A n n a l s o p C l i n i c a l a n d L a b o r a t o r y S c i e n c e , Vol. 4 , No. 6Copyright © 1974, Institute for Clinical Science

Biochemical Abnormalities in Reye’s Syndrome

MARIANO M. ALVIRA, M.D.* AND DONALD T. FORMAN, Ph.D.

Department of Pathology and Laboratory Medicine,Evanston Hospital,

Evanston, IL 60201 and

Departments of Biochemistry and Pathology, Northwestern University Medical School,

Chicago, IL 60611

ABSTRACT

Reye’s syndrome has been characterized by cerebral edema without inflam­matory cell infiltration or demyelination, and fatty degeneration of the viscera, especially the liver.19 Serum from nine children (ages 7 months to 8 years) with Reye’s syndrome confirmed at autopsy were examined prior to death. From our chemical studies this entity is defined biochemically as a severe metabolic acidosis presenting with decreased glucose concentrations in blood and cerebrospinal fluid, high concentrations of serum uric acid, urea nitrogen, ammonia and associated with marked increase of aspartate and alanine amino­transferase, lactic dehydrogenase and creatine phosphokinase (C P K ).

Biochemically, these changes may be explained by a sudden insult to intra­cellular respiration causing an increased tissue catabolism resulting in an elevated endogenous nitrogen load. Elevations of CPK in all subjects studied implies a severe impairment of oxidative metabolism. Since CPK isoenzymes in two patients showed marked elevations in skeletal muscle CPK ( M M ) and minor increases in heart ( M B) CPK, it may be postulated that this is a result of the primary hypoxemia and hypocapnia which these children exhibit. Serum gamma-glutamyl transpeptidase activity and bilirubin concentrations in the same patients were not increased and it is possible that liver damage in Reye’s syndrome is not so important a factor as has been reported.22 A point under study at this time is the relationship of the increased CPK con­centration and the time of demise. Preliminary findings indicate a progressive rise in CPK suggests a grave prognosis.

In tro d u ctio n

D ata are presented on the clinical and pathological features of Reye’s syndrome as seen in a series of nine patients. Reye’s

* Present address: Department of Pathology, University of Cincinnati Medical Center, Cincin­nati, OH 45221.

syndrome has been characterized by cere­bral edema without inflammatory cell infil­tration or demyelination, and fatty degen­eration of the viscera, especially the liver.19

Included are additional biochemical data found in nine fatal cases of Reye’s syn­drome at Evanston Hospital between July 1966 and October 1973.

477

4 7 8 A L VIRA AND FO RM A N

A presumptive diagnosis of Reye’s syn­drome is made in a pediatric patient pre­senting with acute encephalopathy of no apparent cause. Certain clinical laboratory tests tend to support this diagnosis.9 The cerebrospinal fluid is free of inflammatory cells, and hypoglycemia are severe met­abolic acidosis with hyperkalemia are usu­ally present. Most striking are biochemical abnormalities suggestive of severe hepatic disease.5

M ateria l an d M ethods

Sera from nine children (ages 7 months to 8 years) with Reye’s syndrome con­firmed at autopsy were examined prior to death.

Serum glucose was measured by a mod­ification of the method of Hoffman7 and serum potassium values were determined by flame photometry.22 Serum osmolality was measured by a freezing-point depres­sion apparatus3 and bilirubin was deter­mined by a modification of the method of Jendrassik and Grof.11 Serum urea nitrogen and creatinine were determined by a ref­erence autoanalyzer* method.14’17 Uric acid was measured by the method of Liddle12 and plasma ammonia was deter­mined by an ion-exchange technic as de­scribed by Forman.4 Serum aminotrans­ferases were assayed according to Henry et al6 and lactic dehydrogenase following the method of Amador.1 Alkaline phos­phatase was determined by the optimized technique described by McComb and Bowers15 and creatine phosphokinase (C PK ) by the method of Rosalki.21 CPK isoenzymes were separated by agarose electrophoresis at pH 8.6 and their migra­tion patterns determined by fluorescence assay. Serum gamma-glutamyl transpepti­dase activity was determined according to Lum and Gambino.13

* Technicon Instruments Corp., Tarrytown, NY 10591.

Post-mortem examinations were per­formed in all nine cases. Post-mortem de­lay before fixation of tissues ranged from two hours to 18 hours. All tissues were fixed in 10 percent phosphate buffered formalin. In three more recent cases, tissue was fixed in 2 percent gluteraldehyde in sodium cacodylate buffer followed by post fixation in osmium tetroxide and 812 Epon embedding. The fixation time delays ranged two to four hours. Sections were studied with a Siemens Elmiskop 101 elec­tron microscope. Hematoxylin and eosin, PAS, PTAH, Sudan IV, silver carbonate and gold sublimate stains were used to study tissue sections by light microscopy.

R esu lts

In table I are summarized the biochem­ical data obtained from nine patients with autopsy confirmed Reye’s syndrome. Four out of eight patients presented with serum hypoglycemia on admission and their cer­ebrospinal fluid (C S F ) glucose was low­ered proportionally. Serum potassium was elevated in all these cases and mean values were also increased above normal. Serum urea nitrogen and creatinine analyses showed elevations in seven and five cases, respectively, and serum uric acid and plasma ammonia levels were elevated in five patients. Serum osmolality was mea­sured in five cases and found to be very elevated in four subjects. Arterial blood gas analyses are summarized in table II. All subjects demonstrated a negative base excess, and decreased pH was found in five cases. Forty percent of subjects studied presented with mild hypoxemia prior to oxygen treatment. In eight cases, hypo- capnia was the rule.

In table III are illustrated the serum enzyme changes in the last five subjects studied. All patients showed marked ele­vations in serum lactic dehydrogenase (L D H ), aspartate aminotransferase (A ST) and alanine aminotransferase (A L T ). CPK

B IO C H EM IC A L A B N O R M A LIT IES IN R E Y E S SYNDROM E 4 7 9

T A B L E IC l i n i c a l B i o c h e m i c a l D a t a o n N i n e P a t i e n t s w i t h R e y e ’s S y n d r o m e

ChemicalConstituent

5 Yr. W.M.

7 Yr. 5 Yr. W.F. W.F.

A g e , 7 Yr. W.F.

Race and 7 Mo. W.F.

Sex 8 Yr. W.M.

4 Yr. W.F.

8 Yr. W.M.

6 Yr. W.M.

Blood glucose* 20 74 98 52 146 35 62 114mg per 100 mlCSF glucose* - 52 55 24 96 20 56 82mg per 100 ml

Potassiumf 5.9 5.3 6.0 5.6 5.5 5.6 5.8 4.9 5.1mEq per liter (4.7) (4.9) (5.8) (5.1) (4.7) (4.6) (5.2) (4.7) (4.9)

BUN+ 98 291 24 73 97 96 27 48 68mg per 100 ml (48) (179) (19) (43) (49) (60) (22) (43) (54)

Creatininet mg per 100 m l

1.0 9.6(7.1)

3.8(2.4)

4.2(3.4)

— 1.5 2.5(2.3)

Bilirubin, totalt mg per 100 ml

0.14 _ — “ “ 1.2 2.0(1-3)

1.1 2.8(2.3)

Ammonia (plasma)t Ug per 100 ml

“ _ _ “ 215 148(98)

426(282)

623(532)

875(409)

Uric acidt - 32.4 17.1 >12 9.8 - - 15.6mg per 100 mlSerum osmolalityt mOsm per Kg

333 485(378)

300 363(341)

348(340)

*Before treatment with intravenous fluids. ( ) = Mean value during hospital course.

tHighest values during hospital course.

T A B L E II Arterial Acid-Base Values

5 Yr.Age

7 Y r . 5 Yr. 7 Yr., Race and Sex

7 Mo. 8 Yr. 4 Yr. 8 Yr. 6 Yr.Acid-Base Constituent W.M. W.F. W.F. W.F. W.F. W.M. W.F. W.M. W.M.

Arterial pH* 7.43 7.32 7.45 6.91 6.93 7.43 7.22 7.51 7.24Arterial P02 (mm Hg)t - - - 90 76 92 90 70Arterial PC02 (mm Hg)f 16 35 8 17 30 20 10 14 15Base Excess mmol per 1* -10 -5.8 -16 -18 -17 -11 -22 -9 -9

C-8.5) (-3.9) (-13.5) (-8.7) (-8.3) (-3.2) (-13.1) (-5.5) (-2.5)

*Lowest values including aggressive treatment with intravenous sodium bicarbonate.fwithout oxygen treatment. ( ) = Mean, value during hospital course.

T A B L E III Serum E nzyme Values

Age, Race and Sex7 Mo. 8 Yr. 4 Yr. 8 Yr. 6 Yr. (Normal

Serum Enzyme* W.F. W.F. W.F. W.M. W.M. Range)

LDH/ Lactic dehydrogenase (U per liter) 320 1640 2460 765 (30-115);.AST, Aspartate aminotransferase (U per liter) 139 268 1525 - 910 (5-28).ALT, Alanine aminotransferase (U per liter) 73 - 175 1605 885 (5-26)'ALP, Alkaline phosphatase (U per liter) - 243 463 359 234 (40-200)'CPK, Creatine phosphokinase (U per liter) - - 3600 554 620 (5-50)GGT, ^-Glutamyl transpeptidase (U per liter) “ 34 “ 8 (9-45)

♦Highest values during hospital course.

480 A L VIRA AND FO RM AN

was assayed in the last three subjects and was remarkably elevated. In four patients studied, the serum alkaline phosphatase (ALP) was only slightly elevated. Serum y-glutamyl transpeptidase (G G T) was within normal limits in the two cases stud­ied; in five cases examined, total bilirubin (table I) was within the normal range.

All the nine cases showed fatty degen­eration of liver cells and peculiar cerebral edema without inflammatory changes as described.19 Seven cases had varying de­grees of fatty degeneration of kidneys and heart. All the cases showed extensive acute neuronal necrosis and early astrocytosis of the central nervous system. The latter was best shown with silver carbonate and gold sublimate stains. Ultrastructural features in three cases showed severe mitochondrial round swelling of liver, kidney, heart and brain (figures 1 and 2). The matrix space was increased and coarsely granular matrix substance was present. The mitochondrial cristae were remarkably well preserved and the intracristae spaces were not ex­panded. Intramitochondrial dense bodies were always present. The outer mitochon­drial membrane was generally well pre­

served. Ameboid mitochondrial shapes were generally absent.18 Organelle changes were also present as shown in figure 2. Proliferation of ribosomes and smooth endoplasmic membranes were very prom­inent, particularly in the central nervous system.

D isc u ss io n

Reye et al19 described the syndrome of acute encephalopathy and fatty degenera­tion of the viscera in children. In addition to the clinical and pathological findings, elevations of serum aspartate aminotrans­ferase and alanine aminotransferase were reported in seven patients. Hypoglycemia, low glucose in cerebrospinal fluid and ele­vated serum urea nitrogen were also re­ported. Five years later, Huttenlocher et al8 reported plasma ammonia elevations in three children in whom blood ammonia levels were serially determined. Hutten­locher et al suggested that ammonia intox­ication might be a causative factor in the onset of the acute encephalopathy. Since these reports, further elucidation of Reye’s syndrome has been lacking.

F ig u r e 1. Representa­tive section from myo­cardium, left ventricle. Numerous swollen mito- chondriae disrupt myo­cardial cross striation. The Z bands are well preserved. Some mito­chondria contain elec­tron dense granules. (X 2,400)

BIO C H EM IC A L A B N O R M A LIT IES IN R EY E’S SYNDROME 481

F ig u r e 2. Portion of a granular cell of the cer­ebellum. Numerous ribo­somes bound to the outer nuclear membrane and to the endoplasmic retic­ulum. A swollen mito- chondrium contains elec­tron dense granules and fioccular matrical sub­stance. Islands of smooth endoplasmic reticulum ( arrows) are present in the cytoplasm. ( X 12,000)

From our own chemical studies in nine fatal cases of Reye’s syndrome, this entity can be defined biochemically as a severe metabolic acidosis presenting with de­creased glucose values in blood and cere­brospinal fluid, high levels of serum uric acid, urea nitrogen, ammonia and marked elevations of AST, ALT, LDH and CPK.

Biochemically, these changes may be ex­plained by a sudden insult to intracellular respiration causing an increased tissue ca­tabolism resulting in an elevated endoge­nous nitrogen load. Elevations of CPK in all the subjects studied implies a severe impairment of oxidative metabolism.2 In order to maintain an adequate amount of intracellular ATP for oxidative phosphory­lation, CPK is required to release energy from phosphocreatine. Tissues that are de­pendent on oxidative phosphorylation are brain, heart and liver. A severe insult to oxidative metabolism can result in elevated tissue catabolism and a concomitant rise in secondary nitrogen load. Since CPK iso­enzymes in two patients studied showed marked elevations in muscle CPK (M M ),20 and minor increases in heart (MB), it may be postulated that this is a result of the pri­

mary hypoxemia and hypocapnia which these children exhibited. It is also conceiv­able that the disproportionate rise in mus­cle CPK can be related to the seizure ac­tivity seen in most of these cases. Another possibility is that the bulk of the cathodic CPK isoenzyme (presumably MM) is not of muscle origin but is of mitochondrial origin (figure 3). Intramitochondrial cre-

F ig u r e 3. Photograph of fluorescent CPK iso­enzyme pattern on agarose gels (pH 8.6) of a mixture of pure MM enzyme from rabbit muscle (slides 1 and 3 ). CPK-3 (MM) seems to be the predominant band for patient with Reye’s syn­drome (slide 2 ). Note minor CPK-2 (MB) band in same patient.

482 A L V IR A AND FORM AN

F ig u r e 4. Representa­tive mitochondrium from the molecular layer of cerebellum. The matrix space ( m ) is expanded, and particulate matrical substance is clumped. Mitochondrial cristae (arrows) are intact. The outer membrane is well preserved. Cytoplasmic ribosomes are numerous. (X 20,000)

atine kinases of various organs show uni­formity and do not migrate on gel electro­phoresis as demonstrated by Jacobs et al.10 Reye’s syndrome has been recently classi­fied as a potentially reversible mitochon­drial injury.18 Three of our cases show extensive mitochondrial damage (figure4 ). The presence of severe mitochondrial injury supports a mitochondrial origin of creatine phosphokinase in these patients. Such a possibility is presently being inves­tigated. Elevations of serum AST and po­tassium were also observed in these pa­tients and may reflect this mitochondrial damage.

Serum GGT activity and bilirubin con­centration in the same patients were not increased and it is possible that liver dam­age in Reye’s syndrome is not as important a factor as has been reported.8’19 Evidence for increased brain tissue catabolism was demonstrated in three cases by electron microscopy. The severe brain tissue dam­age and the poor prognosis of the patients seem to be directly related.

A point under study at this time is the relationship of the CPK elevation in these children and the time of demise. Prelim­

inary findings indicate a progressive rise in CPK forecasts a grave prognosis.

In summary, nine fatal cases of children with Reye’s syndrome demonstrate a se­vere metabolic acidosis associated with in­creased nitrogen load and elevation of se­rum enzymes. Studies of CPK isoenzymes suggest that mitochondrial damage in skel­etal muscle and heart may be a major complication of this syndrome.

R eferen ces1. A m a d o r , E., D o r f m a n , L. E., a n d W a c k e r ,

W. E. C.: Serum lactic dehydrogenase activ­ity: A n analytical assessment of current assays. Clin. Chem. 9:391-399, 1963.

2. E p p e n b e r g e r , H. M., D a w s o n , D . M., a n d K a p l a n , N. O.: The comparative enzymology of creatine kinases. Isolation and properties. J. Biol. Chem. 242:204-209, 1967.

3. F o r m a n , D. T. a n d C h a n g u s , G. C .: A n a u to m a te d o sm o m e te r . C lin . C h e m . 14:38-46,1968.

4. F o r m a n , D. T.: Rapid determination of plasma ammonia by an ion-exchange technic. Clin. Chem. 10:497-508, 1964.

5. G l a s g o w , A. M., C o t t o n , R. D ., a n d D h i e n - s i r i , K.: I. Blood ammonia and consideration of the non-histologic diagnosis. Amer. J. Dis. Child. 124:827-833, 1972.

6. H e n r y , R. J., C h ia m o r i , N., G o l u b , O. J., a n d B e r k m a n , S.: Revised spectrophotometric methods for the determination of glutamic-

BIO C H EM IC A L A B N O R M A LIT IES IN REY e ’s SYNDROM E 4 8 3

oxaloacetic transaminase, glutamic-pyruvic transaminase, and lactic dehydrogenase. Amer. J. Clin. Pathol. 34:381-388, 1960.

7. Hoffman , W. S.: A rapid photoelectric method for the determination of glucose in blood and urine. J. Biol. Chem. 120:51-55, 1937.

8. Huttenlocher, P. R., Schwartz, A. D., and Klatskin , G.: Reye’s syndrome: ammonia in­toxication as a possible factor in the enceph­alopathy. Pediatrics 43:443-454, 1969.

9 . H u t t e n l o c h e r , P. R.: Reye’s syndrome: Re­lation of outcome to therapy, j . Pediat. 80: 8 4 5 - 8 5 0 , 1 9 7 2 .

10. J acobs, H., Heldt, H. W., and Klingenberg, M.: High activity of creatine kinase in mito­chondria from muscle and brain and evidence for a separate mitochondrial isoenzyme of creatine kinase. Biochem. Biophys. Res. Com- mun. ¿6:516-521, 1964.

11. J endrassik, L . and Grof, P.: Vereinfachte Photometrische Methoden zur Bestimmung des Blutbilirubins. Biochem. Ztschr. 297:81- 89, 1938.

12. L iddle, L ., Seegm iller , J. E., and L aster, L .: The enzymatic spectrophotometric method for determination of uric acid. J. Lab. Clin. Med. 54:903-913, 1959.

13. L um , G. and Gambino, S. R.: Serum gamma- glutamyl transpeptidase activity as an indi­cator of disease of liver, pancreas, or bone. Clin. Chem. 18:358-362, 1972.

14. Marsh, W. H., F ingerhut, B., and K irsch, E.: Determination of urea nitrogen with the diacetyl method and an automatic dialyzing

apparatus. Amer. J. Clin. Pathol. 28:681-688, 1957.

15. McComb, R. B. and B owers, G. N .: Study of optimum buffer conditions for measuring alkaline phosphatase activity in human serum. Clin. Chem. ¿8:97-104, 1972.

16. Olson, L. C., Bourgeois, C. H., J r ., Cotton, R. B., Harikul, S., Grossman, R. A., and Smith , T. J . : Encephalopathy and fatty de­generation of the viscera in Northeastern Thailand. Clinical syndrome and epidemiol­ogy. Pediatrics 47:707-716, 1971.

17. Owen, J. A., Iggo, B., Scandrett, F. J., and Stewart, C. P.: The determination of cre­atinine in plasma and serum, and in urine; a critical examination. Biochem. J. 58:426-437, 1954.

18. Partin, J. C., Schubert, W. K., and Partin, J. S.: Mitochondrial ultrastructure in Reye’s syndrome (Encephalopathy and fatty degen­eration of the viscera). New Eng. J . Med. 285:1339-1344, 1971.

19. Reye, R. D. K., Morgan, G., and B aral, J.: Encephalopathy and fatty degeneration of the viscera: a disease entity in childhood. Lancet 2:749-752, 1963.

20. Roe, C.: Duke University Medical Center, Personal Communication.

21. Rosalki, S. B.: An improved procedure for serum creatine phosphokinase determination. J. Lab. Clin. Med. 69:696-702, 1967.

22. T ietz, N. W.: Electrolytes.. Fundamentals of Clinical Chemistry, Tietz, N. W., ed. W. B. Saunders and Co., Philadelphia, pp. 613-621,1970.

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