Creatine kinase brain isoenzyme in infantile osteopetrosis

Creadne nzyme in Irffandle Osteopetrosis Yoshifumi Himyama, MD, Teruhisa Miike, MD, Shigeto Sugino, MD, and Keiichi Taku, MD

A 14-month-old girl with infantile osteogmm~is had hematologic and neumlogic complicadous with severe brain atrophy. Although serum conmiaed high creatine kinase brain isoenzyme activity (CK.BB), CK-BB activity was not detected on repeated ccrebmspinal fluid examinations. After frequent blood tranffusions and steroid therapy, hematologic involvement improved gradually and disappeared finally at age I1 months; serum CK-BB tended to show a concomitant proportional increase in activity. A t'lIndium chloride scan was performed at age 4 weeks when the patient had relatively low serum CK-BB activity. It indicated acdve extramedullary hematopoiesis in the liver and spleen. The second scan was performed at age 12 months when she had high serum CK-BB aodvity and indicated active medullary hematopoiesis in the cranium. The tests disclosed that the elevated serum CK-BB activity was the result of bone marrow serum leakage, and not leakage from brain tissue. This fmding may be a good marker of medullary hematopoietic activity in patients with osteopetrosis. Meanwhile, biopsied sural nerve revealed storage of cellular debris, including myelin figures in the Schwann cells, which suggested i n ~ degradation process in the cells or lysosomal enzyme defidency.

Introduction Infantile osteopetrosis is a rare, recessively inherited bone disorder resulting from defective osteoclastic function and is often associated with hematologic and neurologic involvement. The principle features are osteosclerosis, hepatosplenomegaly, and anemia; however, early symptoms are often neurologic. Although cranial nerve disorders are attributed to peripheral entrapment, mental retardation and motor disturbances cannot be readily explained by either mechanical factors or anemia, Recent studies have demonstrated that the concentrations of crearine kinase brain isoenzyme (CK-BB) [1] and neuron-specific ,enolase (NSE) [2] are reliable indicators of brain damage. This report documents the presence of CK-BB and NSE in the serum and cerebrospinal fluid (CSF) of a patient with infantile osteopetrosis who had severe mental retardation and motor dysfunction with severe brain atrophy.

Case Report A 14-month-old female was horn to a 35-year-old female after an uneventful pregnancy, labor, and delivery; the parents are first cousins. The patient's sister suffered from osteopetrosis and died of pneumonia at age 8 months. At 7 days of age, a diagnosis of osteopetrosis was made on the basis of radiographs and family history. She was well until age 26 days, when she developed nasal bleeding and was admitted to our hospital for further treatment. Her subsequent course is depicted in Figure 1. On examination she was pale and apathetic, but hypersensitive to sound. She had multiple joint ankylosis, bilateral ankle clonus with exaggerated tendon reflexes, a high-arched palate, and hepato-

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From the Department of Child Development; Kumamoto University Medical School; Kumamoto,Japan.

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Communications should be addressed to: Dr. Hiroyama; Department of Child Development; Kumamoto University Medical School; 1-1-1 Honjo; Kumamoto 860,Japan. Received May 2, 1986; accepted November 24, 1986.

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Figure 2. (A) CT at age 4 weeks depicts mild brain atropby. (B) CT at age 6 months depicts severe brain atrophy. (C) "*lndium chloride scan at age 4 weeks demonstrates increased uptake in the liver and spleen. (D) ~1qndium chloride scan at age 12 months reveals active hematopoiesis in the cranium and decreased uptake in the liver and spleen.

splenomegaly. Funduscopic examination revealed degenerative retinal changes and pale optic discs. Laboratory examinations revealed anemia, thrombocytopenia, leukocytosis, and abnormal blood chemistry results, including: alkaline phosphatase, acid phosphatase, glutamic oxaloacetictransaminase (GOT), glutamic-pyruvic transaminase (GPT), and lactic dehydrogenase (LDH). The most outstanding feature was high serum CK activity of 914 U/L (normal: - 175), mostly of the brain isoenzyme (MM 34%, MB 0%, BB 66%). Other examinations were normal: parathyroid hormone, urinary amino acid analysis, and chromosome studies. 1"Indium chloride scan at age 4 weeks (Fig 2C), demonstrated virtually no uptake in the long bones, pelvis, or spine, and increased uptake in the liver and spleen, which suggested extramedullary hematopoiesis. Cranial computed tomography (CT) (Fig 2A) demonstrated mild frontal atrophy. Because of severe hematologic involvement, including anemia and

thrombocytopenia, frequent platelet and occasional red cell transfusions were performed during the first few months of life. At age 2 months, steroid therapy (prednisolone: 2 mg/kg/day) was initiated. At age 2 months, she developed marked hepatosplenomegaly; total CK and CK-BB levels were relatively low (Fig 1). At age 4 months, she developed convulsions with an abnormal electroencephalogram (EEG), demonstrating spike activity in the left temporal region. Antiepileptic drug therapy was not successful, and she had several brief tonic seizures daily. At age 6 months, CT (Fig 2B) revealed severe brain atrophy, especially in the left hemisphere, but there was no evidence of intracranial hemorrhage. She could not smile or support her head. Thereafter, blood transfusion requirements decreased and the hepatosplenomegaly became less prominent. Concurrently, the total serum CK and CK-BB activities began to increase, but CSF CK and CK-BB activities were not detected on repeated examinations. On the other hand, NSE was continuously,

Hiroyama et al : CK-BB in Osteopetrosis

55

mildly elevated in both the serum and CSF (Table 1). At age 9 months, anemia and thrombocytopenia had almost disappeared, but neurologic problems had worsened. At age 12 months, 11qndium chloride scan (Fig 2D) showed decreased uptake in the liver and spleen and high uptake in the cranium which suggested active skull hematopoiesis. The patient was emaciated, weighing only 4 kg with a length of 52 cm. She did not respond to light and did not track objects; the pupils did not react to light and the optic discs were pale. Corneal reflexes were absent. There were no spontaneous movements of the extremities. Tendon reflexes were brisk. Biopsied sural nerve at age 13 months revealed storage of cellular debris including myelin figures in the Schwann cells (Fig 3). Although the diameter of myelinated and unmyelinated nerve fibers tended to be more hypertrophic than that of the age-matched normal control [3], no obvious changes were observed in axons. Total fascicular area and density of both myelinated and unmyelinated fibers were normal compared with the control [3].

Table 1.

Serum

CSF

NSE levels

NSE Ievel (ng/ml)

Age (Months)

26.5 24.6 33.6 22.2 21.2 39.9 8.1

1.5 2.5 4.5 5.5 8.0 6.5 8.5

Normal NSE level in serum: 5.39 - 1.86 ng/ml.

Discussion Osteopetrosis is characterized by radiopaque sclerotic bones and diagnosis is confirmed radiographically. Osteoclastic dysfunction is believed to cause this disease; defective bone resorption results in the accumulation of excessive mineralized osteoids and cartilage. Low-calcium diets [4] and high-dose calcitriol

[5] are administered with varying results. Bone marrow transplantation has been used recently [6]; however, this treatment is not always possible because of the lack of appropriate donors. Anemia and thrombocytopenia are common hematologic problems and therapy consists of blood replacement, splenectomy, and corticosteroid administration. Although the mechanism of action is unknown, corticosteroid therapy results in increased bone marrow hematopoiesis and decreased extramedullary hematopoiesis [7]. Our patient also demonstrated a positive response to prednisolone. Furthermore, neurologic involvement also is significant in this disease. Klintworth [8] reported six patients with osteopetrosis who had macrocephaly, optic atrophy, facial palsy, anosmia, proptosis, impaired hearing, and trigeminal nerve involvement. Symptoms were attributed to mechanical encroachment on cranial nerves and venous obstruction. He also suggested that macrocephaly was secondary to hydrocephalus, although the mechanism of the latter remains unknown. In 1977, Lehman et al. [9] reviewed seven patients with infantile osteopetrosis and neurologic complications and concluded that not all the neurologic complications could be explained by mechanical compression. They also demonstrated brain atrophy on CT and pneumoencephalography. Our patient had severe brain atrophy with slight ventricular dilatation as determined by CT. Radiographic examination revealed that the optic canal diameters were not narrow enough to compress the optic nerves. Recent studies have demonstrated that the concentration of CK in CSF, especially that of CK-BB, is a reliable indicator of brain damage. Worley et al. {1] found CK-BB in both neurons and astrocytes, and suggested that the quantity of CK-BB in CSF correlated with the amount of brain tissue damage. Moreover,

Figure 3. (A) Schwann cell isfilled with degradation products, including myelin figures (arrows)and glycogen particles (G), xl 4, 000. (B) Myelin figures and vesicular inclusions (arrows)also are observed in the area of node of Ranvier, x7, 000.

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Royds et al. [10] measured aldolase, enolase, pyruvate kinase, LDH, and CK in CSF of patients with central nervous system disorders and concluded that enolase is the most sensitive marker of neurologic disorders. Enolase is a dimeric enzyme with three subunit types: ol, 3, o~3, oLT, and 3'3'. Glial cells contain only oto~ enolase, whereas the 3'3'form, confined to the neurons, is called NSE. The presence of the ot and 3' isoenzymes appeared to reflect damage to glia and neurons, respectively [2]. Therefore, we studied concentrations of CK-BB and NSE in serum and CSF in our patient to determine the origin of the serum CK-BB. In spite of the high serum CK level, no CK activity was detected in CSF. The results indicated that brain damage was very gradual, indicating the effect of hemolysis in our patient. It has been reported that brain atrophy is commonly observed in West syndrome treated with ACTH. In addition, Bentson et al. [11] reported that long-term corticosteroid administration induced brain atrophy, even with the usual dosage. These facts suggest that steroid therapy might have played an important role in our patient's brain atrophy. Furthermore, Hoch-Ligeti et al. [12] reported that in bone marrow, serum LDH, GOT, alkaline phosphatase, and acid phosphatase activities are higher than those of cubital vein serum. It has been reported that CK-BB is high in bone marrow serum as well as in brain tissue [13]. These results suggest that high serum levels of CK, LDH, GOT, alkaline phosphatase, and acid phosphatase were derived from bone marrow serum through possible leakage. In bone marrow, cells may enter the sinusoids through fenestration of the endothelial cells that line the sinusoids; however, the exact mechanism is not completely understood [14]. In osteopetrosis, abnormal calcification in the bone marrow destroys the barrier mechanism between the hematopoietic parenchyma and sinusoids. Components of bone marrow leak into peripheral blood in proportion to the hematopoietic activity stimulated by prednisolone. Therefore, we conclude that the serum CK-BB activity in osteopetrosis originates from bone marrow serum, and not from brain tissue, and suggest that the concentration of CK-BB might reflect the degree of compensated hematopoiesis. With regard to the mechanism of neurologic involvement, we must make careful observations and search for a useful indicator. A recent report suggested that the neuronal [15] and axonal changes [15,16] play important roles in the

neurologic involvement of this disease. In our patient, biopsied sural nerve demonstrated storage of degradation products such as myelin figures and vesicular inclusions in Schwann cells suggesting an accelerated degradation process or lysosomal enzyme deficiency that dispose of the degradation products. Although the diameter of myelinated and unmyelinated nerve fibers in biopsied sural nerve tended to be hypertrophic in comparison to the normal control, no axonal changes were observed morphologically. Further investigations, includ!ng autopsy study, are needed to confirm the pathogenesis of the condition.

References

[1] Wodey G, Lipman B, Gewolb IH, et al. Creatine kinase brain isoenzyme. Pediatrics 1985;76:15-21. [2] Royds J, Davis-Jones GAB, Lewtas NA, et al. Enolase isoenzyme in the cerebrospinal fluid of patients with disease of the nervous system. J Neurol Neurosurg Psychiatry 1983 ;46:1031-6. [3] Origuchi Y. Quantitative histological study in the sural nerves of children. Brain Dev 1981 ;3: 395-402. 14] Yu JS, Oates RK, Walsh KH, et al. Osteopetrosis. Arch Dis Child 1971;46:257-63. [5] Key L, Carnes D, Cole S, et al. Treatment of congenital osteopetrosis with high-dose calcitriol. N Engl J Med 1984;310: 409-15. [6] Coccia PF, Krivit W, Cervenka J, et al. Successful bonemarrow transplantation for infantile malignant osteopetrosis. N Engl J Med 1980;302:701-8. [7] Reeves JD, Huffer WE, August CS, et al. The hematopoietic effects of prednisone therapy in four infants with osteopetrosis. J Pediatr 1979;94:210-4. [8] Klintworth GK. The neurologic manifestations of osteopetrosis. Neurology 1963; 13: 512-9. [9] Lehman RAW, Reeves JD, Wilson WB, et al. Neurological complications of infantile osteopetrosis. Ann Neurol 1977;2:378-84. [10] Royds JA, Timperley WR, Taylor CB. Levels of enolase and other enzymes in the cerebrospinal fluid as indices of pathological change. J Neurol Neurosurg Psychiatry 1981 ;44:1129-35. [11] Bentson J, Reza M, Winter J, et al. Steroids and apparent cerebral atrophy on computed tomography scans. J Comput Assist Tomogr 1978;2:16-23. [12] Hoch-Ligeti C, Jasen FJ. Enzymes in peripheral and bone marrow serum in patients with cancer. Cancer 1976;38:1336-43. [13] Silverman LM, Caruso LM, Irwin LE, et al. Creatine kinase BB isoenzyme activity in bone-marrow serum. Clin Chem 1978; 24:1423-5. [14] Wintrobe MM, Lee GR, Bitbell TC, et al. The normal hematopoietic system. In: Wintrobe MM, ed. Clinical hematology, 8th ed. Philadelphia: Lea and Febiger, 1981 ;33-525. [15] Ambler MW, Trice J, Grauerholz J, et al. Infantile osteopetrosis of infantile neuronal storage disease. Neurology 1983;33:437-41. [16] Fitch N, Carpenter S, Lachance RC. Prenatal axonal dystrophy and osteopetrosis. Arch Pathol 1973 ;95: 298-301.

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