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Peroxidase in ceroid-lipofuscinosis
Journal of the Neurological Sciences, 1978, 38: 191-193 © Elsevier/North-HollandBiomedicalPress
191
PEROXIDASE IN CEROID-LIPOFUSCINOSIS
W. R. DEN TANDT1 and J. J. MARTINz 1Medical Genetics, 2Neurology, Antwerp University Medical School, B-2610 Wilrijk (Belgium)
(Received 24 January, 1978) (Accepted 15 May, 1978)
SUMMARY Peroxidase determination in leucocyte homogenates of 3 patients with ceroidlipofuscinosis the infantile, late-infantile, and juvenile form, was not different from normal control values.
INTRODUCTION Ceroid-lipofuscinosis is a group of diseases comprising infantile (Hagberg-Santavuori), late-infantile (Jansky-Bielschowky), juvenile (Spielmeyer-Vogt), and adult (Kufs) forms. There have been several reports on the deficiency of a peroxidase (Armstrong, Dimmitt and Van Wormer 1974; Armstrong, Dimmitt, Boehme, Leonberg and Vogel 1974; Armstrong, Van Wormer, Neville, Dimmitt and Clingan 1975; Clausen and Jensen 1975; Aswasthi, Morris, Schochet, Powell, Schmalstieg and Srivastava 1977) in the last 3 forms. An animal ceroid-lipofuscinosis in English setters is also characterized by the same enzyme deficiency (Patel, Koppang, Patel and Zeman 1974). All of these reports except one (Awasthi et al. 1977) have measured the activity of this enzyme in the supernatant of leucocyte homogenates. The last report also could demonstrate the deficiency with other hydrogen donors than p-phenylenediamine which was used in the previous studies. In contradistinction with the finding of deficient peroxidase by some investigators, other workers did find normal leucocyte values for this enzyme in proven cases of ceroid-lipofuscinosis (Pilz, Goebel and O'Brien 1976; Pronk and Koster 1976). The peroxidase of both the supernatant and total homogenate proved to be normal. Pilz, O'Brien and Heipertz (1976) also concluded that salivary peroxidase and isoelectric fractionation of this enzyme was normal in cases with neuronal ceroid-lipofuscinosis. We have carried out peroxidase determination on leucocyte homogenates of 1 patient with the infantile, late-infantile, and juvenile form of the disease, respectively.
192 MATERIAL AND METHODS Heparinized blood was drawn and the supernatant collected after sedimentation of the red blood cells for about 2 hr. Hemolysis of the contaminating red blood cells was achieved by osmotic shock. The pellet was frozen once and the activity ofperoxidase determined not later than one week after the blood was taken. The homogenate was made in bi-distilled water using a Potter-Elvejhem homogenizer. Peroxidase of the whole homogenate was determined as follows: 0.7 ml of 1 M potassium phosphate buffer pH 7.3, 2.2 ml of 0.041 M p-phenylenediamine, 0.1 ml of a 32 mM solution of hydrogen peroxide and 0.010 ml of the leucocyte homogenate were mixed in a cuvette. The optical density was recorded immediately at 485 nm. The reaction was followed for 15 min but the actual enzyme activity was calculated for the first 2 rain of the reaction by comparing the optical density with that of a known solution ofp-phenylenediamine in the oxidized form. RESULTS AND DISCUSSION In Table 1 are recorded the activities of peroxidase in the 3 patients. It is clear from these data that this enzyme is not deficient. Other lysosomal enzymes were also determined on each homogenate and had normal activity. We have repeatedly observed that solubilization of peroxidase is very difficult. Less than 5 ~o of the total amount of this enzyme remained in the supernatant after 15 rain ofcentrifugation at 12,000 × g of the homogenate obtained by the following procedures: (1) homogeneization after repeated freezing and thawing (up to 6 times), (2) ultrasonication, (3) homogeneization in 0.1 ~ Triton X-100 followed by repeated freezing and thawing (6 times). This observation is in agreement with the results of Awasthi et al. (1977) who could solubilize peroxidase only after homogeneization in Triton X-100 followed by ultrasonication. It appears that solubilization of peroxidase is not easily performed unless special care is taken and that differences in the activity in the supernatant of leucocyte homogenates could also reflect differences of release of peroxidase. In our hands, p-phenylenediamine peroxidase determination on a whole leucocyte homogenate did not distinguish the 3 mentioned forms of ceroid-lipofuscinosis from normals. TABLE l ACTIVITY OF PEROXIDASE IN LEUKOCYTES OF PATIENTS AND NORMAL CONTROLS The activity is expressed in ~tmoles of paraphenylenediamine oxidized per minute and per mg of protein. Infantile form: W. VER (Centerick, Martin, Casaer and Edgar 1976) Late infantile form: G.D. Father of G.D. Mother of G.D. Juvenile form: H.S. Normal controls Mean: Range: Number:
1930 1208 896 963 2060 1533 670-2700 8
193 ACKNOWLEDGEMENTS P a t i e n t G . D . was referred to us by D r . J. R a d e r m e c k e r (Bunge Institute, BerchemA n t w e r p e n , Belgium). U l t r a - s t r u c t u r a l e x a m i n a t i o n by one o f us (J.J.M.) has confirmed the diagnosis o f p a t i e n t W . V E R . a n d G . D . H . S . was s t u d i e d by Dr. H. C a r t o n ( D e p a r t m e n t N e u r o l o g y , M e d i c a l School, U n i v e r s i t y o f Leuven, Belgium). W e like to express o u r t h a n k s for the o p p o r t u n i t y to study these patients.
REFERENCES Armstrong, D., S. Dimmitt and D. E. Van Wormer (1974) Studies in Batten disease, Part 1 (Peroxidase deficiency in granuloeytes), Arch. Neurol. (Chic.), 33 : 144. Armstrong, D., D. E. Van Wormer, H. Neville, S. Dimmitt and F. Clingan (1975) Thyroid peroxidase deficiency in Batten-Spielmeyer-Vogt disease. Arch. Path., 99: 430. Armstrong, D., S. Dimmitt, D. H. Boehme, S. C. Leonberg and W. Vogel (1974) Leukocyte peroxidase deficiency in a family with a dominant form of Kufs disease, Science, 186: 155. Awasthi, Y. C., H. H. Morris, S. S. Schochet, G. F. Powell, F. C. Schmalstieg and S. K. Srivastava (1977) Studies in neuronal ceroid-lipofuscinosis - - Leukocyte peroxidase deficiency in a patient with neuronal ceroid-lipofuscinosis (Jansky-Bielschowsky type), J. Lab. clin. Med., 89: 770. Ceuterick, Ch., J. J. Martin, P. Casaer and G. W. F. Edgar (1976) The diagnosis of infantile generalized ceroid-lipofuscinosis (type Hagberg-Santavuouri) using skin biopsy, Neuropiidiatrie, 7: 250. Clausen, J. and G. E. Jensen 0975) Acid proteinase and peroxidase activity in Spielmeyer-Vogt's syndrome. (Batten's syndrome-Stengel's syndrome), Clin. chim. Acta, 65 : 283. Patel, V., N. Koppang, B. Patel and W. Zeman (1974)p-Phenylenediamine-mediated peroxidase deficiency in English setters with neuronal ceroid-lipofuscinosis, Lab. Invest., 30: 366. Pilz, H., H. H. Goebel and J. S. O'Brien (1976) Isoelectric enzyme patterns of leukocyte peroxidase in normal controls and patients with neuronal ceroid-lipofuscinosis, Neuropiidiatrie, 7: 261. Pilz, H., J. S. O'Brien and R. Heipertz (1976) Human saliva peroxidase - - Microanalytical isoelectric fractionation and properties in normal persons and in cases with neuronal ceroid-lipofuscinosis, Clin. Biochem., 9: 85. Pronk, J. C. and J. F. Koster (1976) Leukocyte peroxidase in Spielmeyer-Vogt's disease, Hum. Genet., 34: 73.
191
PEROXIDASE IN CEROID-LIPOFUSCINOSIS
W. R. DEN TANDT1 and J. J. MARTINz 1Medical Genetics, 2Neurology, Antwerp University Medical School, B-2610 Wilrijk (Belgium)
(Received 24 January, 1978) (Accepted 15 May, 1978)
SUMMARY Peroxidase determination in leucocyte homogenates of 3 patients with ceroidlipofuscinosis the infantile, late-infantile, and juvenile form, was not different from normal control values.
INTRODUCTION Ceroid-lipofuscinosis is a group of diseases comprising infantile (Hagberg-Santavuori), late-infantile (Jansky-Bielschowky), juvenile (Spielmeyer-Vogt), and adult (Kufs) forms. There have been several reports on the deficiency of a peroxidase (Armstrong, Dimmitt and Van Wormer 1974; Armstrong, Dimmitt, Boehme, Leonberg and Vogel 1974; Armstrong, Van Wormer, Neville, Dimmitt and Clingan 1975; Clausen and Jensen 1975; Aswasthi, Morris, Schochet, Powell, Schmalstieg and Srivastava 1977) in the last 3 forms. An animal ceroid-lipofuscinosis in English setters is also characterized by the same enzyme deficiency (Patel, Koppang, Patel and Zeman 1974). All of these reports except one (Awasthi et al. 1977) have measured the activity of this enzyme in the supernatant of leucocyte homogenates. The last report also could demonstrate the deficiency with other hydrogen donors than p-phenylenediamine which was used in the previous studies. In contradistinction with the finding of deficient peroxidase by some investigators, other workers did find normal leucocyte values for this enzyme in proven cases of ceroid-lipofuscinosis (Pilz, Goebel and O'Brien 1976; Pronk and Koster 1976). The peroxidase of both the supernatant and total homogenate proved to be normal. Pilz, O'Brien and Heipertz (1976) also concluded that salivary peroxidase and isoelectric fractionation of this enzyme was normal in cases with neuronal ceroid-lipofuscinosis. We have carried out peroxidase determination on leucocyte homogenates of 1 patient with the infantile, late-infantile, and juvenile form of the disease, respectively.
192 MATERIAL AND METHODS Heparinized blood was drawn and the supernatant collected after sedimentation of the red blood cells for about 2 hr. Hemolysis of the contaminating red blood cells was achieved by osmotic shock. The pellet was frozen once and the activity ofperoxidase determined not later than one week after the blood was taken. The homogenate was made in bi-distilled water using a Potter-Elvejhem homogenizer. Peroxidase of the whole homogenate was determined as follows: 0.7 ml of 1 M potassium phosphate buffer pH 7.3, 2.2 ml of 0.041 M p-phenylenediamine, 0.1 ml of a 32 mM solution of hydrogen peroxide and 0.010 ml of the leucocyte homogenate were mixed in a cuvette. The optical density was recorded immediately at 485 nm. The reaction was followed for 15 min but the actual enzyme activity was calculated for the first 2 rain of the reaction by comparing the optical density with that of a known solution ofp-phenylenediamine in the oxidized form. RESULTS AND DISCUSSION In Table 1 are recorded the activities of peroxidase in the 3 patients. It is clear from these data that this enzyme is not deficient. Other lysosomal enzymes were also determined on each homogenate and had normal activity. We have repeatedly observed that solubilization of peroxidase is very difficult. Less than 5 ~o of the total amount of this enzyme remained in the supernatant after 15 rain ofcentrifugation at 12,000 × g of the homogenate obtained by the following procedures: (1) homogeneization after repeated freezing and thawing (up to 6 times), (2) ultrasonication, (3) homogeneization in 0.1 ~ Triton X-100 followed by repeated freezing and thawing (6 times). This observation is in agreement with the results of Awasthi et al. (1977) who could solubilize peroxidase only after homogeneization in Triton X-100 followed by ultrasonication. It appears that solubilization of peroxidase is not easily performed unless special care is taken and that differences in the activity in the supernatant of leucocyte homogenates could also reflect differences of release of peroxidase. In our hands, p-phenylenediamine peroxidase determination on a whole leucocyte homogenate did not distinguish the 3 mentioned forms of ceroid-lipofuscinosis from normals. TABLE l ACTIVITY OF PEROXIDASE IN LEUKOCYTES OF PATIENTS AND NORMAL CONTROLS The activity is expressed in ~tmoles of paraphenylenediamine oxidized per minute and per mg of protein. Infantile form: W. VER (Centerick, Martin, Casaer and Edgar 1976) Late infantile form: G.D. Father of G.D. Mother of G.D. Juvenile form: H.S. Normal controls Mean: Range: Number:
1930 1208 896 963 2060 1533 670-2700 8
193 ACKNOWLEDGEMENTS P a t i e n t G . D . was referred to us by D r . J. R a d e r m e c k e r (Bunge Institute, BerchemA n t w e r p e n , Belgium). U l t r a - s t r u c t u r a l e x a m i n a t i o n by one o f us (J.J.M.) has confirmed the diagnosis o f p a t i e n t W . V E R . a n d G . D . H . S . was s t u d i e d by Dr. H. C a r t o n ( D e p a r t m e n t N e u r o l o g y , M e d i c a l School, U n i v e r s i t y o f Leuven, Belgium). W e like to express o u r t h a n k s for the o p p o r t u n i t y to study these patients.
REFERENCES Armstrong, D., S. Dimmitt and D. E. Van Wormer (1974) Studies in Batten disease, Part 1 (Peroxidase deficiency in granuloeytes), Arch. Neurol. (Chic.), 33 : 144. Armstrong, D., D. E. Van Wormer, H. Neville, S. Dimmitt and F. Clingan (1975) Thyroid peroxidase deficiency in Batten-Spielmeyer-Vogt disease. Arch. Path., 99: 430. Armstrong, D., S. Dimmitt, D. H. Boehme, S. C. Leonberg and W. Vogel (1974) Leukocyte peroxidase deficiency in a family with a dominant form of Kufs disease, Science, 186: 155. Awasthi, Y. C., H. H. Morris, S. S. Schochet, G. F. Powell, F. C. Schmalstieg and S. K. Srivastava (1977) Studies in neuronal ceroid-lipofuscinosis - - Leukocyte peroxidase deficiency in a patient with neuronal ceroid-lipofuscinosis (Jansky-Bielschowsky type), J. Lab. clin. Med., 89: 770. Ceuterick, Ch., J. J. Martin, P. Casaer and G. W. F. Edgar (1976) The diagnosis of infantile generalized ceroid-lipofuscinosis (type Hagberg-Santavuouri) using skin biopsy, Neuropiidiatrie, 7: 250. Clausen, J. and G. E. Jensen 0975) Acid proteinase and peroxidase activity in Spielmeyer-Vogt's syndrome. (Batten's syndrome-Stengel's syndrome), Clin. chim. Acta, 65 : 283. Patel, V., N. Koppang, B. Patel and W. Zeman (1974)p-Phenylenediamine-mediated peroxidase deficiency in English setters with neuronal ceroid-lipofuscinosis, Lab. Invest., 30: 366. Pilz, H., H. H. Goebel and J. S. O'Brien (1976) Isoelectric enzyme patterns of leukocyte peroxidase in normal controls and patients with neuronal ceroid-lipofuscinosis, Neuropiidiatrie, 7: 261. Pilz, H., J. S. O'Brien and R. Heipertz (1976) Human saliva peroxidase - - Microanalytical isoelectric fractionation and properties in normal persons and in cases with neuronal ceroid-lipofuscinosis, Clin. Biochem., 9: 85. Pronk, J. C. and J. F. Koster (1976) Leukocyte peroxidase in Spielmeyer-Vogt's disease, Hum. Genet., 34: 73.