Tohoku J. exp. Med., 1984, 142, 239-248

A Successful Trial of Enzyme Replacement in a Case of Argininemia

TAKESHI SAKIYAMA,HIROKI NAKABAYASHI,HISASHI SHIMIZU,WAKIO KONDO, SEIDO KODAMA* and TERUO KITAGAWA Department of , Nihon University School of , Tokyo 101 and *Department of Pediatrics, Nagano Red Cross Hospital, Nagano 380

SAKIYAMA,T., NAKABAYASHI,H., SHIMIZU,H., KONDO,W., KODAMA,S. and KITAGAWA,T. A Successful Trial of Enzyme Replacement Therapy in a Case of Argininemia. Tohoku J. exp. Med., 1984, 142 (3), 239-248 A 5-year-old boy with severe mental retardation and spastic quadriplegia accompanied by tonic and was diagnosed as having argininemia due to an deficiency in his erythrocytes. His motor and mental abilities began to deteriorate at the age of 3 years. Thereafter, he lost his ability to stand alone, to sit and even to crawl by himself. After he was diagnosed as argininemia, a restricted diet was given as therapy, which was accompanied with a supplement of essential amino acids. However, his clinical condition had not improved very much. The erythrocytes in a normal person was found to have the ability to decrease the patient's elevated plasma level to normal when they are mixed in vitro. First we tried replacing his red cells by a transfusion. Then we replaced them with the aid of an IBM 2997 blood cell separator. Following this his clinical and biochemical condition improved, and as a result so did his sitting and crawling abilities. It appears that the replacement of red blood cells improves not only the clinical and biochemical conditions, but the general condition of the patient as well. argininemia ; enzyme replacement therapy

Argininemia results from a deficiency of arginase, the enzyme in the final step of the cycle. The first probable case was noticed by Peralta Serrano (1965) and was later described in detail by Terheggen et al. (1969). Thereafter, 13 patients of different ethnic origins had been reported (Terheggen et al. 1969, 1975a; Snyderman et al. 1977, 1979; Cederbaum et al. 1977, 1979; Michels and Beaudet 1978; Qureshi et al. 1981; Yoshino et al. 1982; Walser 1983). An arginase deficiency can be characterized by clinical features or biochemical conditions such as a predominantly elevated level of serum arginine, a marked degree of spastic diplegia and severe mental and neurological deterioration. These occur after fairly normal development through the first few years of life. Receivedfor publication,May 25, 1983. 239 240 T. Sakiyama et a1.

There were some unsuccessful attempts at treatment. One was to induce arginase activity by an injection of the Shope papilloma virus (Terheggen et al. 1975b). Another was to reduce hyperargininemia with the use of a diet (Terheg- gen et al. 1970). A therapeutic trial of enzyme replacement using erythrocytes transfusion also failed to decrease significantly serum arginine or to show any clinical improvement (Michels and Beaudet 1978). On the other hand, the treatment of this disease with a restricted protein intake and a mixture of essential amino acids succeeded in preventing episodic hyperammonemia, reducing hyper- argininemia and improved the mental capacity of two children affected with the disease (Snyderman et al. 1977). This was the first real success in dealing with this diasease. Nevertheless, in our case, the clinical condition had not improved enough by the use of a diet which consisted of moiety as a mixture of essential amino acids. In this paper, we present our first successful attempt at treating argininemia. We replaced the patient's erythrocytes with the aid of an IBM 2997 blood cell separator.

METHODS Clinical studies. Amino acid levels in plasma, cerebrospinal fluid and urine were determined by a Hitachi model 835 automatic amino acid analyzer. was deter- mined by a modification of the method used by Okuda and Fujii (1966). During the time that a laboratory was not available, Amitest (Tada et al. 1979) was used to detect and monitor the level of blood ammonium. Orotic acid in urine was determined by the Roger and Porter method (1968). Enzymatic studies. arginase (EC 3.5, 3.1) was determined in hepariniz- ed specimens by using the method of Tomlinson and Westall (1964). Blood specimens were collected in heparin then processed and diluted with twice the volume of distilled water to make hemolytic enzyme source. The enzyme solution in 5 mM MnC12was preincubated at 50°C for 30 min. The reaction mixture (0.25 M buffer pH 9.5, plus 0.25 M L-arginine and the enzyme solution) was incubated at 3TC for 30 min. The reaction was stopped by adding 1 M perchloric acid. It was then centrifuged and the supernatant was mixed with a-isonitrosopropiophenon and phosphorus-sulfate reagent in order to detect the color at 0D54° with a Hitachi spectrophotometer. The concentration of hemoglobin was determined by the cyanmethemoglobin method. The application of a cell separator IBM 2997 in the trials of enzyme replacement therapy is as follows : Normal packed red blood cells were substituted for the patient's red blood cells, mixed with the patient's plasma through the cell separator, then returned to the patient.

CASE REPORT' Patient T.M., a 5-year-old male is the second of two children born to healthy nonconsanguineous parents. He was born at term with a body weight of 3,500 g after a near miscarriage at the gestational age of 3 months. He was born with asphyxia and was attacked by a due to Iiypocalcemia (2.2 mEq/liter) at 3 days old. His weak cries and tonic clonic convulsions disappeared after an injection of calcium. At 7 days old, he was treated for neonatal jaundice by Enzyme Replacement for Argininemia 241 phototherapy. He had an operation on his left inguinal hernia at the age of 3 months, but his wound did not cure completely for several months. He had hyperirritability at 6 months old, so he was examined by spinal tap and electroen- cephalography, but all findings were normal. He appeared to be slightly re- tarded. His head control was not obtained until 5-6 months of age. He could not sit alone until 8 to 10 months old, stand alone and utter words until 1 year 6 months and walk until 2 years. His teeth also began forming late. However, there were no seizures. He had chicken pox at 2 years old and mumps at 2 years 10 months old, however, these did not cause any unfavorable effects on his clinical manifestation. After he caught a cold at the age of 3 years 2 months, he suddenly became unpleasant and hyperirritable and fell into somnolence. His motor and mental abilities also began to deteriorate at this time. He lost his ability to stand alone, to sit and even to crawl by himself. Occasionally tonic clonic convulsions without fever were noticed and he was put on anticonvulsants. He began to vomit and also had convulsive attacks of 5 to 6 min at the age of 4 years 7 months. The elevated serum transaminase (GOT 246 mIU, GPT 184 mIU) showed a disturbance in his . In addition, a high value of blood ammonia (278-288 1ug/ 100 ml) was noticed. He was admitted to a local hospital and was fed with a high-protein-diet which was followed by frequent vomiting and also with a low- protein-diet which was followed by improvement of his clinical signs and sym- ptoms. Amino acid analysis in his serum showed an elevated level of arginine (10.1 mg/100 ml under high-protein-diet and 8.0 mg/100 ml under low-protein- diet). For further examinations and therapeutic trials, he was transferred to our

Fig. 1, a : Spaspic quadriplegia. Note the scissored position of his legs before the enzyme replacement therapy. b : Reduced spasticity and clinical improvement were observed after the enzyme replacement therapy. 242 T. Sakiyama et al, hospital. His height (93 cm), weight (14.2 kg) and head circumference (50.3 cm) were the third, tenth and fiftieth percentile, respectively. The nutritional status of the patient was good. His consciousness was clear, but he could not speak and could barely move by himself. He could not stand alone and not even roll over by himself. His legs were held in a rigid, scissored position and were difficult for us to move (Fig. la). His facial characteristic was a prominent beaked nose and an apathetic gaze. Strabismus of the eyes and hypoplasia of teeth enamel were noticed. On physical examination, his liver was palpable 2 to 3 cm under the costal margin and his spleen was not palpable. Spastic quadriplegia, an accel- erated deep tendon reflex of the knee and a positive Babinski reflex were bilateral- ly found. There were no apparent sensory defects. His IQ was estimated to be 27 at 5 years old. Laboratory data. The serum -oxaloacetic transaminase (160 mIU normal 11-35 mIU, SCOT), serum glutamic-pyruvate transaminase (130 mIU, normal 4-30 mIU, SGPT), and lactic dehydrogenase (712 mIU, normal 220-440 mIU) levels were all elevated. BUN (5.7 mg/100 ml) and uric acid (3.1 mg/100 ml) levels were low. A blood ammonium level of 297 u g/ 100 ml was noticed. The amino acid chromatogram of the patient's paasma under feeding with approxi- mately 1.5 g of protein per kg/day showed hyperargininemia (7-9 mg/loo ml). Even under feeding with a low protein diet of 0.75 g of protein per kg/day, a high level of serum arginine (7 mg/100 ml) was observed. Other amino acids, however, were almost within normal level (see Table 1). The urinary amino acid chroma- togram showed increased excretion of arginine (over ten-fold of normal), ornithine (over ten-fold of normal), (over six-fold of normal) and ornithine (over three-fold of normal) but argininosuccinate was undetectable. The excretion of orotic acid in urine was elevated (1 mg/ml, normal less than 1.4 mg per day). A CSF sample was obtained while the patient was consuming 1.5 g of protein/kg/ day. Arginine was increased 3-5 times of normal but the levels of other amino acids were within normal limits. Red blood cell arginase was invariably undetectable in the lysates of the patient's erythrocytes. The level of erythrocyte's arginases of his parents was in between the normal range and the patient's level which denoted heterozygocity. The arginase activity of his sister's erythrocytes was just below the normal range (Table 2). His serum calcium level was subnormal at 8.1-8.6 mg/100 ml (normal 8.8-10.8 mg/100 ml). Other routine laboratory data were within the normal range. The electroencephalograms taken during the anticonvulsant therapy showed spontaneous sharp waves without prominent paroxysmal discharge. The electromyogram and nerve conduction velocity were normal. His bone age was normal. Computerized axial tomography of the brain at the time of admission revealed marked cerebral atrophy with enlargement of the lateral ventricles and sulci. Enz yme Replacement for Argininemia 243 244 T. Sakiyama et al.

TABLE 2. The arginase activity of erythrocytes in his f amily

RESULTS Diet therapy The plasma ammonia level rose as the protein intake was increased. In addition, when the patient had a cold, plasma ammonia also rose and irritability and convulsions appeared even while on a restricted protein diet with an essential amino acid supply. Initially he was fed a diet of a total of 1,500 kcal per day including 1.5 g of protein per kg/day. Later, the protein intake was reduced to 0.75 g/kg/day. The essential amino acid mixture (EAA), which was prepared in our laboratory from L-amino acids according to the prescription of Snyderman et al. (1977), was introduced in the regimen. This mixture corresponded to a protein level of 0.85 g/kg/day. Two months of therapy with protein restriction and essential amino acid supply seemed to have no effect on his clinical conditions. He still could not roll over by himself and continued to show difficulty even to sit down on a toilet because of marked spasticity.

Trials of enzyme relacement therapy In vitro experiments. Blood specimens were collected in heparin from the patient, his mother and a healthy control. They showed all identical blood types. After taking blood samples, the whole blood was kept at room temperature, then the amino acid level of the plasma and red blood cells were determined. These were determined first on the sample taking day, then 2 days after and again at 4 days after. Next the red blood cells separated from the patient's mother and from the healthy control were mixed with the patient's plasma respectively. Then the levels of amino acids in the pretreated plasma and the red blood cells were determined at the same time intervals. These procedures were all done in sterile conditions. The levels of arginine in the patient's plasma and red blood cells increased gradually after standing for a testing period of 0-4 days. However, those of the patient's mother and control plasma decreased with the passage of time. Normal- ization of arginine levels in plasma and red blood cells was observed by mixing with the mother's or control's red blood cells (Fig. 2). Enzyme Re placement for Argininemia 245

Fig. 2. Enzyme replacement therapy in vitro. o , patient's blood ; A patient's plasma+mother's RBC ; • , patient's plasma+control RBC ; o , moth er's blood ; o , control blood.

Fig 3. The levels of blood arginine, ammonia and arginase during his hospitalization and subsequent treatment.

In vivo experiments. The total volume of the patient's blood was estimated at about 1,100 ml. The patient received a 400 ml blood transfusion. After the blood transfusion of 400 ml, the estimated arginase in his body should be about 0.36% of the total body arginase in a normal person. Although a very small amount of red blood cell arginase was temporarily changed, his spasticity seemed to improve slightly. Through the treatment using the blood 246 T. Sakiyama et al. cell separator, his replaced arginase could be estimated to be about 1 to 1.5 percent of normal. The results showed not only a marked decrease in the level of arginine in serum and CFS but also serum ammonia reduction. Moreover, his clinical improvement was obvious in that he became able to sit by himself with reduced spasticity and to roll over by himself (see Fig. lb). The activity of the patient's arginase in erythrocytes reached normal level after the enzyme replacement, and in addition, the level of arginine decreased remarkably. The second trial of replacement was not quite completed because of an urticaria eruption on his skin, but the erythrocyte's arginase rose to its normal level (Fig. 3).

DISCUSSION In cases of argininemia reported previously, all patients were severely retard- ed. However, mental and neurologic symptoms developed after a nearly normal development in the first few years of life (Terheggen et al. 1969, 1975a ; Snyder- man et al. 1977 ; Cederbaum et al. 1977, 1979 ; Michels and Beaudet 1978; Walser 1983). In addition, poor growth, , spastic diplegia, hyper- reflexia and seizures were predominant clinical manifestations. Clinicobiochemi- cally, hyperammonemia was highly dependent on dietary intake and was nor- malized by protein restriction. An elevated level of arginine in the blood and in the cerebrospinal fluid was almost always observed. Hyperargininemia was not normalized successfully by a diet alone (Terheggen et al. 1970), however, it was improved by a protein restricted diet accompanied with a complement of essential amino acids (Snyderman et al. 1977). This nutritional approach succeeded in preventing episodic hyperammonemia and also in keeping a normal level of serum arginine in the affected patient from its birth (Snyderman et al. 1979). In our case, the patient had already developed slight mental and physical retardation as a result of hypocalcemia and convulsions during his neonatal period. Except for these episodes, he had had no vomiting or ataxia before his abrupt unconscious- ness at the age of 3 years 2 months. Since that time, he had developed tonic clonic convulsions and spasticity of his lower extremities leading to the deteriora- tion of his motor and mental abilities. His disability became so great that he lost his ability to crawl. A protein restricted diet as therapy was introduced to him when hyperammonemia was discovered. A controlled diet accompanied by a supplement of essential amino acids was tried after argininemia was diagnosed. However, the diet therapy was not enough to improve his clinical condition. Arginase activity in his red blood cells was undetectable. Arginase activity of his parents showed intermediate levels between controls and the patient. This result seems to agree with the autosomal recessive trait of inheritance (Walser 1983). The plasma arginine of the patient was in vitro normalized by mixing with normal erythrocytes or his mother's red blood cells. Arginases in red blood cells and in the liver are thought to be encoded with the same gene. This was Enzyme Replacement for Argininemia 247 confirmed by the coordinated lack of the enzyme in both the erythrocytes and liver from a patient with argininemia (Cederbaum et al. 1979) and was also proven by kinetic and immunological similarities of purified arginase from human ery- throcytes and (Cabello et al. 1961, 1965). Gene replacement therapy was initially introduced for this disorder by injecting the Shope papilloma virus (Terheggen et al. 1975b), which is known to code for arginase. However, this trial failed to increase the level of arginase in the patients or to improve their clinical manifestations. Michels and Beaudet (1978) attempted enzyme replacement by blood transfusions but it also resulted in a lack of clinical or biochemical improvement. The replaced red blood cells, however, could be calculated up to 10% of the total blood, which was only 0.1 to 0.2% of the total body arginase activity. Those transfusions were of less volume than our trials using blood transfusion. Our trials by transfusion also resulted in an insignificant decrease of blood arginine, but showed slight clinical improve- ment. But through the cell separator, our patient's red blood cells could have been replaced up to 1 to 2% of the total arginase activity in the normal liver and red blood cells. exists only in the liver and not in the brain. It has been reported that neurological manifestation in urea cycle disorders is mainly due to the elevated ammonia and amino acids in the brain which are produced by a deficiency of enzyme activity in the liver (Walser 1983). In patients with argininemia, blood ammonia is not so high. Therefore, elevated arginine in the brain may be mainly responsible for the development of neurological manifesta- tions (Terheggen et al. 1975a; Michels and Beaudet 1978). If blood arginase activity was elevated significantly and serum arginine was normalized by the enzyme replacement therapy, neurological manifestation should improve to some extent. It was apparent that the red blood cell exchange by the cell separator was significantly effective in decreasing the serum arginine level, which was similarly decreased in in vitro experiments. In addition, clinical improvements such as the loosening of spasticity in his legs and the ability to sit without aid were observed after the red blood cell replacement. The increased level of arginine in CSF before the treatment reduced after the treatment. Although additional trials and longitudinal studies are necessary, it appears that the replacement of red blood cells improves the clinical and biochemical condition. These results might encourage us to try bone marrow transplants in order to replace permanent intact red blood cells in future.

Acknowledgments

We are grateful to Mr. Ken Suzuki for assistance with amino acid analysis and to Miss Hiroko Nakano for help in preparation of the manuscript. This research was supported in part by grans from the Ministry of Health and Welfare (82-08-06) and from IBM under the title of "Enzyme Replacement Therapy", Japan. 248 T. Sakiyama et al.

References 1) Cabello, J., Basilio, C. & Prejoux, V. (1961) Kinetic properties of erythrocyte and liver arginase. Biochim. biophys. Acta, 48, 148-152. 2) Cabello, J., Prajouv, V. & Plaza, M. (1965) Immunodiffusion studies on human liver and erythrocyte arginase. Biochim. biophys. Acta, 105, 583-593. 3) Cederbaum, S.D., Shaw, K.N.F. & Valente, M. (1977) Hyperargininemia. J. Pediat., 90, 569-573. 4) Cederbaum, S.D., Shaw, K.N.F., Spector, E.B., Venty HA., Snodgrass, P.J. & Suger- man, G.I. (1979) Hyperargininemia with arginase deficiency. Pediat. Res., 13, 827- 833. 5) Michels, V.V. & Beaudet, AL. (1978) Arginase deficiency in multiple tissues in argininemia. Gun. Genet., 13, 61-67. 6) Okuda, H. & Fujii, S. (1966) Colorimetrical assay of blood ammonia. Saishin Igaku, 21, 622-627. (in Japanese) 7) Peralta Serrano, M. (1965) Cited from Walser M. (1983), Reference 19. 8) Qureshi, IA., Letarte, J., Quellet, R., Lelievre, M. & Laberge, C. (1981) Ammonia metabolism in a family affected by hyperarginiriemia. Diabet. Metab., 7, 5-11. 9) Roger, L.E. & Porter, F.S. (1968) Hereditary orotic aciduria. Pediatrics, 42, 423- 428. 10) Snyderman, SE., Sansaricq, C., Chen, W.J., Norton P.M. & Phaneoalkav, S.V. (1977) Argininemia. J. Pediat., 90, 563-568. 11) Snyderman, SE., Sansaricq, C., Norton, P.M. & Goldstein, F. (1979) Argininemia treated from birth. J. Pediat., 95, 61-63. 12) Tada, K., Okuda, K., Watanabe, K., Iimura, T. & Yamada, S. (1979) A new method of screening for hyperammonemia. Europ. J. Pediat., 130, 105-110. 13) Terheggen, HG., Schwenk, A., Lowenthal, A., Van Sande, M. & Colombo, J.P. (1969) Argininemia with arginase deficiency. Lancet, 2, 748-749. 14) Terheggen, HG., Schwenk, A., Lowenthal, A., Van Sande, M. & Colombo, J.P. (1970) Hyperargininemie mit Arginasedefekt, eine neue familiare Stoffwechselstorung. I. Klinische Befunde. II. Bhiochemische Untersuchungen. Z. Kinderheilk., 107, 298- 312. 15) Terheggen, HG., Lowenthal, A., Lavinha, F. & Colombo, J.P. (1975a) Familial hyperagininemia. Arch. Dis. Child., 50, 57-62. 16) Terheggen, HG., Lowenthal, A., Lavinha, F., Colombo, J.P. & Rogers, S. (1975b) Unsuccessful trial of gene replacement in arginase deficiency. Z. Kinderheilk., 119, 1- 3. 17) Tomlinson, S. & Westall, R.G. (1964) Argininosuccinic aciduria, argininosuccinase and arginase in human blood cells. Clin. Sci., 26, 261-269. 18) Walser, M. (1983) Urea cycle disorder and other hereditary hyperammonemic syn- drome. In: Metabolic Basis of Inherited Metabolic Diseases. 5th edition, edited by J.S. Stanbury et al., McGraw-Hill Book Company, New York. pp. 402-438. 19) Yoshino, M., Murakami, T., Kubota, K., Yoshida, I., Yamashita, F., Eto, Y., Yama- moto, M., Nakashima, H. & Hashimoto, T. (1982) A new case of argininemia. Acta pediat. jpn., 86, 30-37. (in Japanese)