toxin under conditions designed to separate (32) with changes in water content ranging from 1-4%. There- FFAs according to their degree of unsaturation. Alternatively, fore, in light of the fact that no evidence of cerebral edema was while longer chain unsaturated FFAs, may distill in the proper demonstrated by light microscopy (H+E), we are confident that range they do not tend to be very stable at the temperatures or none, in fact, was present. The reason for this remains unclear oxidizing (i.e. hydrogen peroxide) conditions to which they were but may be attributable to the fact that the animals may expire subjected. We are currently pursuing the identity of the toxin in before they have a chance to develop measurable cerebral edema. an effort to clarify this. Possibly if they were supported by a respirator they may survive In conclusion, the acute (less than 24 h) exposure of the long enough for edema to form. This would be analogous to the laboratory rat to MO gives rise to a RLS which has many of the situation seen in children who have consumed MO and, possibly, clinical, pathological, and biochemical features of RS including to children with RS (33). encephalopathy and a fatty hepatopathy. However, the enceph- The characteristic decreases in hepatic mitochondrial enzyme alopathy appears to have no relation to the development of activities seen in RS also were not seen in this model. Further cerebral edema and neither the encephalopathy nor the hepato- evidence for both the mitochondrial enlargement and the lack pathy seems to have a relation to mitochondrial enzymatic of decrease of mitochondrial enzyme activity comes from the activity. These findings may have important implications for the LADNADH histochemical staining which demonstrated coarser understanding of the pathogenetic mechanisms involved in RS but not decreased staining. Since there was no necrosis or inflam- insofar as this dissociation has been demonstrated in the labo- mation of the liver and mitotic figures were rarely seen, it is ratory rat but has not been heretofore documented in the case of possible that not enough time had elapsed for decreased mito- human MO poisoning. What has been demonstrated in human chondrial enzyme activity to be seen. In light of the fact that the RS, however, is that there appears to be no relationship between half-lives of mitochondrial enzymes is on the order of days (34), either the hepatic mitochondrial enzyme activities and degree of a prolonged exposure to MO may be necessary to produce encephalopathy or outcome (8) or between the degree of intra- measurable effects. This would also be analogous to the situation cranial hypertension and encephalopathy (33). The identity of seen in RS where there is a prodromal illness and a period of the toxic component of MO is unknown at this time but its several days may elapse before the syndrome proper manifests isolation and purification may yield an important research tool itself. While this speculation assumes some constancy in the rates with which to investigate the pathogenetic mechanisms of this of protein synthesis in the light of an accelerated protein degra- novel animal model of a RLS and RS itself. dation or a decreased synthesis in the presence of normal rates of degradation, pilot studies in our laboratory wherein we ad- Acknowledgment. The authors are deeply indebted to the late ministered a sublethal dose of MO to laboratory rats daily for 2 Harry B. Neustein, M.D. for the electron microscopy. wk have shown that this, in fact, may be the case. In these studies monoamine oxidase levels were decreased 80% and glutamate dehydrogenase levels were decreased 50% (35). 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N Engl J Med 309:133-139 4. Arias IM 1980 Needs for animal models of human diseases of the gastrointes- preclude normal neuronal function and may thus give rise to tinal system. Am J Pathol 101:S56-S57 encephalopathy, the function of neuronal mitochondria in these 5. Pollack JD 1979 Models of chemical and virus interaction and their relation acute animals may not be so severely compromised as to lead to to a multiple etiology of Reye's syndrome. In: Crocker JFS (ed) Reye's a collapse of neuronal ionic gradients. Syndrome 11. Grune & Stratton, Inc., New York, pp 341-360 6. Sinniah D, Baskaran G 1981 Margosa Oil poisoning as a cause of Reye's While MO has been used for centuries as a panacea in the syndrome. Lancet 1:487-489 Indo-Malaysian regions of the world (37), its potential toxicity 7. Sinniah D, Baskaran G, Looi LM, Leong KL 1982 Reye-like syndrome due to has only recently come to the attention of the medical commu- Margosa oil poisoning. Report of a case with postmortem findings. Am J nity (a situation bearing certain similarities to the discovery of Gastroenterol 77: 158- 16 1 8. Mitchell RA, Arcinue EL. Partin JC, Partin JS, Ram ML, Chang CH, Smialek unripe Ackee fruit as the cause of Jamaican vomiting sickness) J, Samiak A 1985 Quantitative evaluation of the extent of hepatic enzyme (38). It is the crude oil that tends to have toxic properties and changes in Reye syndrome compared with normal liver or with non-Reye further purification such as by alkali treatment and treatment liver disorders: objective criteria for animal models. Pediatr Res 19:19-22 with activated charcoal and diatomaceous earth render the oil 9. Mitchell RA, Ram ML. Arcinue EL, Chang CH 1980 Comparison of cytosolic and mitochondrial hepatic enzyme alteration in Reye Syndrome. Pediatr nontoxic. The composition of the oil has been studied by a Res 14:1216-1221 number of investigators and is not unlike that of many other 10. Loveioy FH, Smith AL, Bresnan MJ. Wood JN, Victor Dl, Adams PC 1974 commonly used seed oils (39). Its major components are triglyc- ~lhicalstaging in Reye Syndrome. Am J Dis Child 128:36-41 erides composed of long-chain saturated fatty acids and some I 1. Romshe CA. Hiltv MD. McClunr HJ. Kerzner B. Reiner CB 198 1 Amino acid unsaturated fatty acids. Our preliminary studies of the oil (un- pattern in eye's syndrome. comparison with clinically similar entities. J Pediatr 98:788-790 published data) have shown that the toxin is hydrogen peroxide 12. Ogburn PL, Sharp H, Lloyd-Still JD, Johnson SB, Holman RT 1982 Abnormal and heat stable, does not steam distill, co-chromatographs with polyunsaturated fatty acid patterns of serum lipids in Reye's syndrome. Proc monounsaturated FFAs under two different thin-layer chroma- Natl Acad Sci USA 79:908-9 11 13. 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