mars or COPPER WWW”: m0 mmwmmwsfla ONZTHE * , {-‘f-«i; :5 meme RAT lama, ‘ _ . j 2,; 35"5:1}???1';rijjif:53. Thesis for the Degreegf Ph. D. ‘ , MECHIGRH STATE :UNWERSWY ; - 1035??! ROBERT PRGHRSKA ' y 1m » ‘ 0—7 639 Luna» " ' ‘1 Y": 2:; Michigan 1”" Unraersxw ? This is to certify that the '1?" thesis entitled EFFECTS OF COPPER DEFICIENCY AND HYPERPHENYLALANINE ON THE DEVEEOPING RAT BRAIN presented by Joseph Robert Prohaska has been accepted towards fulfillment of the requirements for _Eh.D_._ degree in Jinchemistry .ili L IBRARY BINDERS Anti ,..”W V ABSTRACT EFFECTS OF COPPER DEFICIENCY AND HYPERPHENYLALANINE ON THE DEVELOPING RAT BRAIN By Joseph Robert Prohaska It was the initial intent of this thesis to test the possibility that high levels of phenylalanine could affect copper metabolism. Experiments producing hyperphenylalaninemic conditions in rats were carried out by feeding L-phenylalanine (5%) to male weanling rats or by subcutaneous injection of L-phenylalanine with or without DL-p-chlorophenylalanine to neonatal rats. When a diet adequate in copper was fed to the rats or to the dams whose offspring were injected, hyperphenylalanine induction did not affect plasma or brain copper levels, brain cytochrome c oxidase, plasma diamine oxidase, or fecal copper output. The activity of plasma diamine oxidase (ceruloplasmin) from patients with phenylketonuria, an inherited disease in which hyperphenylalaninemia is characteristic, was equivalent to age matched controls. Injections of phenylalanine to offspring from dams fed a low copper diet suggested retention of body copper based on a delayed fall in the plasma diamine oxidase level and on an increased brain capper content and cytochrome oxidase level. The hyperphenylalaninemic pups from dams fed a diet adequate in copper and the copper-deficient offspring injected with saline both developed abnormally and further studies were carried Joseph Robert Prohaska out on the two groups, independently, as model systems for the genetic disorders Phenylketonuria and Menkes' steely-hair disease. Since it was known that both copper deficiency and hyperphenyl— alaninemia resulted in hypomyelination of the central nervous system, a marker to follow this process was investigated. The myelin enriched protein 2',3'—cyclic nucleotide 3'-phosphohydrolase was employed as an indicator of myelination in the experimental models. An improved assay for this protein was devised resulting in an increased precision - a coefficient of variation of 1.7% for six trials. This was partially the result of optimizing the temperature (30°C) and the pH (6.2) for the reaction. The concentration of this protein in whole brains of three neurological mutant mice (quaking, jimpy, and myelin synthesis deficiency) was reduced five-fold when compared to littermate controls. There were no differences among these mice in plasma diamine oxidase, brain copper content, or cytochrome c oxidase activities suggesting that copper metabolism was grossly normal in these mice despite several similarities to the copper-deficient rat. Since prior work on the effects of hyperphenylalaninemia on the developing rat brain had emphasized the reduction in myelin lipids, a study was carried out to investigate brain protein development. It was found that subcutaneous injections of L-phenylalanine and DL—p-chloro- phenylalanine into developing rats resulted in reduced body and brain weights and a small but significant decrease in 2',3'-cyclic nucleotide 3'-phosphohydrolase. These reductions were less marked when injections were reduced by half. When animals were allowed to recover after 13 days of age, no differences were observed at 40 days of age suggesting Joseph Robert Prohaska recovery when treatment was discontinued before hyperplasia was complete. Brain hexokinase and cytochrome c oxidase development was not changed by the injection treatments. This is consonant with the idea that the reduction in the myelin protein 2',3'-cyclic nucleotide 3'-phosphohydrolase represents less myelin and that the reduction was not a general feature of all brain proteins. Copper deficiency was produced in developing rats by feeding a low copper diet (0.3 parts per million) to rats during gestation and lactation and providing the offspring the same diet or by preconception depletion followed by marginal supplementation during gestation. The progeny showed an appreciable decrease in body growth, a slight decrease in whole brain and cerebellar growth and a significant reduction in the concentration of 2',3'-cyclic nucleotide 3'-phosphohydrolase. Specific effects of a five-fold decrease in copper content were reductions in the metalloproteins cytochrome oxidase (to 20% of control) and superoxide dismutase (to 70% of control). A similar reduction in norepinephrine concentration was also found. Zinc levels of control and copper deficient brains were equivalent; there was some evidence of a slight reduction in iron concentration. Electron microscopic examination of the cerebral cortex revealed the presence of enlarged mitochondria from the copper-deficient animals. When isolated, these mitochondria oxidized succinate and glutamate at 70% the rate control mitochondria did; oxygen consumption in the presence of glutamate was not appreciably increased by the addition of adenosine diphosphate to the reaction. Mitochondrial difference spectra verified the large reduction in cytochrome oxidase and demonstrated slight increases in cytochromes Joseph Robert Prohaska b, c1, and c. Enzyme analysis of isolated mitochondria showed slight increases in succinic dehydrogenase and fumarase, and a small decrease in hexokinase. Malondialdehyde production was equivalent in controls and copper-deficient brain homogenates suggesting no abnormal lipid peroxidation. Determination of metabolites suggested that the copper-deficient brain was in a more reduced state since both lactate/ pyruvate and a-glycerol-phosphate/dihydroxyacetone-phosphate ratios doubled. However, creatine-phosphate, adenosine triphosphate, and adenosine diphosphate concentrations were not different. Therefore, the tenet that hypomyelination in copper deficiency results from a decrease in cytochrome oxidase and therefore a reduction in energy production should be questioned until further investigation is performed. EFFECTS OF COPPER DEFICIENCY AND HYPERPHENYLALANINE ON THE DEVELOPING RAT BRAIN By Joseph Robert Prohaska A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1974 ACKNOWLEDGEMENTS The author would like to thank Professor William W. Wells for his enthusiastic suggestions, generous financial support, and unselfish flexibility. Doctors Richard Luecke, Steven Aust, Loran Bieber, Clarence Suelter and Duane Ullrey, members of my guidance committee, have my gratitude for their cooperative manner of instruction, suggestion and criticism. I would also like to acknowledge Douglas Clark for his technical assistance and Don Musick whose penetrating discussions facilitated my thesis. This work could not have transpired without the totally unselfish encouragement of my parents and my loving wife, Carol. Lastly, I wish to acknowledge the financial help I received from the National Science Foundation. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . LIST OF FIGURES O I O O O O O O O O 0 LIST OF ABBREVIATIONS . . . . . . . . INTRODUCTION Organization . . . . . . . . Approach . . . . . . . . . Literature Review '. . . . . . Copper-Amino Acid Interaction . 2' ,3'-Cyclic Nucleotide 3'-Phosphohydrolase . Hyperphenylalanine . . . . . Menkes' Steely-Hair Disease . . Copper and Human Brain Metabolism Copper Deficiency in Animal Brain References . . . . . . . . . Chapter I. EFFECTS OF HYPERPHENYLALANINE ON COPPER Abstract 0 O O O O O O O 0 Introduction . . . . . . . . Materials and Methods . . . . . Materials . . . . . . . . Animal Treatments and Diets . . Copper Analysis . . . . . . Tissue Preparation . . . . . Enzyme Assays . . . . . . . Chemical Methods . . . . . . Statistical Methods . . . . . Results . . . . . . . . . . Discussion . . . . . . . . . Acknowledgments . . . . . . . References . . . . . . . . . METABOLISM Page vi viii uih‘hl O‘U‘IU'IbJ-‘UJ 11 12 l3 l4 14 15 16 16 l7 18 18 19 22 23 24 Chapter II. III. IV. Page EFFECT OF PHENYLALANINE AND p-CHLOROPHENYLALANINE ADMINIS- TRATION ON THE DEVELOPMENT OF RAT BRAIN 2',3'-CYCLIC NUCLEOTIDE 3'-PHOSPHOHYDROLASE . Introduction . . . . . . . Materials and Methods . . . . Results . . . . . . . . . Discussion . . . . . . . . Summary . . . . . . . . . References . . . . . . . . COPPER DEFICIENCY IN THE DEVELOPING RAT BRAIN: MODEL FOR MENKES' STEELY-HAIR DISEASE . . Abstract . . . . . . . . Introduction . . . . . . . Materials and Methods . . . . Materials . . . . . . . Animal Care and Diets . . . Tissue Preparation . . . . Cytochrome c Oxidase . . . Superoxide Dismutase . 2' ,3'-Cyclic Nucleotide 3'-Phosphohydrolase . . Copper Analysis . . . . . NOrepinephrine . . . . . Statistics . . . . . . . Re8u1ts O O O O O O O O O Behavioral and Growth Effects Brain Development . . . . Development of Copper Dependent Copper Metalloproteins . . . Adult Rats . . . . . . . Discussion . . . . . . . . References . . . . . . . . Systems COPPER DEFICIENCY IN THE DEVELOPING RAT BRAIN: ABNORMAL MITOCHONDRIA . . . . Abstract . . . . . . . . Introduction . . . . . . . Materials and Methods . . . Animal Care and Diets . . Enzyme Assays . . . . . . Electron Microscopy . . . . Mitochondrial Studies . . . iv . . . . 38 . . . . 39 . . . . 39 . . . . 41 . . . . 42 . . . . 45 . . . . 46 A POSSIBLE O O O O 58 . . . . 59 . . . . 59 O O 0 6O 0 O O O 60 . . . . 61 O O O O 62 O O O O 63 O O O 63 . 63 O O O O 64 . . . . 64 . . . . 64 O O O I 65 O O I O 65 O O O O 65 . . . . 66 O O O O 66 . . . 67 O O O O 67 O I I O 72 EVIDENCE FOR . . . . 88 . . . . 89 . . . . 90 . . . . 91 O O O 91 O O 91 O O O O 92 . . . 93 Page Metabolite and Metal Analysis . . . . . . . 93 Lipid Peroxidations . . . . . . . . . . . 94 Results . . . . . . . . . . . . . . . . 94 Induced Copper Deficiency . . . . . . . . . 94 Mitochondrial Morphology . . . . . . . . . 95 Mitochondrial Function . . . . . . . . . . 96 Energy Metabolites . . . . . . . . . . . 97 Trace Element Levels . . . . . . . . . . 98 Lipid Peroxidation . . . . . . . . . . . 98 Discussion . . . . . . . . . . . . . . . 99 Acknowledgment . . . . . . . . . . . . . 104 References . . . . . . . . . . . . . . . 105 DISCUSSION . . . . . . . . . . . . . . . . . . 121 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase . . . . 121 Copper Deficiency . . . . . . . . . . . . 122 Hyperphenylalanine . . . . . . . . . . . . 124 References . . . . . . . . . . . . . . . 127 APPENDIX - IMPROVED RAPIDITY AND PRECISION IN THE DETERMINATION OF BRAIN 2',3'-CYCLIC NUCLEOTIDE 3'-PHOSPHOHYDROLASE . . 130 Abstract . . . . . . . . . . . . . . . 130 Introduction . . . . . . . . . . . . . . 130 Materials . . . . . . . . . . . . . . . 131 Methods . . . . . . . . . . . . . . . . 131 Preparation of Tissue Homogenates . . . . . . 131 Final CNP Assay Conditions . . . . . . . . 131 Chemical Analyses . . . . . . . . . . . 132 Results . . . . . . . . . . . . . . . . 132 Discussion . . . . . . . . . . . . . . . 134 References . . . . . . . . . . . . . . . 137 LIST OF TABLES Table Page Chapter I 1. Effect of Dietary Phenylalanine on Fecal and Plasma Copper and Diamine Oxidase . . . . . . . . . 26 2. Plasma Diamine Oxidase Levels of Phenylketonuric Patients 0 O O O O O O O O O O O O O O 27 3. Effect of Injections of L-Phenylalanine on Plasma and Brain Copper Levels of Neonatal Rats . . . . . . 28 4. Effect of Injections of L-Phenylalanine and DL-p- Chlorophenylalanine on Cytochrome Oxidase and Copper Levels of Neonatal Rat Brain . . . . . . . . 29 5. Brain Copper Levels, Cytochrome Oxidase and Diamine Oxidase Activities of Myelin Deficient Mice . . . 30 6. Activity of 2',3'-Cyclic AMP 3'-Phosphohydrolase in Myelin Deficient Mice . . . . . . . . . . . 31 Chapter II I. Plasma Tyrosine Ratios in Developing Rats Following Injection of L-Phenylalanine and DL-p-Chloro- phenYJ-alan ine I O O O O O O O O O O O O 48 II. Body, Whole Brain, and Cerebellar Weights Following In- jection of L-Phenylalanine DL-p-Chlorophenylalanine . 49 III. Effect of Injections of L-Phenylalanine and DL-p-Chloro- phenylalanine on Development of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Rat Brain . . . . . . . 50 IV. Effect of Injections of L-Phenylalanine and DL—p-Chloro- phenylalanine on Development of Hexokinase and Cytochrome c Oxidase in Rat Brain, Experiment III . . . . . 51 Chapter III 1. Effect of a Low Copper Diet Upon Body, Whole Brain, and Cerebellum Growth in the Developing Rat . . . . . 76 vi Chapter II. III. VI. DISCUSSI Adult Rat Values for Brain and Cerebellar Weights: Brain Copper and Norepinephrine Content: Cerebellar Cytochrome c Oxidase, 2',3'-cAMP 3'-Phosphohydrolase, and Superoxide Dismutase Activities . . . . . . IV Rat Brain Development-Recovery From Perinatal Copper Deficiency . . . . . . . . . . . . . . Copper Deficiency-Brain Mitochondrial Oxygen Consumption . . . . . . . . . . . . . . Copper Deficiency-Brain Mitochondrial Enzymes and cytOChromeS O O O O O O O O O O O O O 0 Copper Deficiency—Brain Energy Metabolites . . . . Copper Deficiency-Brain Copper, Zinc, and Iron Levels 0 O I O O O O O O O O O O O l 0 Copper Deficiency-Brain Lipid Peroxidation . . . . ON 1. Activity of 2',3'-Cyclic AMP 3'-Phosphohydrolase in APPENDIX 1. 2. Developing Rat Cerebella Following Experimental Treatments . . . . . . . . . . . . . . Comparison of Whole Brain 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase Activities . . . . . . . . Comparison of Current Procedures for Measuring 2',3'- Cyclic Nucleotide 3'-Phosphohydrolase . . . . . vii 77 109 110 111 112 113 114 129 135 136 Figure Chapter 1A. 1B. 4I Chapter 1. 2B. Chapter LIST OF FIGURES Page I Formation of Product with Increasing Plasma Content, Diamine Oxidase Activity . . . . . . . . . 33 Formation of Product with Increasing Time, Diamine Oxidase Activity . . . . . . . . . . . . 33 Velocity of the Diamine Oxidase Reaction with Increasing Substrate . . . . . . . . . . . 34 Relationship Between Dietary Copper Level and Rat Plasma Diamine Oxidase Activity . . . . . . . 35 Diamine Oxidase Activity of Neonatal Copper-Deficient Rats Injected with Phenylalanine or Saline . . . 37 II The Postnatal Growth (Body Weight (A) and Whole Brain Weight (3)) of Rats Injected Twice Daily From Six Days of Age with Either Saline or Phenylalanine and p-Chloro- phenylalanine, Experiment I . . . . . . . . 53 The Postnatal Development of Whole Brain 2',3'-cAMP 3'-Phosphohydrolase in Saline Injected and in Phenylalanine, p-Chlorophenylalanine Injected Rats . . . . . . 55 The Postnatal Development of Whole Brain Cytochrome c Oxidase in Saline Injected and in Phenylalanine, p- Chlorophenylalanine Injected Rats . . . . . . 57 III The Postnatal Development of Cerebellar 2',3'-cAMP 3'- Phosphohydrolase in Control and Copper Deficient Rats I I I I I I I I I I I I I I I I 79 Concentration of Copper in Fresh Tissue in Developing Control and Copper Deficient Whole Rat Brains Minus cerebella I I I I I I I I I I I I I I 81 viii 5. Chapter 1. 2. The Postnatal Development of Cerebellar Cytochrome c Oxidase in Control and Copper Deficient Rats . . . 83 The Postnatal Development of Cerebellar Superoxide Dismutase in Control and Copper Deficient Rats . . 85 Concentration of Norepinephrine in Developing Control and Copper Deficient Whole Rat Brains Minus Cerebella 87 IV Electron Micrographs of 30-Day Old Rat Cerebral Cortex From Control and Copper-Deficient Animals . . . . 116 Difference Spectra of Reduced (Ascorbate + NaZS 04) Versus Oxidized (K3Fe(CN)6) Rat Brain Mitochondria in the Presence of Deoxycholate . . . . . . . . 118 Copper—Deficient Rat Brain Mitochondrial Components From 30-Day Old Rats as a Per Cent of Control Rat Data . 120 Effect of pH on Adult Rat Brain CNP Activity . . . 132 Effect of Temperature on Adult Rat Brain CNP . . . 133 Effect of 2',3'-cAMP Concentration on the Activity of Adult Rat Brain CNP . . . . . . . . . . 134 ix 2',3'-cAMP AMP, ADP, ATP CNS CNP CoA DAO EDTA p-Clphe RCR sc SD Tris LIST OF ABBREVIATIONS adenosine 2',3'-cyclic monophosphate adenosine 5'-mono-, di-, or triphosphate central nervous system 2',3'-cyclic nucleotide 3'-phosphohydrolase Coenzyme A diamine oxidase ethylenediaminetetraacetic acid Michaelis constant nicotinamide adenine dinucleotide parachlorophenylalanine respiratory control ratio subcutaneous standard deviation tris(hydroxymethyl)aminomethane INTRODUCTION ORGANIZATION Each chapter in the thesis text is presented in a form found in biochemical journal articles. Each one includes its own abstract or summary, introduction, materials and methods, results, discussion, acknowledgements and references. Style varies somewhat depending on the particular journal for which the chapter was written. Chapter II, under its title, is presented as it was accepted for publication in the Proceedings of the Society for Experimental Biology and Medicine. Chapter III has been published under the chapter title in the Journal of Neurochemistry (1974) 23, 91-98. Chapter IV is in review, Archives of Biochemistry and Biophysics. These were all accepted or submitted under my authorship and that of W. W. Wells. For the reader's convenience, a reprint of an additional article (Analytical Biochemistry (1973) 56, 275-282.) of some interest to the thesis has been inserted as an APPENDIX. A DISCUSSION section has been included to consider material not specifically dealt with in the preceeding chapters and to offer some overview on the project's results. APPROACH Despite a multitude of hypotheses concerning the underlying cause of the mental retardation associated with the untreated phenylketonuric patient, no single concept seemed satisfactory. There are several superficial similarities between the phenylketonuric and the copper— deficient animal. Both are characterized by hypomyelination of the central nervous system (CNS) and had pigmentation abnormalities and behavior changes that could be rationalized by catecholamine alterations. Since it was also known that amino acids had a high affinity for Cu2+ (1) and that Cu-amino acid complexes existed in serum (2), a study was initiated to determine whether the induction of hyper— phenylalaninemia could result in a change in copper homeostasis. These experiments were performed with albino rats which served as models throughout the course of this research. Results indicated that under normal conditions phenylalanine did not significantly alter copper metabolism. However, both the production of hyperphenylalanine and copper deficiency resulted in abnormal development of the offspring. It has been estimated (3) that during the peak of myelination a single oligodendroglial cell synthesizes greater than 3-fold its own weight of myelin per day. This endergonic process is, therefore, most susceptible to metabolic insults. In order to monitor myelination a marker protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase, was employed during rat brain development (4). An improved assay method for this enzyme made the handling of multiple samples more feasible. In some studies the development of the cerebellum was emphasized because of its higher risk of injury in postnatal development (5). Experiments producing hyperphenylalanine were designed to see if myelin protein levels were changed by this treatment since prior work had involved only myelin lipid constituents. Another objective was to learn if this retardation in myelination was reversible upon discontinuation of the injection treatment, since it was known that dietary restriction of phenylalanine, if applied at an early age, was effective in the amelioration of the phenylketonuric. While this research was in progress Danks et a1. (6) reported that the genetic disorder referred to as Menkes' disease was characterized by copper deficiency. Severe neuropathology is also characteristic of this disorder. The developing rat brain was, therefore, chosen as a model for this disease. Dietary production of copper deficiency was carried out and the offspring were used to follow myelination and the development of the copper metalloenzymes, cytochrome oxidase and superoxide dismutase. Norepinephrine was measured to evaluate the status of another copper protein, dopamine-B-hydroxylase. Subsequent work revealed that the brains of copper-deficient rats had abnormal mitochondria. So further work dealing with oxygen consumption and enzyme components of isolated mitochondria was performed. These studies were accompanied by metabolite analyses in an attempt to correlate the in vitro studies of isolated mitochondria and enzyme assays with the in vivo brain energy status. LITERATURE REVIEW Copper-Amino Acid Interaction. In 1950 Albert (1) reported quantitative results on the stability constants of bivalent amino acids with trace metal ions. He found Cu2+ was bound most tightly of the eight ions studied. Fifteen years later the first reports of amino acid-copper complexes in serum appeared (2, 7). This bound copper was in equilibrium with a pool bound to serum albumin. There is good evidence that a ternary complex of albumin - Cu2+ - amino acids exists in vivo (8). This amino acid pool is thought to be involved in the biological transport of copper (7) and thus under pathological situations where the elevation of an amino acid occurs there may be adverse effects (2). 2[,3'-Cyclic Nucleotide 3'-Phosphohydrolase. Drummond g£_§1, (9) first reported the activity of this protein from brain which was later (10) shown to be associated with the myelin. The use of this protein as an enzyme marker for myelin as well as a detailed summary of the many techniques available for its detection have been described before (11) (see APPENDIX). Newer methods for the detection of CNP (2',3'-cyclic nucleotide 3'-phosphohydrolase) include potentiometric analysis (12), a fluorometric assay (13) using NG-ethenoadenosine- 2',3'-cyclic monophosphate as substrate and a spectrophotometric technique (14) using 2',3'-cyclic cytosine monophosphate. CNP activity can also be detected after electrophoresis on polyacrylamide gels (15). This technique demonstrated that CNP was not one of the major myelin proteins. It is reported to have a molecular weight of approximately 100,000 (14). Its continued useage as a myelin marker is evidenced by reports on studies of spinal cord tissue cultures (16), wallerian degeneration (l7), and human brain tumors (18). Hyperphenylalanine. The hyperphenylalaninemic state is used in experimental models to learn more about this condition in man, since it is a consequence of the disease phenylketonuria (PKU). PKU was first described by Félling (19) in 1934 and has since been characterized as an autosomal disorder resulting in the failure to convert phenylalanine to tyrosine (20), thus, creating the hyper- phenylalanemic state. It has been suggested by many authors that this condition results in the neuropathological changes seen in PKU specimens. These defects are usually hypomyelination in the young patient and progressive demyelination in the older cases (21). These conditions vary somewhat and the reader is referred to an excellent discussion on this topic (22). Another characteristic of PKU brain tissue is a reduction in the catecholamine content, dopamine and norepinephrine, (23); due to the combined effects of an inhibition of tyrosine hydroxylase and to competitive inhibition of tyrosine transport. The pigmentation abnormality in PKU can be attributed to inhibition of tyrosinase by phenylalanine (24) and to reduced substrate, tyrosine. Of the many models producing hyperphenyl- alaninemia, dietary induction (25) or subcutaneous injection techniques (26, 27) are the most common. Menkes' Steely-Hair Disease. In 1962 Menkes et a1. (28) first reported cases of a sex-linked recessive disorder accompanied by neural degeneration. Ten years later Danks g£_§l, (6, 29) reported that this disease was characterized by copper deficiency due to defective intestinal absorption. It is a rare disorder and only limited biochemical studies have been made. Preliminary data based on reduced myelin lipids such as cerebroside and sulfatide (30) and on lower yields when myelin was isolated (31) suggested that the disorder was associated with hypomyelination. A recent publication (32) reported the presence of cholesterol esters in a brain specimen suggestive of active demyelination. One positive argument as to the implication of copper's role in the etiology of this disease has been strengthened by the finding of low brain copper in three cases of this disorder (33). Copper and Human Brain Metabolism. A lack of copper can be detrimental to the development of the CNS as in the case of Menkes' disease. An excess of copper can also be detrimental to the CNS and a genetic disease in which this occurs is known, Wilson's disease or hepatolenticular degeneration (34). This is an autosomal recessive disorder which results in brain damage due to an accumulation of copper because of an abnormal binding protein in the liver (35). Besides these genetic disorders of copper, many more subtle roles of this element in brain metabolism can be found in a cursory perusal of Chemical Abstracts. Some of these include Parkinson's disease, schizophrenia, epilepsy, reflex reactions of the spinal cord, meningeal affection and multiple sclerosis. Copper homeostasis in the mammalian system has been recently reviewed (36) and will not be covered here. Copper Deficiency in Animal Brain. One of the earliest observations concerning the copper-deficient brain was a reduction in cytochrome A (a + a3) and oxidase activity (37). This finding was later verified by Gallagher EE_El3 (38) in the most extensive work to date on copper- deficient rats. Reduced cytochrome oxidase associated with low copper levels has been reported for other animals besides rats and also for yeast (39). This loss of cytochrome oxidase was thought to be partially responsible for the hypomyelination seen in swayback lambs (38, 40). Gallagher g£_al, (40) also reported a reduced rate of phospholipid synthesis in copper-deficient rats which was due to a failure of condensation of acyl CoA with a-glycerol phosphate. Others have been unable to repeat this (41). Despite the fact that the original work (40) found normal 32P labeling of brain phospholipids and that all evidence suggests that copper deficiency has no effects on brain phospholipid distribution (42, 43), current dogma supports the notion that abnormal phospholipid synthesis is responsible for hypomyelination. Most studies concerning the effect of copper deficiency on brain metabolism have been carried out in animals whose CNS was already fully developed. However, prior to this research it had been shown that guinea pigs (44) were severely affected by gestational deprivation of copper. Carlton and Kelly (45) had done a study on the offspring of copper— deficient rats but did not include biochemical evaluations. 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(1970) in Myelination (DAVISON, A.N. and PETERS, A., eds.) pp. 183-190. Charles C. Thomas, Springfield. MC KEAN, C.M. (1972) Brain Res. 3;, 469-476. MIYAMOTO, M. and FITZPATRICK, T. (1957) Nature 179, 199-200. KERR, G.R. and WAISMAN, H.A. (1967) J. Nutr. 22, 10—18. CLARKE, J.T.R. and LOWDEN, J.A. (1969) Can. J. Biochem. 41, 291-295. ANDERSEN, A.E. and GUROFF, G. (1972) Proc. Natn. Acad. Sci._§2, 863-867. MENKES, J.H., ALTER, M., STEIGLEDER, G.K., WEAKLEY, D.R. and SUNG, J.H. (1962) Pediatrics 29, 764-779. DANKS, D.M., CAMPBELL, P.E., STEVENS, B.J., MAYNE, V. and CARTWRIGHT, E. (1972) Pediatrics 29, 188-201. O'BRIEN, J.S. and SAMPSON, E.L. (1966) J. Neuropath. Exp, Neurol. 22, 523-530. FRENCH, J.H., SHERARD, E.S., LUBELL, H., BROTZ, M. and MOORE, C.L. (1972) Arch. Neurol. 26, 229-244. LOU, H.C., HOLMER, G.K., RESKE-NIELSEN, E. and VAGN-HANSEN, P. (1974) J. Neurochem. 22, 377-381. WALKEReSMITH, J.A., TURNER, B., BLOMFIELD, J. and WISE, G. (1973) Arch. Dis. Childhood 48, 958-962. BEARN, A.G. (1972) in The Metabolic Basis of Inherited Disease (STANBURY, J.B., WYNGAARDEN, J.B. and FREDRICKSON, D.S., eds.) 3d edition, pp. 1033-1050. McGraw-Hill, New York. EVANS, G.W., DUBOIS, R.S. and HAMBIDGE, KJM. (1973) Science 181, 1175-1176. EVANS, G.W. (1973) Physiol. Revs. 2;, 535-570. COHEN, E. and ELVEHJEM, C.A. (1934) J. Biol. Chem. 107, 97-105. GALLAGHER, C.H., JUDAH, J.D. and REES, K.R. (1956) Proc. Roy. Soc. London B 145, 134-149. WOHLRAB, H. and JACOBS, E.E. (1967) Biochem. Biophys. Res. Commun. 28, 991—997. 40. 41. 42. 43. 44. 45. 46. 10 GALLAGHER, C.H., JUDAH, J.D. and REES, K.R. (1956) Proc. Roy. Soc. London B 145, 195—205. DI PAOLO, R.V. and NEWBERNE, P.M. (1971) in Trace Substances in Environmental Health - V. Symposium. (HEMPHILL, D.D., ed.) pp. 177-191. Univ. Missouri Press, Columbia. GALLAGHER, C.H. and REEVE, V.E. (1971) Aust. J. Exp, Biol. Med. SCi 0 59-, 453-461 I DI PAOLO, R.V., KANFER, J.N. and NEWBERNE, P.M. (1974) J. Neuropath. Exp. Neurol. 33, 226-236. EVERSON, G.J., SCHRADER, R.E. and WANG, T. (1968) J. Nutr. g_6_, 115-125. CARLTON, W.W. and KELLY, W.A. (1969) J. Nutr. _91, 42-52. PROHASKA, J.R. AND WELLS, W.W. (1974) J. Neurochem. 22, 91-98. CHAPTER I EFFECTS OF HYPERPHENYLALANINE ON COPPER METABOLISM 11 12 ABSTRACT Hyperphenylalaninemia was produced by either feeding a diet 5% L-phenylalanine or by the subcutaneous injection of L-phenylalanine (L-phe) with or without DL-p—chlorophenylalanine (DL-p-Clphe). These treatments were given to rats fed either a diet adequate or deficient in copper or to offspring from dams fed these diets. This was done to study the chronic effects of an elevated amino acid on copper homeostasis. When a diet adequate in copper was supplied, treatment with L-phe or L-phe with DL-p-Clphe did not affect copper levels in the plasma, diamine oxidase activity, fecal copper output, brain copper concentration, or brain cytochrome c oxidase levels. Likewise, ceruloplasmin (diamine oxidase) activity of phenylketonuric patients and age matched controls were equivalent. However, injection of L—phe and DL—p-Clphe into copper-deficient pups delayed the large fall in diamine oxidase activity and resulted in higher brain copper and cytochrome c oxidase. Three neurological mutations of mice with faulty myelin synthesis (quaking, jimpy and myelin synthesis deficiency) were studied because of similarities to the copper- deficient rat. They were found to have greatly reduced activities of the myelin protein 2',3'-cyclic nucleotide 3'-phosphohydrolase but displayed no differences in comparison to littermates in brain copper, cytochrome c oxidase or plasma diamine oxidase. It was concluded that under normal conditions hyperphenylalanine does not influence copper homeostasis, but that both the copper-deficient and hyperphenylalaninemic rat develop abnormally. 13 INTRODUCTION Plasma copper exists predominantly (over 90%) bound to the glycoprotein ceruloplasmin (1). This copper has a biological half- time of 13 hrs, a maximum tissue uptake of 40 hrs (2) and is not exchangeable in vivo (3). A smaller fraction of copper (less than 10%) is bound to albumin (3) has a half-time of 10 min, a maximum tissue uptake of 1 hr (2) and is rapidly exchangeable in vivo (4). This albumin bound fraction is in equilibrium with a third pool, an amino acid-bound fraction (5), and is thought to have a physiological role in copper transport. In a comparison of the avidity of bivalent Cu2+ complexes were found to amino acids for the ions of trace metals, have the highest stability constants (6). It has been suggested that the cupruria seen in acute viral hepatitis may result from the observed aminoacidemia and aminoaciduria (7). Oral methionine loading of patients with Wilson's disease, which is characterized by high tissue levels of copper, resulted in a two-fold increase in both fecal and urinary capper output (8). Administration of histidine to man has produced hyperzincuria with an associated hypozincemia (9). Sarkar and Kruck have suggested ". . . that in patients with abnormal plasma free amino acid levels there may be abnormal patterns of copper-amino acid complexes that could have pathological consequences." (10). Phenylketonuria is an inherited diesease of man in which plasma phenylalanine may be highly elevated. It had been previously shown that phenylalanine could interact with Cu2+ in reversing the inhibition of (Na++K+)—ATPase (11). Therefore, experimental hyperphenylalaninemia 14 was studied to learn if it could alter copper homeostasis. Since dietary treatment with cuprizone (bis(cyclohexanone) oxaldihydrazone), a potent copper chelator, resulted in a reduction in brain copper as well as a concomitant decrease in cytochrome c oxidase (12), both these parameters were measured during phenylalanine treatment. Plasma copper was also evaluated in experimental animals as well as in phenylketonurics. Although likely fortuitous, both phenylketonuria and copper deficiency are characterized by a neuropathology involving both hypomyelination and demyelination (13, 14). Copper metabolism was also studied in three myelin-deficient mouse mutants: quaking (15), jimpy (15) and myelin synthesis deficiency (16). D1 Paolo, gngl, suggested an investigation of this type based on the similarity between these mice and the copper-deficient rat (17). This would also provide a useful hypomyelination model to compare with both copper deficiency and hyperphenylalaninemia. MATERIALS AND METHODS Materials. Male weanling or female sperm-positive rats were purchased from Holtzman Co. (Madison, WI). Myelin mutant mice of the strain C57BL/6J were obtained from the Jackson Laboratory (Bar Harbor, ME). Frozen phenylketonuric serum and plasma were the generous gifts of Drs. Thomas Egan (Univ. Chicago), Richard Allen (Univ. Michigan), and James Higgins (Michigan State Univ.). Human ceruloplasmin was the gift of Dr. M. Wickerhauser (American Red Cross Blood Res. Lab.). Horse heart cytochrome c was received from Boehringer-Mannheim 15 (New York, NY) and Tween-80 from Nutritional Biochemicals (Cleveland, OH). E. coli alkaline phosphatase type III-S (EC 3.1.3.1), 2',3'-cAMP, DL-O-chlorophenylalanine, L—phenylalanine, and p-phenylenediamine were purchased from Sigma Chemical Co. (St. Louis, MO). Animal Treatments and Diets. The composition of the basal diet has been detailed elsewhere (18) (see chapt. III) and was used as such except that in some cases the casein was not washed with EDTA. Diets containing 5% L-phenylalanine (19) were prepared by a corresponding 5% reduction in the sucrose content. Diets with both adequate and low copper content were used with or without supplemental L-phenylalanine. Animals were maintained in stainless steel cages throughout the feeding experiments and received distilled water and food ad libitum. When hyperphenylalanine was produced by injections, the pregnant rats were fed either adequate or low copper diets beginning on the 8th day of gestation. One day prior to parturition they were placed in plastic cages bedded with wood shavings and remained there throughout lactation. Pups were randomized at birth (day 0), 8 per dam, within each dietary group. Offspring were injected subcutaneously with a 3% solution of Lrphenylalanine at a dose of l mg/g body wt (20) or alternatively with.the same solution containing 0.3% DL-p-chlorophenylalanine (21). An equivalent volume of saline was injected into age matched control rats. All solutions were adjusted to pH 7.4 and were heated to 37°C before injection. Copper analysis of 100 m1 aliquots of each 16 injection solution resulted in equivalent levels all below 0.1 pg Cu/ml. Mice (C57BL/6J) were killed within 24 hours after arrival and included the following genotypes: quaking (qk/qk), jimpy (jp/Y) and myelin synthesis deficiency (msd/Y). Copper Analysis. Copper was determined by atomic absorption spectroscopy (Perkin-Elmer model 303). Tissue, feces and diets were wet-ashed in HNOa—HC101+ acids and the residue was dissolved in hot 1% HCl. Standards were prepared in this solvent. Plasma samples were diluted with 2 volumes of glass distilled water (0.3 ng Cu/ml) and were measured directly. COpper standards for use in plasma measurements were prepared in 5% (v/v) glycerol. To avoid contamination, all glassware used in these experiments was soaked in HNO3 or HCl and was thoroughly rinsed with glass distilled water. Plastic ware used for storage of samples was soaked in 1% EDTA and thoroughly rinsed. TisSue Preparation. Whole brains were homogenized in 9 volumes of 0.32 M sucrose in a Potter-Elvehjem homogenizer fitted with a Teflon pestle. Aliquots were used to measure cytochrome c oxidase (EC 1.9.3.1) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.16b). In some cases brain tissue was stored -85°C, before homogenization. Alternatively, brains were first dryed, 807C, to constant wt or used directly for acid digestion and copper analysis. Plasma was obtained after centrifugation, 3,000 x g for 10 min, of heparinized blood taken by heart puncture from ether anesthetized animals. Feces were collected by placing animals in stainless steel metabolic cages for two 48 hr periods. Fecal material was collected 17 in copper-free plastic containers and was free of dietary contamination (unlike the urine specimens). Enzyme Assays. The assay of 2',3'—cyclic nucleotide 3'—phosphohydrolase (CNP) was performed as described previously (22) (see APPENDIX). Whole mouse brain was assayed without freezing of the homogenates. Cytochrome c oxidase was measured as detailed previously (18) (see chapt. III) by following the oxidation of ferrocytochrome c at 550 nm. It was found the 1% (v/v) Tween-80 released total activity while Triton X-100 was found to be inhibitory (these observations are also true for succinic dehydrogenase activity). The activity of cytochrome c oxidase was found to decrease by as much as 1/3 when stored as a 10% (w/v) homogenate at -20.C for as little as 3 days. Conversely, tissue stored for 1 month lost less than 20% activity. For maximum precision cytochrome c solutions must be prepared fresh. In some experiments detergent treated mitochondria were incubated at 25°C for 20 min in either the presence of 5 mM L-phenylalanine or 5 mM cuprizone (bis(cyclohexanone) oxaldihydrazone) before activity was measured. The diamine oxidase activity of ceruloplasmin (EC 1.16.3.1) was measured by a modification of the method of Levine and Peisach (23). The reaction was run at 37°C in 0.1 M sodium acetate (pH 5.5). The commercial p-phenylenediamine (1,4-diaminobenzene) was purified by repeated sublimation and was employed at 4 mM final concentration. It was dissolved just prior to use and adjusted to pH 5.5 as suggested (23). To avoid interference by traces of iron, 18 5 uM EDTA was also employed (24). Sodium azide, a potent inhibitor of ceruloplasmin (24), was used (0.5 mM) to terminate the reaction and to provide a nonenzymatic oxidation blank when included prior to substrate addition. Routine assays were performed for 30 min with 50 ul of plasma in a 1 m1 final volume. The appearance of the purple oxidation product N,N'-bis-(2,5 diaminophenyl)-p-quinonediimine (Bandrowski's base) (25) was followed at 533 nm. Extrapolation from the data of Rice (25) would yield a value of e = 11.6 X 103 for this product. Based on this constant, ceruloplasmin activity was defined as umoles/min/liter of plasma. Chemical Methods. Protein was measured by the method of Lowry 55:31, (26) using bovine serum.albumin as standard; Tween-80 interference was corrected for in the appropriate cases. Powdered human ceruloplasmin was dissolved in 0.15 M NaCl (pH 7.0) and the absorbance at 280 and 610 nm taken on several samples (Gilford model 24008). The purity was estimated to be greater than 95% based on 280/610 ratio (27). Crystalline Bandrowski's base was prepared according to Rice (25) and was found to have an equivalent melting point (237°C). The visible spectrum (Cary model 15) displayed a broad peak centered at 533 nm. Attempts at solution of this compound, in a media comparable to the assay, were not successful although the molar absorptivity did approach that reported by Rice (25). Statistical Methods. Mean comparisons were evaluated by use of a computer program written to calculate variance ratios and statistical E, Student's Eftest was used and hypothesis testing was done at two levels of 6:0.05 and 0.01. 19 RESULTS Since modifications to the diamine oxidase assay were introduced, conditions for validity were studied. Figures 1A and 1B show that the formation of product, as measured by an increase in absorbance at 533 nm, is proportional to the amount of plasma and to the reaction time used in the final assay, 50 ul and 30 min respectively. A plot of velocity versus substrate is shown in Figure 2. When the data were analyzed by the method of Hofstee (28), the human ceruloplasmin had a Km of 0.5 mM for 1,4-diaminobenzene. Activity was not influenced by inclusion of 2.5 mM L-phenylalanine during the reaction. Dietary induction of hyperphenylalanine was carried out in conjunction with both copper-adequate and copper-deficient diets in three separate experiments of similar design, Table 1. The copper contents of the diets (both control and low) varied somewhat depending on whether the casein was washed with an EDTA solution (i.e. I versus III). The animals fed the low copper diet excreted less than 1/10 the amount of fecal copper that animals fed adequate copper did, irrespective of the presence of dietary phenylalanine, exp. 1. No consistent statistical differences were evident in plasma copper levels or diamine oxidase activites (DAO) when dietary phenylalanine was the sole criterion, Table 1. This is illustrated more clearly in Figure 3 which shows that a strong correlation exists between the diamine oxidase activity and the dietary copper level regardless of the presence of 5% L-phenylalanine. There were no differences in the DA0 activities between phenylketonurics and age matched controls, Table 2, although the younger patients had less activity than the 20 adult cases. Injections of L—phenylalanine were administered to developing rats from dams fed either adequate or low copper diets, Table 3. As can be seen, the offspring from copper adequate dams were not affected by the daily injection regimen in terms of either plasma or brain copper levels. The offspring from copper deficient dams injected with phenylalanine seemed to have higher plasma copper levels especially at age 14 days, Table 3; perhaps brain copper is also elevated at this age. Only these offspring were noticeably reduced in body wt compared to the other three groups. When a more intensive injection sequence was carried out, including DL—p-chlorophenylalanine (21), pups from both control and COpper— deficient dams were severely reduced in size (data not shown). Pups from copper-deficient dams but injected with saline were somewhat smaller than pups similarly injected but of dams fed a diet adequate in copper. Analysis of the plasma DAO activities of the pups from copper-deficient dams injected with either phenylalanine with or without p-chlorophenylalanine is displayed in Figure 4. It can be seen that in both experiments the injection of the amino acid delayed the large fall in DAO. This suggestion of elevated plasma copper is strengthened by the data in Table 4. Injection of phe and p-Clphe to the copper- deficient offspring resulted in higher brain copper levels and a corresponding elevation in brain cytochrome c oxidase content. However, Table 4 also demonstrates that pups from COpper-adequate dams were not affected by the injection treatment in either the 21 concentration of brain copper or the level of cytochrome c oxidase. Cytochrome c oxidase activity was not affected by either the presence of L-phenylalanine or cuprizone in vitro. Plasma DAO was not affected by injections of phe with or without p-Clphe in pups from copper-sufficient dams (data not shown); however, the DA0 levels were lower than the adult values reported in Table 1 in agreement with the results for human plasma, Table 2. An evaluation of copper metabolism in plasma and brain of three neurological mutant mice deficient in myelin along with their corresponding littermates is shown in Table 5. Adult values are also listed and represent data obtained from foster mothers supplied with the suckling mice. No differences exist in DAO, brain copper, or cytochrome c oxidase concentrations between the mutants and their littermate controls. The older mice, adults and quaking mutants, do have higher cepper levels than the younger suckling animals. This increase in copper content with age is also demonstrated for rats, Tables 3 and 4, even for the short period observed. When the mouse brains were analyzed for the CNS myelin associated protein 2',3'-cyclic nucleotide 3'- phosphohydrolase (CNP) (29) a marked reduction in activity was observed, Table 6. The littermates for the jimpy mutants had less activity than the older controls of the other mutants. 22 DISCUSSION The results clearly indicate that with adequate dietary copper, phenylalanine exerts no effects on copper homeostasis. This is true for the most sensitive indicator of copper status, plasma copper, Table l. The linear correlation between diamine oxidase activity and dietary copper, Figure 3, has also been shown to exist between plasma copper levels and activity of DAO (30). The values for DAO, Table 2, for both control and PKU plasma lie in the accepted range for normal adult humans (25) based on a similar assay. The fact that plasma DAO was elevated, Figure 4, by hyperphenyl- alaninemia in the copper deficient pups may be partially explained by the known increase in hepatic copper uptake by amino acids (31). The liver would then have an increased pool of copper for the synthesis of ceruloplasmin, i.e. DAO. Also the faster growth of the saline injected rats would deplete liver copper-stores more rapidly. It would, thus, appear that rather than remove copper from the animal, phenylalanine facilitates its retention, at least under conditions in which the copper supply is limiting, Figure 4 and Table 4. It is perhaps not surpising that phenylalanine elevation did not reduce the copper pool because it would be a weak bivalent ligand in competition with albumin. Indeed, of those amino acids studied, the tridendate ligands histidine and cystine (cysteine) would seem to be the primary copper chelators (5, 10). This also holds true for a computed distribution (32) of amino acid-Cu2+ complexes 23 and for the ligands associated with another trace metal, zinc (33). These experiments did suggest that the hyperphenylalaninemic or copper-deficient offspring did not develop normally. Further investigation into these separate model systems will be the subject of subsequent reports, and will utilize the enzyme marker CNP which was shown to be highly reduced in three genetic mutations, with faulty myelination. ACKNOWLEDGEMENTS The author wishes to thank Professor R. W. Luecke for his generosity in the use of the atomic absorption facility. The technical aid of Mr. Douglas Clark is gratefully acknowledged in the experiment in which phenylalanine and p-chlorophenylalanine were administered. 10. 11. 12. 13. 14I 15. 16. 17. 24 REFERENCES HOLMBERG, C.G. and LAURELL, C.B. (1948) Acta Chem. Scand. 3, 550-556. MARCEAU, N. and ASPIN, N. (1973) Biochim. Biophys. 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LOWRY, 0.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J. (1951) J. Biol. Chem. 193, 265-275. DEUTSCH, H.F., KASPER, C.B. and WALSH, D.A. (1962) Arch. Biochem. Biophys. 33, 132-135. HOFSTEE, B.H.J. (1956) Enzymologia 11, 273-278. KURIHARA, T. and TSUKADA, Y. (1967) J. Neurochem. 14, 1167-1174. MC COSKER, P.J. (1961) Nature 190, 887-889. HARRIS, D.I.M. and SASS-KORTSAK, A. (1967) J. Clin. Invest. 49, 659-667. HALLMAN, P.S., PERRIN, D.D. and WATT, A.E. (1971) Biochem. J. 121, 549-555. GIROUX, E.L. and HENKIN, R.I. (1972) Biochim. Biophys. Acta 273 , 64-72 0 PROHASKA, J.R. and WELLS, W.W. (1974) Proc. Soc. Exp. Biol. Med., in press. 26 TABLE 1 EFFECT OF DIETARY PHENYLALANINE ON FECAL AND PLASMA COPPER AND DIAMINE OXIDASEa Exp. No. Group Dietary Cu Fecal Cu Plasma Cu Diamine Oxidase (ug/g) (us/day) (us/m1) (un1t8/1) I Control 16.0 142 319(4) 1.17:0.02b 26.4:1.0b L-Phe 12.3 136 126(4) 0.87:0.04b 19.1:2.3b -Cu+L-Phe 3.2 12.311.6(4) 0.42:0.16 2.8(1) II Control 11.0 ---- 0.78:0.08(4) 18.115.2(4) L-Phe 12.3 ---- 0.88:0.01 16.7:2.3 IIIc Control 10.5 ---- 0.9610.05 18.8:2.7 aValues are means 1 SD for 3 samples unless indicated (N). Male weanling rats were fed a synthetic diet containing the reported copper levels and 5% L-Phe where indicated for either 4 weeks (exp. I), 2 weeks (II) or 5 weeks (III). Copper was determined by atomic absorption and plasma diamine oxidase by its ability to oxidize 1,4-diaminobenzene as described in MATERIALS AND METHODS. bMeans were significantly different (P<0.01). cMeans are from 3 pools of 3 animals each. 27 TABLE 2 PLASMA DIAMINE OXIDASE LEVELS OF PHENYLKETONURIC PATIENTSa Cases N Plasma L-Pheb Plasma Diamine OxidaseC (mM) (units/1) Adult Controls 5 ---- 41.5110.2 Adult PKU 7 1.81:0.20 38.11 6.6 Neonatal Control 1 —--- 22 Neonatal PKU 6 1.65:0.73 16.6: 4.6 aValues are means 1 SD as determined on plasma and serum received from 3 sources (see Materials). Neonatal refers to cases in which the patients were less than 1 month old. Activities were measured as described in METHODS. b Data was calculated from information supplied with the specimens. cThe normal range for adults based on a similar method is 35-65 (ref. 25). 28 TABLE 3 EFFECT OF INJECTIONS OF L-PHENYLALANINE ON PLASMA AND BRAIN COPPER LEVELS OF NEONATAL RATSa Property Group Age (Days) 7 ll 14 Control 1.10 0.92 0.62 Plasma Copper L—Phe 1.44 1.07 0.85 (pg/ml) -Cu --- 0.75 0.34 -Cu+L-Phe 0.79 0.84 0.78 - - - - - -Con;rol- - - 7:42 - - 8.94- - I0.0- Brain Copper L-Phe 7.21 9.16 10.6 (ug/g dry wt) -Cu 3.45 3.05 2.69 -Cu+L-Phe 3.73 3.79 4.09b aValues are means of duplicate determinations on pools of 4 animals each. Copper was determined by atomic absorption spectroscopy as described in METHODS. Dams were fed either adequate copper diet (control, 11 ppm) or low copper diet (-Cu, 0.44 ppm) and the offspring were injected daily with either L-phenylalanine (L-Phe) or an equivalent volume of saline beginning at 3 days of age. bOnly 1 brain was used. 29 TABLE 4 EFFECT OF INJECTIONS OF L-PHENYLALANINE AND DL-p-CHLOROPHENYLALANINE ON CYTOCHROME OXIDASE AND COPPER LEVELS OF NEONATAL RAT BRAINa Property Group Age (Days) 10 14 20 Controlb 14.3 10.22 15.3 11.29 30.7 13.61 Cytochrome L-Pheb 13.8 11.56 14.1 11.31 29.7 11.25 Oxidase (units/g) -Cu 9.9710.99 7.9110.49 9.4412.13 -Cu+L-Phe 12.1 10.73c 13.2 13.13c 19.4 13.06d Control 1.5610.24 1.7210.22 1.9710.34 Copper L-Phe 1.6610.22 I.8210.09 2.0210.35 (ug/ 8 wet wt) -Cu 1.2010.18 0.6010.30 0.1410.06 -Cu+L-Phe 1.1610.35 0.9610.20 0.7710.11d aValues are means 1 SD for four samples. Dams were fed either normal copper diets (control, 10 ppm) or low copper diets (-Cu, 0.53 ppm) and the offspring received twice daily injections of either saline or L-Phe DL-p-Clphe (L-Phe) from 6 days of age. b Data taken from (34) (see chapt. II). cMeans are significantly different than -Cu group P<0.05. dMeans are significantly different than -Cu group P<0.01. 30 TABLE 5 BRAIN COPPER LEVELS, CYTOCHROME OXIDASE AND DIAMINE OXIDASE ACTIVITIES OF MYELIN DEFICIENT MICEa Species Age Sex Brain Cu Cytochrome Oxidase Diamine Oxidase (Days) (pg/g wet wt) (units/mg protein) (units/l) CS7BL/6J Adult F 4.4610.44(3) 0.311:0.034 ---- Jimpy - — - 14:15 - M - 2.56:0:21 - - 0:303I0.028 - I0.4-10.73(2) Littermates 14-15 M 2.2710.26(3) 0.309:0.04l(3) 9.5(1) MyelIn Syn. Def.— 18:22 - M - 2.I410.07 - -0.34110:111(5) _ 9:95:I.36(3) Littermates 18-22 M 1.99:0.10(2) 0.272:0.044(5) 8.84:1.05 OuakIng - - — 64:75 - F - 3.60:0:16(3) - 0:43810.1I8(3) I1.4—10.57(2) Littermates 64-75 F 3.44:0.21 0.402:0.097 10.5 11.31(3) aValues are means 1 SD for four animals unless indicated (N). Mice were of the strain C57BL/6J and of the indicated mutations. Copper was determined by atomic absorption on wet-ashed brain samples. Cytochrome oxidase was measured on whole brains spectrophotometrically as described in METHODS. Diamine oxidase was measured on plasma as described in METHODS. 31 TABLE 6 ACTIVITY OF 2',3'—CYCLIC AMP 3'-PHOSPHOHYDROLASE IN MYELIN DEFICIENT MICEa Species Age Activity % Control (Days) (units/mg protein) C57BL/6J Adult 2.6610.05(4) -- OuakIng - - - - 64-75- - - 076110.08(3)b- - - - 26- Control 64-75 2.30:0.29(4) Myelin Syn. Def.- - 18-22- - - 073010.07(4)b- - - - 12- Control 18-22 2.52:0.72(4) Jimp; - - - - 14-15- - - 072210.00(3)b- - - — 22- Control 14-15 0.9910.39(3) aValues are means 1 SD for the indicated number of whole brains (N). A unit was defined as l umole/min and was determined on 10% (w/v) homogenates as described under METHODS. bMeans are significantly different than littermate controls P<0.01. 32 Figure 1A. Formation of product with increasing plasma content, diamine oxidase activity. Varying amounts of rat plasma were incubated with 4 mM 1,4-diaminobenzene for 30 min as described in METHODS. Figure 1B. Formation of product with increasing time, diamine oxidase activity. Reactions were carried out with 4 mM 1,4- diaminobenzene using 10 ug of human ceruloplasmin in a 1 m1 final volume as described in METHODS. 33 1 #:‘Q Plasma U") 1 25 50 Time (min) Assn/30mm. 34 l l J 0. 1 a s 1,4-Dummoaeuzeue (mill Figure 2. Velocity of the diamine oxidase reaction with increasing substrate. Reactions were carried out with 10 ug of human ceruloplasmin as described in METHODS. 35 N 9 III 9 Diamine Oxidase(units/I) l 1 l 0 6 12 DIETARY Cu(PPm) Figure 3. Relationship between dietary copper level and rat plasma diamine oxidase activity. Points are means of triplicate determinations from pools of at least 3 male rats fed synthetic diets of varying copper content (0), in some cases supplemented with 5% L-phenylalanine (0). Details of diet composition and enzyme assay can be found under METHODS. 36 .somo mamsaom q mo mHoom Scum «Human co mammmm ounceamfiuu mo momma osu mum muofioa wouuoam .mmomHmz nova: vooom on one amafiwmu GOHuOOnoH vow mudwmooum zmmmm onu mo mHHmuwo .AOIIIQV mafiamm no AOIIIOV maaomamamnmsmouoaula vow mcwamamahamsa sues kafimw mowau Amy Ho “Anillov oceans Ho Aalllov wofiomamahomsm sues Afiuum ommefixo oaHEMHQ .e muowfim 37 l l 00 v (l/suun) asspgxo anguwm 10 20 0 AGE (Days) 10 G CHAPTER II EFFECT OF PHENYLALANINE AND p-CHLOROPHENYLALANINE ADMINISTRATION ON THE DEVELOPMENT OF RAT BRAIN 2',3'-CYCLIC NUCLEOTIDE 3'-PHOSPHOHYDROLASE 38 39 INTRODUCTION Attempts to explain the mental defect associated with the untreated phenylketonuric patient have encompassed a wide range of proposals, but as yet, a clear explanation has failed to emerge. Alvord o£_a1, (1) first suggested that the neuropathology of phenylketonuria (PKU) was characterized by an arrest of myelination in young patients; chemical evidenCe (2,3) indicating lower concentrations of cholesterol, cerebrosides, and proteolipid protein supported this view. However, other theories have Been suggested involving demyelination in the adult or absence of pathological changes of myelin (for a review, see (4)). Prior work on the effects of experimental PKU on the developing rat Brain have emphasized the reduction in myelin lipids (5-8). This report deals with_the development of the myelin associated protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.16b) and of the ‘mitochondrial proteins, hexokinase (EC 2.7.1.1) and cytochrome c oxidase (EC 1.9.3.1) under conditions employing injection of both phenylalanine and p-chlorophenylalanine (9), since neuronal protein sythesis is disrupted by this treatment (10). MATERIALS AND METHODS Pregnant rats were obtained from the Holtzman Co. (Madison, WI) and were fed either a synthetic control diet described previously (11) or‘a commercial pellet diet purchased from Allied Mills, Inc. (Chicago, IL). DL-p-Chlorophenylalanine, L-phenylalanine, and L-tyrosine were the products of Sigma Chemical Co. (St. Louis, MO). l-Nitroso-Z—naphthol was oBtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Treatment was induced in rat pups by ac injection of L-phenylalanine 40 (30 mg/ml, pH 7.4) at a dose of 1 mg/g body weight (12). The injection solution also contained DL-p-chlorophenylalanine (3 mg/ml) so that a dose of 0.1 mg/g body weight was achieved. Pups receiving 33 ul/g body weight of 0.2 M NaCl, pH 7.4, served as injection controls. In injection experiment I, dams were fed the synthetic diet and pups were injected twice daily from age 6-20 days. In injection experiments II and III, dams were fed the pellet diet and pups were injected once daily between ages 6-20 (exp. II) and ages 3-13 (exp. III). These pups were weaned at 21 days of age and were fed the pellet diet until 40 days of age. In all injection experiments, pups were randomized at 1 day of age and distributed at 8 per litter. PUps were killed either by exsanguination (experiments I and II) or by decapitation (experiment III). In experiment I, plasma tyrosine was fluorometrically determined by the method of Ambrose'gs4a1, (13). .Brain 2',3'-cyclic nucleotide 3'-phospbohydrolase (CNP) (14) and cytochrome c oxidase (11) were measured on tissue stored for various periods at -903 CNP activity was stable to storage; however, loss of cytochrome c oxidase activity on storage is sufficient to permit only relative comparisons between experiments. For injection experiment III, cerebellar -heXORinase was’measured on crude homogenates of fresh tissue according to the procedure of Knull 41141, (15). Details of these procedures including statisticaIJmethods are reported elsewhere (11). Enzyme activities were defined on a unit/g fresh weight tissue basis with one unit defined as one umole of product formed/min. 41 RESULTS During injection experiment I, measured tyrosine ratios indicated an initial rise in plasma tyrosine of the treated pups, which was followed by a drop to levels found in pups injected with saline (Table I). Pups which received no injections and whose dams were on a commercial diet had plasma tyrosine levels equivalent to the saline injected pups (Table I). Attempts to quantify plasma phenylalanine levels by the fluorometric method of McCaman and Robins (16) were unsuccessful due to interference by DL-p-chlorphenylalanine, which when analyzed under optimal conditions for L-phenylalanine, resulted in an increased relative fluorescence. Twice daily injections in experiment I were accompanied by high mortality and marked differences in growth of whole body and brain (Fig. 1). In comparison (Table II), although pups from experiment II, in which injections were halved, demonstrated differences in body and brain weights even after 20 days of recovery, absolute differences between groups were less than in experiment I. When pups were allowed to recover after day 13, experiment III, only cerebellar weights were different at 40 days of age. Differences between the groups were also less reduced than in experiment I. In experiment (I), the concentration of whole brain 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), an enzyme enriched in CNS myelin (17) was much lower in animals receiving the L-phenylalanine DL-p-chloro- phenylalanine; mean differences were highly significant on days l4, 16, 18, and 20 (Fig. 2A). In experiment II in which injections were given once daily, whole brain CNP concentration was reduced at day 20 but 42 equivalent at day 40 following recovery (Table III). At this age, however, CNP content per brain was significantly higher in the saline injected rats (682 i 32.4 units versus 596 i 49.8 units (a = 0.05)). For injection experiment III in which treatment began earlier, day 3 versus day 6, but was terminated earlier, day 13 versus day 20, CNP concentration.in the cerebellum.and in the whole brain less the cerebellum demonstrated a different pattern (Table III). At 7 days of age, there was a significant decrease in beth cerebellar and whole brain (less cerebellum) CNP concentrations. At 20 days of age, a week after treatment, onlvahole brain (less cerebellum) CNP concentration was higher in the 3311?? inJSCtion Pups; at 40 days Of age. no differences were measurable in either region. Like CNP, both hexokinase and cytochrome c oxidase increased with increasing age (Fig. 2B and Table IV). However, there were no consistent differences in concentration of cytochrome c oxidase between saline injected and Lrphenylalanine DL—p-chlorophenylalanine injected pups for either experiment I or III. Cerebellar hexokinase also failed to show a difference between groups in injection experiment III. DISCUSSION Experimental animal models of PKU in which administration of excess phenylalanine was used have been criticized (9) for failing to duplicate the high ratio of phenylalanine/tyrosine found in PKU patients. These authors have developed a model in which p-chloro- phenylalanine, an in vivo inhibitor of rat liver phenylalanine hydroxylase (18), is administered in conjunction with phenylalanine. This 43 approximates the plasma phenylalanine/tyrosine ratios found in PKU (19). Since injection of either phenylalanine or tyrosine into neonatal rats produced equivalent effects on reduced body weight, brain weight, total brain lipid, and incorporation of 35S-sulfate into brain (6), the model system of Andersen and Guroff (9) employing both phenylalanine and p-chlorophenylalanine was used in these experiments. The absence of elevated plasma tyrosine levels during the last week of injections in experiment I would imply reduction in phenylalanine hydroxylase activity. The injection of p-chlorophenylalanine alone causes no changes in serum tyrosine levels (20). The chemical composition of isolated myelin from experimental PKU rat brain was reported to be normal although less myelin was present (8). These same conclusions were reached when comparing myelin from 4 PKU and 5 normal human brains (21). However, other evidence suggests that the myelin lipid from experimental PKU rat brain appears to be different, i.e., there is a reduction in unsaturated and long chain fatty acids (22). Evidence of abnormal myelin based on whole brain or white matter specimens from PKU patients also includes reductions in unsaturated and long chain fatty acids (23) and changes in proteolipid fractions (24). The concomittant increase in 2',3'-cyclic nucleotide 3'-phospho- hydrolase concentration in rat whole brains with the period of myelination (Fig 2A) agrees with the work of Olafson.o£_a1, (25). The decrease in enzyme content in treated pup brain from experiment I suggests a deficit in myelin protein concentration. Others have also suggested a decrease 44 in myelin levels, in studies in which pups were injected with L-phenyl- alanine alone, based on reduced cholesterol and galactolipid (5), sulfatide (6), or proteolipid (7). Furthermore, Clarke and Lowden (5) found lower concentrations of cholesterol and galactolipid from brains of rats 40 days of age. These animals had been injected twice daily between ages 2 to 20 days before recovery until age 40 days. In the present study, experiment II, when pups injected between ages 6 to 20 days were allowed to recover, although body and brain weight differences persisted, the concentration of the myelin protein CNP showed no differences at 40 days. However, the amount of CNP per brain was higher in the saline injected rats. When recovery was begun earlier, experiment III, body and brain weight difrerences were not Observed at 40 days of age. The degree and duration of the initial insult seem to dictate whether recovery will occur. Fish and Winick (26) report that DNA synthesis continues until 17 days of age in various regions of rat brain; thus, potential for recovery would be reduced if the abuse were carried beyond this period. Geison and waisman (27) found little change in lipid distribution in rats that were fed excess phenylalanine beginning at 3 weeks of age. Recently, Adelman o£_§1, (28) have reported that injections of phenylalanine to neonatal rats resulted in lesions to both cerebellar Purkinje and granule cells. Although we found no decreases in cerebellar cytochrome c oxidase or hexokinase two mitochondrial proteins presumably enriched in neuronal cells (29), neuronal cell dysfunction may exist and contribute to the reduced myelin level. The molecular events giving rise to the neuropathology of PKU, hypomyelination and demyelination, remain an area for further inquiry. 45 SUMMARY Subcutaneous injections of L-phenylalanine and DL-p-chloro- phenylalanine in developing rats resulted in reduced body weight, brain weight, and concentration of 2',3'-cyclic nucleotide 3'-phos- phohydrolase -— a myelin enriched protein. These reductions were less marked when injections were reduced by half; when pups were allowed to recover after age 13 days, no differences were observed at 40 days of age. Brain hexokinase and cytochrome c oxidase develop- ment was not affected by the injection treatments. 46 REFERENCES l. Alvord, E.G., Stevenson, L.D., Vogel, F.S., and Engle, R.L., J. Neuropath. Exp. Neurol. 3, 298 (1950). 2. Crome, L., Tymms, V., and WOolf, L.I., J. Neurol. Neurosurg. Psychiat. 33, 143 (1962). 3. Prensky, A.L., Carr, 8., and Moser, H.W., Arch. Neurol. 13, 552 (1968). 4. Knox, W.E., in "The Metabolic Basis of Inherited Disease" (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, eds.), 3rd edition, pp. 266-295. McGraw-Hill, NEW'YOIR (1972). 5. Clarke, J.T.R., and Lowden, J.A., Can. J. Biochem. 41, 291 (1969). 6. Chase, H.P., and O'Brien, D., Pediat. Res. 4, 95 (1970). 7. Prensky, A.L., Fishman, M.A., and Daftari, B., Brain Res. 33, 181 (1971). 8. Shah, S.N., Peterson, N.A., and McKean, C.M., J. Neurochem. 13, 479 (1972). 9. Andersen, A.E., and Guroff, G., Proc. Nat. Acad. Sci. 33, 863 (1972). 10. Copenhaver, J.H., Vacanti, J.P., and Carver, M.J., J. Neurochem. 31, 273 (1973). ll. Prohaska, J.R., and Wells, W.W., J. Neurochem., 33, 91 (1974). 12. Lowden, J.A., and La Ramee, M.A., Can. J. Biochem. 41, 883 (1969). 13. Ambrose, J.A., Sullivan, P., Ingerson, A., and Brown, R.L., Clin. Chem. 13, 611 (1969). 14. Prohaska, J.R., Clark, D.A., and Wells, W.W., Anal. Biochem. _5_6_, 275 (1973). 47 15. Knull, H.R., Taylor, W.F., and Wells, W.W., J. Biol. Chem. 343, 5414 (1973). 16. McCaman, M.W., and Robins, E., J. Lab. Clin. Med. 33, 885 (1962). 17. Kurihara, T., and Tsukada, Y., J. Neurochem. 14, 1167 (1967). 18. Koe, B.K., and Weissman, A., J. Pharmac. Exp. Ther. 134, 499 (1966). 19. Lipton, M.A., Gordon, R., Guroff, G., and Udenfriend, S., Science 133, 248 (1967). 20. Hole, K., Dev. PyschObiol. 3, 157 (1972). 21. Shah, S.N., Peterson, N.A., and McKean, C.M., J. Neurochem. 13, 2369 (1972). 22. Johnson, R.C., and Shah, S.N., J. Neurochem. 31, 1225 (1973). 23. Gerstl, B., Malamud, N., Eng, L.F., and Hayman, R.B., Neurology 11, 51 (1967). 24. Menkes, J.H., Neurology 13, 1003 (1968). 25. Olafson, R.W., Drummond, G.I., and Lee, J.F., Can. J. Biochem. 41, 961 (1969). 26. Fish, 1., and Winick, M., Pediat. Res. 3, 407 (1969). 27. Geison, R.L., and Waisman, H.A., J. Neurochem. 11, 469 (1970). 28. Adelman, L.S., Mann, J.D., Caley, D.W., and Bass, N.H., J. Neuropath. Exp. Neurol. 33, 380 (1973). 29. Seller, N., in "Handbook of Neurochemistry" (A. Lajtha, ed.), Vol. 1, p. 328. Plenum Press, New York (1969). 48 use use: mass smog: use mcowuummcw o: cm>_m moon on sweet mass _ocecoo 3.96 .323 E81955“, 23. () .mmsa e swam” so mo mecca :3 mcowpm:_51mumn mpmoepazu mo mmsfim> ocemomxu some so nmceacuomo mom: moemems mm.o .. no «P., mm.o om No.0 Fm.o on mo._ mm.o o_ mo._ me._ e_ .. Pm.e m_ .. mm.m on .1 mm._ m u- o._ o wee—mm m:e_mm mmxmov e_ocucou mceceFm—xcmcmmuo_cuum mcwcm_m~»cmem mmq e.wcecmFm_xcm;qoco_cuuauno use m:_:e_m_xcm:mne Co consumncH mcwzo—POm mesa mcrmm_m>mo cw monumm m:_mocxe «amend .H mamqe .po.o .mo.o a an umummu can: mpogucou mew—mm scum acmgmmmwu mcomzu a an cmummu awn: upogucou m:_pmm sog$ ucmgmmmwu mcmmzn .HAp=~u_$_=mpm »_;m_;v .o.o .Aucmu,*_=m,mv mo.oH a we m->m_ an pmuu-u.uo__uu-ozu .mucuuaum new ovum; mucuwg~>-m us» we man an umgmanu mgm: mama: .ucmsumogu uaoguwz ao—m>mu o» uwzo—po mgm: awn» we?» su_gz mewm .AHHH .axmv mom mo mxmu mp-m Lo AHH .quv can yo mxau omum :wwzumn x_pmu um umuuonc_ mgw: mpmsw=< .mpmmn unmwmz um: a co ago muzm_o3 mamm_u mmFoswcm v Lo» .o.m.H memos mg” umumwp mmzpm>c n¢.m~H _o~ m._mwvm~ mo.qu mm._ mo.QH~m.F m.mmfl om. ~.mmu ¢N_ Q¢ om._mfl mm_ o.omummp o__.qw m... mo.qum¢.P um.m.fl.~.nm ¢.¢.va.mm ON om.~.H¢._¢ ~.m.unm omo.qu .m.o No.QH~N.o om._.u.~.¢_ ¢N.o.u m.a_ N HHH -- -- oko.qu Km._ mo.qumm._ em.o&H m__ m.e.u _m. ow -- -- cop.QH mo._ mo.qumm._ um.¢.u om F.m.u we om -- -- ammo.qu~mm.o No.QHw_._ a~.m.u. mm ~.m.H mm mp HH mcnboua «naugu‘ mcwrmm mca—oua mzqu4 «cwwmm m;a_uua usa-4 wc_me Amxmov .oz AmEM uzmwmz gmppmnmgmu ldmv ugm_m2 cwmgm “my uzmwm: anon mm< acmswgoaxm umcvcopoFAcmnaogopsu -a-4o m:_=a_a_»=mgm-4 $o =o_pumn=H mcwzo_Pom mugm_mz g~__mgmgou van .=*mgm m_o;= .auom .HH m4m3 :o «5.5:. -_»gueaoco_gu-a-ao can o=v=._..»:aga-4 co aeo.uuun=~ co uuuccm .>~ m4m + .ucmgmwmpu xpucauwmwcmvm no: mcmms on mgmmog m: mummy-p vm_+ou-ozp m.ucmu=um mg» uca.owumg euee_ee>-e axe ee mempe an mfixeeUEEPeaem »_xaexv _o.o .Aeemuxeeeueev mo.oH e *6 exp-mp N e. c..-e568 pee; ecemz . .ugmwm: up: so woman vougoaog «so mos—m> «sum-p .mpoewco e ea .o.m.H mcams an» acmmogamg nmummp manpo> 2. 5.: H 9:: 3 H :8 , 2 ~85 H :2 ~85 H 3; 8. 3: H ~.8 mg n. 0.8. 8 8. 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NN 6 2. «an H v.3 «6 .+. fig 2 a; H 3; Rod .+. a; 8. 3.x u EN 2; H 0.3 2 .e o.- + e.mx ~.e_ + m.ee we _~_.o + osm.o cho.o + no.- me .~.m + m.o~ ~m.m + m.m~ PP - - ¢~.o + x.om - - moo.o.u m_e.o - - ~m.o.u o.u_ o -- - -- me ~ spo.o.u.mom.o moo.o.n Nwm.o - .m.~ +m.m N - w - - - -M ~_o.o.n .-.o - -- - m- «mucuupw Lunaou so; pogucoEII. «cocoa-u guqaou zed pogucou «cacao-m Lmaaou :oA Fogucou Ammwov .-xemem “may pea..= Lm_peeeeeo -vea.m “my agave: e-mLm -eea.m . “av use-6: seem < h<¢ uz~m64w>ma mzh zu zhzomw zaagmmmxwu az< .z~<¢m m4°zz .>oom 20a: puma mummou 3o; < no humumm11.p m4m uamwuwwov Monaco can mwmusmmoa mums some mcfimun oaons N unmoa um mo mHooa q .AN.m1V m%mw um mesa Houucoo onu pom .woaaamm Hmovfi>fiwaa know do zaoo Imouuooam cowuauomnm uaaoum hp wouammoa mmB uoamou van vamMIums mums mac-nommmamx mmmamxmumum .Amm .NN .ea .HH .ev meme emumommen one be .mHHwnwuoo mocHB mcwmun umu mHoca nelllov unmfiofimmw Monaco cam AOIIIOV Houuaou mafiaoao>mv ca mammfiu :mmuw a“ menace mo coaumuuaoucooll.~ .UHm 81 ] 2.0 - l n. 0. (anssub/brr) HdeOO l “'2 0 IS 20 24 28 l2 AGE (Days) 82 FIG. 3.-—The postnatal development of cerebellar cytochrome c oxidase in control (O-——|) and copper deficient (O---O) rats. At the indicated ages, four pups from each group were decapitated, cerebella separated, homogenized, and after storage overnight (-20°C), activity was measured on detergent treated homogenates by following the decrease in reduced cytochrome c at 550 nm as detailed under METHODS. Values shown are of four cerebella i S.D. for fresh tissue. 83 _ _ _ _ O 8 O 6 33......- o\m=e2 33.x O 4. o o m20¢100k>o O 2 IS 20 24 28 I2 AGE (Days) 84 FIG. 4.——The postnatal development of cerebellar superoxide dismutase in control (0——-0) and copper deficient (O---O) rats. At specified times, four pups were killed by decapitation, cerebella separated, homogenized, and centrifuged (40,000 x g, 15 min.). Activity was measured in the supernatant spectrophotometrically by its ability to inhibit the xanthine oxidase linked reduction of ferricytochrome c as described under METHODS. Values are means of four cerebella i S.D. based on fresh tissue weight. 85 _ s o. 5 o. 3 3 2 2 mexzeme: {:22 33:23 mexommaam I2 IS 20 24 28 AGE (Days) 8 86 .uzmfio3 mammwu smoum co comma .n.m H mafimun know mo mcmoa wan ohm monam> .ocfiusamaaaouoc1Au:fiv :ufia use vofiuumo mums mmfiuo>ooou maaamm stva>wvaH .maomemz nova: vmafimuow mm hHHmofiuuosouosHm venomous can vfiom ofiauow locouoom nuHB vmuomnuxm mmB ocfiunaocfiawuoz .mafiu mo mnuwcofl waahum> How Aueomlv monoum mam moo cHHom do sououm who? maOfiuommwaon umHHopoumom man can wmumufiamoow muoz asouw Sumo mo mesa uaom .mowm woumofiwnfi mnu u< .mHHonmuoo msafia mcfimpn umu oHon3 AOIIIOV uaofioamov Monaco cam Aolllov Houucou wawaoao>ov ca ocaunaoafimouoc mo aowumuucooaoUII.m .on 87 '_L 1 1 ° 0. o F) 01 .; (9118311. 6/ 9010111") amquaNIdauoN IS 20 24 28 I2 AGE (DOVS) CHAPTER IV COPPER DEFICIENCY IN THE DEVELOPING RAT BRAIN: EVIDENCE FOR ABNORMAL MITOCHONDRIA 88 89 ABSTRACT Copper deficiency was produced in developing rats by feeding a low copper diet to rats during gestation and lactation and providing the offspring the same diet. The progeny developed similar to those of an earlier model based on preconception depletion followed by marginal supplementation during gestation. Copper levels were greatly reduced in the brain, iron levels were slightly depressed, and no differences in zinc content were found. Electron microsc0pic examination of brain tissue revealed the presence of enlarged mitochondria from copper-deficient animals. Isolated mitochondria from copper—deficient rats showed a 30% reduction in the rate of both succinate and glutamate oxidation, and for glutamate, the respiratory control ratio (RCR) was decreased by 60%. Difference spectra displayed a four-fold reduction in cytochrome a + a3 and slight increases in cytochrome b, c1, and c. Enzyme analysis of isolated mitochondria revealed a five-fold decrease in cytochrome oxidase, slight increases in succinic dehydrogenase and fumarase, and small decreases in hexokinase and monoamine oxidase. No difference in peroxidation of brain lipids was evident. Determination of metabolites from fast frozen tissue suggested that the copper-deficient brain was in a more reduced state based on a doubling of both the lactate/pyruvate and a-glycerol-P/dihydroxyacetone-P ratios. Creatine-P, ATP, and ADP levels were not different. 90 INTRODUCTION Copper deficiency in the neonate is accompanied by neurological manifestations and can occur in domestic animals such as sheep, can be produced in the laboratory using guinea pigs or rats, and may occur in human infants due to either a nutritional oversight or the genetic disorder Menkes' steely-hair disease. Biochemically, the deficient rat brain accumulates only one-fifth the normal copper content (1,2) and contains lower concentrations of myelin (2, 3) and cytochrome c oxidase than controls (2). The classical work of Cohen and Elvehjem (4), 40 years ago, demonstrated that copper was essential for both oxidase activity and the A component of cytochrome. This observation was further verified by the work of Gallagher g£_§l, (5, 6) who included studies on isolated mitochondria. They found no differences in oxidative phosphorylation of liver mitochondria nor in the ability of brain mitochondria to oxidize pyruvate. thlrab and Jacobs (7) concluded that interchain electron transport played a role in copper-deficient liver mitochondria. Goodman g£_gl, (8) found normal mitochondrial function in both liver and heart mitochondria from copper-deficient rats although morphological changes were seen. Since abnormal mitochondria from the cerebellar Purkinje cells of a young boy with Menkes' disease had also been reported (9) as well as abnormal liver mitochondrial function (10), brain mitochondria from copper-deficient rats were studied as an extension of the model for Menkes' steely-hair disease (2). Measurement of energy related compounds was also performed to correlate morphology with metabolic status. 91 MATERIALS AND METHODS Pyridine and adenine nucleotides, cytochrome c (type III), 2,6-dichlorophenolindophenol, 2-amino-2dmethyl-l-propanol, 2—thiobarbituric acid, and glutamic-pyruvic transaminase (EC 2.6.1.2) were obtained from Sigma. Other enzymes for metabolite determinations were purchased from Boehringer-Mannheim. Malondialdehyde bis-(dimethyl acetal) and 2,6-di-tert-butyl-4-methy1-phenol were products of Aldrich. Kynuramine was obtained from Regis and Tween-80 from Nutritional Biochemicals. Sperm positive female rats were purchased from Holtzman. Animal care and diets. The composition of the basal diet was described previously (2) and contained either 10 ug Cu/g in the control diet or 0.3 ug Cu/g in the low copper diet as determined by atomic absorption spectroscopy (Perkin-Elmer model 303). The experimental design was modified from a preconception capper depletion period of 9 weeks followed by marginal supplementation during gestation (2) to one in which females, sperm positive for 1 day, were fed the low copper diet throughout gestation. Other details of randomization, tissue sampling, and statistical treatments by the Eytest were described previously (2). Enzyme assays. Most enzyme determinations were performed at 25°C in a Gilford 2400-3 spectrophotometer. Cytochrome c oxidase (EC 1.9.3.1) was determined on crude cerebella or on isolated mitochondria after treatment with 1% (v/v) Tween-80 as described previously (2). Superoxide dismutase (EC 1.15.1.1) was measured by the method of 92 McCord and Fridovich (11) as detailed before (2). The myelin associated protein 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.16b) was assayed on crude cerebella as reported previously (12, 2). Succinic dehydrogenase (EC 1.3.99.1) was measured on cerebellar homogenates or mitochondrial preparations after treatment with 1% (v/v) Tween-80. The method of Earl and Korner was employed (13) initiating the reaction with potassium succinate. A value of e = 2.19 x 101+ (14) for dichlorophenolindophenol (DCPIP) was used for calculation of units. Mitochondrial preparations were assayed for hexokinase (EC 2.7.1.1) (15) after treatment with Triton X-100 0.5% (v/v). Monoamine oxidase (EC 1.4.3.4) was assayed after treatment with Triton X-100 by following the loss of kynuramine (e = 3.94 x 103) at 360 nm, 30°C, pH 8.0, by the method of Weissbach.g§;§l, (16). Fumarase (EC 4.2.1.2) was assayed by the method of Racker (17) after treatment of the mitochondria with Triton X-100, 0.5% (v/v) using a value of e = 2.22 x 103 for sodium fumarate at 240 nm for calculations. All enzyme units (except superoxide dismutase) were expressed as umoles/min and were either based on a g of fresh tissue or a mg of mitochondrial protein. Electron microscopy. Thin slices of the right cerebral cortex were fixed for 2-1/2 hrs in 5% (v/v) glutaraldehyde in 0.1 M potassium phosphate (pH 7.2), postfixed 1-1/2 hrs in buffered 1% 030“ and dehydrated in graded aqueous ethanol prior to embedding in Epon 812. Thin sections were stained with uranyl acetate and lead citrate and examined with a Phillips EM 300 microscope. 93 Mitochondrial studies. Mitochondria were isolated in 0.3 M mannitol, 0.1 mM EDTA (pH 7.4) from pools of 2 brains, excluding the medulla, by combining F1 and F2 fractions as detailed by Ozawa SEJEL° (18). Oxidative phosphorylation was carried out by the methods of Milstein §£_al. (19) except substrate concentrations were 4 mM. Oxygen consumption was monitored using a Yellow Springs Instrument MOdel 53 oxygen monitor at 25°C. Calculations of respiratory control ratios (RCR) and ADP/0 ratios were made according to the methods of Estabrook (20). The estimation of the contents of cytochromes a + a3, b, c], and c were made following the calculations of Williams (21). The isolated mitochondria were treated with deoxycholate and the oxidized (K3Fe(CN)6) versus reduced (sodium ascorbate + NaZSZOu) difference spectrum of ca, 1 mg of protein in 0.25 ml (1 cm cuvettes) was taken with a Cary Model 15 spectrophotometer between 500-630 nm. Metabolite and metal analysis. Tissue used for energy metabolite determination was obtained from animals which were immersed into liquid N2 and stored at -85°C until the brains were chipped out, powdered, and extracted as described previously (22). The neutralized extracts were analyzed spectrophotometrically for creatine phosphate, ATP, ADP, AMP, and lactate, and fluorometrically for pyruvate, a—glycerol phosphate, and dihydroxyacetone phosphate by the methods of Lowry and Passonneau (23). The fluorometric measurement of brain norepinephrine was as described previously (2). COpper analysis as well as zinc and iron determinations were performed using single element cathodes and atomic absorption spectroscopy (2). Protein was measured by the procedure of Lowry g£_31. (24) using bovine serum albumin as standard. 94 Lipid peroxidations. Evidence for peroxidation was taken as the formation of malondialdehyde as judged by reaction with 2-thiobarbituric acid according to the method of Pederson (25). To a 0.5 m1 aliquot is added 1.0 ml containing 0.375% 2-thiobarbituric acid, 15% trichloroacetic acid, both (w/v), in 0.25 M HCl to which had been added just prior to use 0.01 vol of 2,6—ditertbuty1-4- methylphenol in CZHSOH to prevent further peroxidation (26). The mixture is heated (95°C) for 15 min, cooled, centrifuged at 1000 x g, and the absorbance determined at 535 nm. Using 9.6 nmoles of malondialdehyde bis-(dimethylacetal) as standard through the above procedure, 1.0 A is obtained. Whole brains were homogenized in 4 vols of 0.15 M KCl, 0.05 M Tris (pH 7.4), in a Potter-Elvehjem homogenizer. Aliquots were used to study autooxidation of lipids in a shaking water bath (37°C) buffered with 0.1 M Tris (pH 7.4) or metal catalyzed peroxidation at 23°C containing 2 mM ADP and 0.12 mM Fe(NHu)2(Sou)2 (pH 7.5). RESULTS Induced copper deficiency. Two equivalent experiments were designed, each consisting of twenty sperm positive females divided equally and fed either control or copper deficient diets from the second day of the presumed gestation. Offspring were randomized within each group within 24 hrs of birth and thereafter at weekly intervals to maintain a constant litter size. Evidence that copper depletion had occurred during gestation was suggested by the fact that the copper content (Hg CU/g body wt) of day old pups was 2.95 i 0.06 95 in controls and 0.60 i 0.25 in the deficient group (mean 3 SD of 4 pools of 2 pups each). Offspring from the first experiment were used to compare with a previous model (2) in which copper deficiency prior to the lactation period was induced by a preconception depletion period and marginal gestational copper supplementation. The data in Table I demonstrate that the present model system produced equivalent results with the former (2). A five-fold reduction in copper concentration was accompanied by reductions in the copper proteins cytochrome c oxidase and superoxide dismutase, by reductions in norepinephrine and in the myelin protein 2',3'-cyclic nucleotide 3'-phosphohydrolase; body, brain, and cerebellar weights were also reduced as reported previously (2). Several 28-day old rats of each group were fed a commercial pellet diet (15 ug Cu/g) for four weeks and the results of this repletion period are shown in Table I. Body weight of the deficient group (data not shown) did not reach that of the controls. Mitochondrial morphology. When the copper deficient animals were 30 days old, severe signs of deficiency were apparent (anorexia and hyperventilation). Electron microscopic examination of 2 brains from each group revealed the presence of enlarged mitochondria in the cerebral cortex (Fig. 1). Although mitochondria of normal appearance were also present, about half appeared to be abnormal in shape and less electron dense. There appeared to be fewer mitochondria in the observed fields from the copper deficient preparations, but no quantitative estimate was made; no other cytological differences were noted. 96 Mitochondrial function. Because of the abnormal appearance of the mitochondria from copper deficient brain, Fig. 1, and the alteration in two mitochondrial proteins (an increase in succinic dehydrogenase and a decrease in cytochrome c oxidase, Table I), offspring from the second experiment were used to investigate mitochondria from developing rat brain. Mitochondrial oxygen consumption was measured at 2 ages using both succinate and glutamate as substrates, Table II. Statistical differences were evaluated although only 2 preparations were used for each mean; differences between the two groups are present at both ages. Mitochondria from copper-deficient rats are reduced (30%) in their ability to oxidize both substrates; however, they appear to be more normal in their ability to use succinate as opposed to glutamate (Table II) when comparing respiratory control ratios. Each preparation for the oxygen uptake studies was run separately and completed within 1-1/2 hrs from the time of decapitation. Figure 2 shows a representative mitochondrial difference spectrum from a control and a copper-deficient brain. The striking reduction of the cytochrome a + a3 peak at 605 nm is quantitatively shown in Table III. At both ages, the cytochrome a + a3 content was significantly reduced (72 and 80%) while cytochromes b, c1, and c showed a slight increase. Table III also shows the specific activities of five enzymes associated with brain mitochondria: hexokinase and monoamine oxidase —- the outer membrane, succinic dehydrogenase and cytochrome c oxidase —- the inner membrane, fumarase —- the matrix. A significant decrease 97 in cytochrome c oxidase is apparent at both ages with only slight Changes in the other enzymes. Both hexokinase and monoamine oxidase may be slightly reduced and both succinic dehydrogenase and fumarase slightly increased. These enzyme changes were also quantitatively seen in crude homogenates (data not presented), suggesting that the mitochondria were not drastically altered during isolation. The data for 30-day old animals were calculated on a per cent control basis and are shown in Fig. 3. There is good agreement in the reduction in cytochrome oxidase measured enzymatically or calculated from difference spectra (20 and 25% of control). Energy metabolites. Since it is essential to the measurement of high energy phosphates that tissue is of a size that can be frozen rapidly, the copper-deficient rat presented a problem because it is smaller than an age matched control. Weight matched animals were, therefore, included as a third control group for this study, Table IV. Metabolite levels are reported in Table IV as well as lactate/pyruvate and a-glycerol phosphate/dihydroxyacetone phosphate ratios to evaluate the cytoplasmic NAD+/NADH levels. No differences in the levels of ATP, ADP, or creatine-P were found between any of the groups. AMP was statistically different in the weight matched control group from either age matched control or copper-deficient groups. Examination of the glycolytic intermediates, however, revealed significant differences between the copper-deficient group and both control groups. Large increases in lactate and a-glycerol phosphate accompanied by small increases in pyruvate and dihydroxyacetone-phosphate resulted in a 2- to 3-fold increase in the 98 lactate/pyruvate and a-glycerol-P/dihydroxyacetone-P ratios when compared with control groups, Table IV. When compared with the weight matched controls, the larger controls (age matched) had moderate increases in lactate, o-glycerol-P and dihydroxyacetone-P reflected in small increases in lactate/pyruvate and a-glycerol-P/dihydroxyacetone-P. Trace element levels. Brains from five-week old rats were grossly divided into three brain regions: the cerebellum, the cerebral cortex, and the remainder (brainstem + cerebral peduncle); and the tissues were rinsed with thrice distilled water to remove surface blood. Since blood Fe was not determined nor the contamination by blood in each region determined, values for Fe are not corrected and, thus, the differences in brain Fe seen in Table V between control and copper-deficient rats are only suggestive. However, the large reduction in brain copper in all three regions, Table V, agrees with that of the whole brain less cerebella, Table I. No differences in brain zinc due to copper deficiency were noted. The reduction in brain copper was equivalent in all three regions; however, 2 of the 3 samples from the cerebral cortex were much lower than the mean reflects. Lipid peroxidation. Four-week old rats were used to investigate the possibility that the enlarged mitochondria seen at 30 days of age, Fig. 1, could be due, in part, to lipid peroxidative swelling. Table VI shows the production of malondialdehyde in both groups with and without ADP-Fe; no differences were observed. The inclusion of the antioxidant 2,6-ditert-butyl-4dmethylphenol (0.9 mM) during incubation completely prevented malondialdehyde formation. Zero time values for 99 malondialdehyde were low, variable, and no differences existed between groups and were thus treated as tissue blanks rather than as true endogenous malondialdehyde levels. Addition of 5 uM Cu2+ to the incubation, an amount equivalent to the total copper in the control contributed by the tissue, produced no dramatic difference in malondialdehyde production, Table VI. DISCUSSION Tissue response to low copper levels results in variable changes to the morphology of the mitochondria. Figure 1 clearly shows that brain mitochondria from copper-deficient rats are enlarged and abnormal in shape. Others have shown that the number of mitochondria increase in cardiac tissue without an appreciable size change (8, 27), while the liver may produce some mitochondria ten times the normal size (27). This same response is seen in mouse tissue after feeding cuprizone (biscyclohexanone oxaldihydrazone), a copper chelator (28). Enlarged mitochondria were also reported in copper-deficient rat erythroblasts (27). Aberrant mitochondrial function during copper deficiency is also variable. Oxygen consumption in the control mitochondria, Table II, agrees with that reported by others (18, 19) for uptake ADP/O ratios and respiratory control ratios (RCR). However, the mitochondria from Cu deficient brains did show a definite reduction in glutamate oxidation, although no differences in tissue glutamate dehydrogenase activity were seen (unpublished data). The copper deficient liver mitochondria studied by thlrab and Jacobs (7) displayed small 100 differences when succinate was used, in agreement with our results, but no differences with glutamate. Goodman.g£_al, (8) found no differences with either substrate for either liver or heart mitochondria between controls and copper-deficient rats. Mice fed cuprizone had normal heart mitochondrial function, but the greatly enlarged liver mitochondria were markedly reduced in all aspects of oxidative phosphorylation (28). The moderate reduction in both succinate and glutamate oxidation (30%) with a much larger reduction in cytochrome oxidase (80%) supports the observations of others (5, 7, 8). The mitochondrial difference spectrum, Fig. 2, from capper deficient rat brain is in agreement with those reported for copper-deficient rat liver (29) and heart (8, 30) and also with that reported from the brain of a boy who died of Menkes' steely-hair disease (31). All contain a striking reduction in the cytochrome a + a3 peak (605 nm) while the remaining spectrum is quite normal. Chemical removal of copper from purified cytochrome oxidase does not appreciably alter its spectrum (32) suggesting absence of the apoenzyme or heme A in copper-deficient mitochondria since the spectrum is changed. The difference spectrum from iron deficient mitochondria is normal (8), although the mitochondria are enlarged. The concentrations of cytochromes from the control rat brain agree reasonably well with those reported by Williams (33) using a similar method. thlrab and Jacobs (29) reported no difference in cytochrome b or c1 levels in copper deficient liver mitochondria but a decrease in cytochrome c. Gubler et a1. (34) first reported an 101 increase in cytochrome c from copper deficient swine heart and this was confirmed by Dallman (30), who also reported that the absorption peak between 550-650 nm was equal to or slightly greater than that from control heart mitochondria. The increase in cytochromes b, c1, and c reported in Table III tend to support this observation for brain mitochondria. A moderate reduction in cytochrome oxidase activity of copper deficient rat brain was reported (4, 5) when deficiency was begun after lactation. A much more striking depletion (to 20—25% of control) is found in the developing brain ((2) and Tables I and III). The slight increase in succinic dehydrogenase activity, Tables I and III, has been detected histochemically from brains of copper-deficient rats (35) and in mouse brain from.animals fed cuprizone (36). Mbnoamine oxidase (MAO), Table III, was found not to change in the copper deficient mitochondria. Chou §£_§l, reported no change in MAO of copper deficient chicken brain (37) while O'Dell 25:21: (38) reported values slightly higher in copper deficient newborn lambs. Hexokinase and fumarase levels in copper-deficient brain have not been reported before. The major difference between the copper deficient and control mitochondrion is an increased size and a reduced amount of cytochrome a + a3 (oxidase). The results of Fig. 3 and Table III taken as a whole (for individual differences in mitochondrial components are slight) would seem to suggest that the mitochondria from copper-deficient brains have an increase in inner membrane components and perhaps matrix and a decrease in the outer membrane. 102 This could arise from individual mitochondria increasing in volume disproportionate to the surface area, by assuming a spherical symmetry (Fig. 1), or from the fusion of mitochondria of different sizes (radii). Either case could explain the enlarged mitochondria seen in Fig. 1 and the change in surface area/volume implied from Fig. 3 and Table III. There does appear to be some correlation between the abnormal functioning of the copper deficient mitochondria in vitro and the energy metabolism of the brain in vivo. The increased lactate/pyruvate and a-glycerol-P/dihydroxyacetone-P ratios in the copper-deficient brain probably reflect lower NAD+INADH ratios, Table IV. However, no differences in creatine-P, ATP, or ADP between groups suggest that high energy phosphate metabolism was grossly normal. Values for ATP and creatine-P are lower than those reported (39) when more rapid freezing of the tissue is possible. Since creatine-P has not dropped below 2 mM (Table IV), the ATP concentration seems to be lower than that predicted from prior studies (40). Perhaps the elevated lactate and a-glycerol phosphate levels reflect a reduced capacity of copper-deficient mitochondria to oxidase NADH as suggested by the impaired glutamate utilization. The concentrations of copper, zinc, and iron in control brain, Table V, agree with those of Kofod (41) for rats of similar age. Feeding excess zinc (4000 ppm) to lactating rats resulted in lower brain copper and iron, but no change in brain zinc in the offspring (42). This_agrees with the pattern seen in Table V when low copper diets were used to induce Cu deficiency. The reduction in brain Fe 103 may have some relevance to the observation that copper deficient brains have less norepinephrine, Table I (2, 38), and dopamine (38). The rate limiting enzyme in their synthesis, tyrosine hydroxylase (EC 1.14.3.2), requires Fe2+ for activity (43). The enzyme catabolizing them, monoamine oxidase, is unchanged. An alternate suggestion for lower norepinephrine based on a reduction of dopamine-B-hydroxylase (EC 1.14.2.1), a copper enzyme, was suggested (2). These studies are currently under investigation, and conclusions are still speculative. In the brains of Menkes' steely-hair disease patients, there was a reduction in unsaturated fatty acids reported (31, 44), and a defect in antioxidant metabolism was suggested. Contrary results have been recently reported (45). However, an autofluorescent pigment, suggestive of lipid peroxidation, has been reported by two authors (44, 46) in the cerebellar Purkinje cells. Since superoxide dismutase activity is lower in copper deficient rat brains, Table I and (2), and has been shown to protect against lipid peroxidation in special systems (47-50), experiments on lipid peroxidation from copper deficient brains were performed, Table VI. These experiments do not appear to provide an explanation for the "aging" effect reported (5) to occur in copper deficient rat liver mitochondria or for the enlarged mitochondria from copper deficient brain (Fig. 1). From the preliminary results in Table I, it appears as though the recovery of brain cytochrome oxidase in copper repleted rats is a slow process. Dallman (30) first studied this process of recovery and concluded that it was governed by the synthesis of new mitochondrial material. Since the turnover of brain mitochondria is 104 on the order of three weeks (51), full recovery would be expected to be slow. 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Chem. 2423 4584-4586. 109 TABLE I RAT BRAIN DEVELOPMENT-RECOVERY FROM PERINATAL COPPER DEFICIENCYa A9e Property Group 14 days 21 days 56 days Brain Weight (g) + Cu 1.24 1.0.03 1.43 :_0.10 1.88 :_ 0.09 - Cu 1.09 1 0.12 1.23 1 0.04” 1.69 1 0.080 Cerebellum Weight (mg) + Cu 143 :_4.7 186 118.5 281 :_19.4 - Cu 120 117.06 159 1 7.00 238 1 20.30 Copper (119/9) + Cu 1.58 1 0.05 2.02 1 0.04 2.32 1 0.20 '- Cu 0.54 1 0.08” 0.43 1 0.08” 1.08 + 0.02” Norepinephrine + Cu 1.44 1 0.22 2.13 1 0.07 2.92 1 0.19 (nmoles/9) - Cu 0.95 1 0.121” 1.53 1 0.11" 3.40 1 0.290 2',3'-cAMP-3'-Phosph0- + Cu 128 1 5.0 264 1 5.0 374 116.5 ”“1359 (“ms/9) - Cu 115 1 7.4" 228 115.9” 367 1 7.8 Succinic Dehydrogenase + Cu 0.588:_0.040 0.901:_0.034 0.965:_ 0.097 (umts/g) - Cu 0.5621 0.057 0.9991 0.0450 1.01 1 0.083 Cytochrome c Oxidase + Cu 28.0 :_2.4 58.9 1 4.0 42.1 1. 4.17 (units/9) - Cu 13.0 11.8” 14.3 1 M" 33.2 1 2.890 Superoxide Dismutgse + Cu 3.37 :_0.24 2.93 1_0.13 2.04 :_ 0.10 (umts/g) x 10 - Cu 3.34 1 0.17 2.06 1 0.0817 1.96 1 0.22 “Values are means :_SD of four animals expressed on fresh weight basis. Copper deficiency was induced during gestation and until 28 days postnatally prior to four weeks of recovery on a commercial pellet diet (56 days of age). Enzyme activities were determined on cerebella, the remainder of the brain was bisected and used for copper and norepinephrine determinations. b Means significantly different p < 0.01. cMeans significantly different p < 0.05. 110 TABLE II COPPER DEFICIENCY-BRAIN MITOCHONDRIAL OXYGEN CONSUMPTIONa Age Substrate Property 23 Days 29 Days + Cu - Cu + Cu — Cu State 3 118 14.9 91.6 18.8 115 115.6 78.6 10.0 . State 4 41.1 16.1 37.4 14.9 31.8 1 6.7 29.5 10.4 5mm“ 1102b 2.9410.56 2.4910.57 3.741 1.28 2.6610.04 ADP/0 1.6810.01 1.5510.13 1.691 0.06 1.6210.04 State 3 51.9 10.3 34.4 10.3** 42.8 1 3.6 30.3 11.6* State 4 13.9 10.7 21.0 12.0* 11.6 1 0.2 22.6 _+_l.8* “Mama“ RCRb 3.7310.24 l.64_+_0.18* 3.671 0.23 l.34_+_0.04** ADP/Dc 2.951007 --- 2.441 0.26 --- “Values are means 1.50 of 2 pools of 2 brains each from either control (+ Cu) or c0pper deficient (- Cu) offspring. Oxygen consumption in the presence (state 3) or absence (state 4) of ADP is expressed as natoms oxygen/min/mg protein. The subStrates employed were 4 mM potassium salts. Details of the procedure are described in Methods. Means were significantly different [*p < 0.05, *f p < 0.01]. bRespiratory control ratio. cLow RCR prevented calculation of ADP/0 ratios for - Cu preparations. 111 TABLE III COPPER DEFICIENCY-BRAIN MITOCHONDRIAL ENZYMES AND CYTOCHROMESa Age Component 21 Days 30 Days + Cu - Cu 1 Cu - Cu Cytochromes (nmoles/mg protein) 0 0.16810.003 0.17210.002 0.25810.035 0.28910.023 01 0.12710.001 0.13210.020 0.11210.010 0.13110.008b c 0.17410.002 0.18410.007 0.23010.020 0.23410.025 a + a3 0.29210.008 0.11210.110 0.43610.044 0.10710.0050 Activities (units/mg protein) Cytochrome oxidase 2.12 10.05 0.60 10.130 2.26 10.23 0.46 10.01c Succinic dehydrogenase x 102 2.58 10.4 2.88 10.2 2.52 10.18 2.83 10.14 Fumarase x 10 8.32 10.04 8.83 10.07b 9.41 11.02 9.48 10.14 Hexokinase x 10 2.55 10.07 2.34 10.14 2.90 10.39 2.65 10.11 Monoamine oxidase x 103 --- --- 4.02 10.18 3.73 10.27 “Values represent means 1.50 for pools of 2 brains each with 2 pools at 21 days and 4 pools at 30 days. determined from the difference spectra (reduced versus oxidized). activity was determined on the same mitochondrial preparations. defined as l umole/min. b Means significantly different p < 0.05. cMeans significantly different p < 0.01. Mitochondria were isolated and the cytochrome content Enzyme One unit is 112 TABLE IV COPPER DEFICIENCY-BRAIN ENERGY METABOLITESa Dietary Groupb Property Control - I Control - II -C0pper - III ' Age (Days) 14 20 20 Body Weight (g) 26.0 15.8 (4) 45.5 10.8 25.8 16.2 Metabolite (nmoles/g tissue) Creatine Phosphate 2.72 10.38 2.23 10.54 2.13 10.57 ATP 1.57 10.59 1.65 10.43 1.58 10.32 ADP 0.40 (10.08 0.52 10.28 ‘0.44 10.08 AMP 0.18 10.01*»+ 0.30 10.06 0.29 10.07 Lactate 1.20 10.46*.++ 2.05 10.21“r 6.07 10.52 Pyruvate 0.101 10.012H 0.110 10.012** 0.167 10.020 a-Glycerol-P 0.046 10.008**-++ 0.110 10,006+ 0.191 10.037 Dihydroxyacetone-P 0.011610.0018** 0.020210.0021 (3) 0.018810.0066 Ratio Lactate/Pyruvate 12.2 16.0ii 19.0 13.7** 35.9 15.5 a-Glycerol-P/Dihydroxy- 4.0 10.6*-+ 5.6 10.6+ (3) 10.8 13.1 acetone-P aAnimals were immersed in liquid N2 and were stored at -85°C until brains were powdered over solid C02. Metabolites were determined in HClO4 extracts by either spectrophotometric 0r fluorometric procedures described in Methods. Values are means 1_SD for 3 animals in control-I 0r 4 animals for control-II 0r -copper-III unless indicated (N). bMean comparisons of the weight matched control-I group were different from the age matched control-II group (*p < 0.05, ** p < 0.01). Mean comparisons of either control group I or II were different from -c0pper-III group (1 p < 0.05, 11 p < 0.01), 113 TABLE V COPPER DEFICIENCY-BRAIN COPPER, ZINC, AND IRON LEVELSa Brain Region Element G (us/9) roup . Cerebral Cortex Cerebellum Brain Stem-Peduncle Copper + Cu 1.88 1_0.13 2.10 1 0.24 1.95 1_0.ll - Cu 0.36 1 0.1219 0.49 1 0.0517 0.45 1 0.0017 Zinc +00 15.7 11.05 15.3 10.91 12.3 10.73 -00 13.8 10.60 15.4 10.20 13.1 11.48 Iron +00 12.0 10.94 17.8 12.51 17.1 10.58 - Cu 8.4 10.59b 20.9 12.10 11.0 12.21b aValues are means 1_SD of 3 pools of 35-day old rat brains (control rats (+ Cu) 2/p001 or copper deficient rats (- Cu) 4/pool). Tissue was wet-ashed and the metal content determined by atomic absorption spectroscopy as described in Methods. Tissue values are uncorrected for blood contamination and expressed on a fresh weight basis. bMeans significantly different p < 0.01. 114 TABLE VI COPPER DEFICIENCY-BRAIN LIPID PEROXIDATIONa Additions ($128) (A51g515) Control Copper Def1c1ent ’ (nmoles malondialdehyde/g tissue) None 40 6 64.5 1_22.8 58.5 1_26.l None 80 6 170 1_55 140 1_38 002+ (5 0M) 40 3 --- 68.4 1_15.0 002+ (5 0M) 80 3 --- 177 1.40 ADP-Feb 5 4 134 1_6 136 1_5 ADP-Feb 10 4 202 + 8 210 1_12 “Values are means 1_SD for the indicated number of 28-day old rats. Incubations were carried out in 1 ml containing 0.1 M Tris (pH 7.4), 80 mg brain tissue in 0.4 m1 of 0.15 M KCl, 0.05 M Tris (pH 7.4) at 37°C with shaking. At the indicated times, aliquots were removed and the content of malondialdehyde was determined colorimetrically as described in Methods. b Fe2 +, 0.12 mM) and the reaction run at room temperature (ca. 23°C). Incubations were carried out as above except that ADP-Fe was included (ADP, 2 mM, 115 .GOHumHomnm oHEoum hp vocaahmumw so msu mam wmummwfiv mm3 :Hmun ecu mo Gowuuom mafiafimaou mnu “whoauoz Ga wonwuomow mm voafimum wow voxam mmB mofiHm mammfiu SASu < .E: H N on cm mafia mamom osu moowumofimwcwma 08mm msu um mum mouona HH< .wa om.o Avv Ho m: 05.3 on mamawomam ucoaofimowiuomaoo coca mauvaonooufia HmHHmBm some m>m£ Anv w: 0H.N was Amv w\oo w: mm.H mafiafimuooo Gamma Houucoo .mamaaam ucmfiowmov1uomdoo mom Houuaoo Boum wouuoo Hounouoo umu wHo howiom mo.m£amuwouowa couuooam .H .wfim 116 117 .muuooam o>onm onu ammuno on com: mums oeaao> Hmofim Ha mm.o :H A11111v dawns uoofiofimow Hoodoo Eoum we wm.a no A v Gamma Houuaoo Soum Gamuoum mo we H¢.H Honufim was mwozumz ca vmnfiuomov mm woumaomfi mama mauvoonooufiz .mumaozohxomv mo mocomoum mnu ca mauvaonooufia camps umu AQAZUmemMV wmufivaxo msmum> Aqommmmz + mumnuoommv voosvmu mo muuoomm mofimuommam .N .me 118 «ES Ihozm4m><3 com com _ _ \\\I // .\ \ I \l \ , x / 1 \ \ ’ \ / \\ I I‘ ’ \ I . \ /.\ , . , 1 // ‘1 ’ ~ , 1 No.0 u< d .\ 119 .HHH manme a“ venom on :mo mumw Hmsww>fiwcfi mom moosuoz a“ vasow on amo mmwavoooua onu mo mHHmumm .woumofiwafi mum mwumconoouaa panama mucmooaaoo Hmocfi>fiwaw mnu mo m:oaumoog .afiouona Hmauvaosooufia we won umwmoumxo mama no momma mm3 owmuamouom .mumw um“ Houuaoo mo uaoo Hon m mm mumu vHo mmwlom Bouw muomaoaaoo Hmanvaozuoufia gamma um“ udoaofimov Hoodoo .m .me 120 INNER MEMBRANE MATRIX 1 oselemng —————————————# osepgxo ow 011190;qu 8 0+1! 0 mo: uooifiq o 0141011490153 1:: 011101 1100149 q 011101 1; oouio oseu 0801 pAqoa olugoons hy———__ ___._ _______ 4.1. m I oseulxoxou z , o: < 11.1 o: 1— m a ' a E r osepgxo oquvouou O In - 2 l O 0 IO 1 OHINOD‘X. DISCUSSION 121 ". . .copper is not a star in the drama of the mind and it cannot provide us with the kind of insight that an Ibsen play provides into human nature. . ." (1). 2',3'-Cyclic Nucleotide 3'—Phosphohydrolase. Although the enzyme 2',3'-cyc1ic nucleotide 3'-phosphohydrolase (CNP) has been detected in nonmyelin associated membranes its usefulness as a CNS myelin marker should not be disregarded. The residual activities found in other membranes may be of embryonic importance but are of no quantitative significance. When myelin was isolated from whole rat brain by the method of Norton and Poduslo (2) the specific activity (umoles/min/mg) of CNP increased 7 fold to 27.3. This agrees well with that reported for human white matter—20.3 (3). A composite of the CNP activities of rat cerebella under various treatments is shown in Table 1. All treatments produced significant depressions in body and brain wt, however only copper deficiency reduced CNP concentration in the development models. Di Paolo g£_§1, (4) have recently shown that there was less myelin in the cerebella of newborn copper-deficient rats by histochemical methods. Table 1 also shows that when weanling rats are fed a low copper diet, CNP is not changed although brain copper is reduced by 40% (unpublished data). This is consonant with the suggestion that copper deprivation is most adverse during brain development. When the zinc deficient (5) or hyperphenylalaninemic (6) insults are more severe the CNP content is reduced accordingly. The cerebellar CNP from the hyperthyroid offspring is greater than the saline injected controls (P<0.05, 122 1-tailed Eftest) suggesting an acceleration of myelination (7) and another case for the usefulness of CNP. However, none of the experi- mental treatments produced the profound decreases seen in the myelin mutant mice and, thus, the consequences of this slight reduction in CNP observed in copper deficiency (8) remain open to speculation. Copper Deficienpy. The developing rat brain provides a useful model in which to study the adverse effects of copper deprivation on brain maturation. This lack of tissue copper may be of etiological significance in Menkes' steely-hair disease. At present, there is a controversy as to whether Menkes' disease is one characterized by hypomyelination (9) or demyelination (10). French g£_§1. (9) found a reduction in long chain unsaturated fatty acids while L0“.EEJEL' (10) reported no differences in fatty acid distribution of white matter lipids. The relatively few documented cases of this disorder prevent firm conclusions. It would seem.worth- while to isolate and chemically characterize the myelin because if synthesized incorrectly (dysmyelination) it could lead to demyelination. Another genetic disease, phenylketonuria, is characterized by hypo- myelination in the young followed by progressive demyelination (11). However, recent use of the hyperphenylalaninemic rat model found (12) that the myelin had a reduction in long chain fatty acids as well as a decrease in unsaturated fatty acids suggesting dysmyelination. It is not clear whether copper deficiency could also lead to dysmyelination. The mechanism for such a reduction in long chain and unsaturated fatty acids remains an enigma. Preliminary attempts to correlate the reduction of superoxide 123 dismutase (8) with an aberrant pattern of lipid peroxidation (i.e. a reduction in unsaturated fatty acids) have failed to provide an answer. Perhaps an anabolic approach based on the altered NAD+/NADH ratio may provide some insight. However, the failure to demonstrate a difference in energy metabolism in copper-deficient brain weakens this hypothesis. The most striking difference associated with the five-fold decrease in brain copper content was the large reduction in cytochrome oxidase (8). This decrease could be demonstrated both enzymatically and spectrally. However, this large decrease (80%) was accompanied by only a 30% reduction in the ability of the mitochondria to oxidize substrates (succinate and glutamate). Of several possibilities for this observation, three seem most likely: l) cytochrome oxidase is not rate limiting; 2) interchain electron transport contributes to the respiratory reaction of copper-deficient mitochondria (13); 3) there may be induction of an additional oxidase similar to that reported for copper-deficient yeast (14). The reduction in the spectral peak at 605 nm (cytochromes a + a3) is an unresolved observation. Current evidence would suggest that COpper is somehow involved in the synthesis of the protoporphyrin moiety of heme A and/or in its combination with the apo-oxidase. This supposition is based on the fact that the levels of the other cytochromes are normal, thus, suggesting no abnormality in the sequence porphyrin + heme + cytochrome. Also, in yeast, copper deficiency has no effect either qualitatively or quantitatively on synthesis of the protein subunits of cytochrome oxidase (14, 15). 124 Not all of the copper in brain is firmly attached to functional proteins (16). The loss of copper and the concomittant reduction in metalloenzyme activity varies between different proteins (8). This would suggest that the brain has several copper ligands of varying affinity for Cu. When weanling rats were fed a low copper diet for 6 weeks their plasma copper level fell to 14% of control however their cerebellar cytochrome c oxidase activity remained unchanged (108% of control). In a similar experiment of five weeks, cerebral cortex activity was also unchanged (112% of control) but brain copper concen- tration had fallen to 61% of control. Again this points out that the developing brain, which has not accumulated its full copper reserves, is more susceptible to metal deprivation be it dietary or genetically produced. "The outstanding problem in phenylketonuria is no longer the effect of the abnormal metabolism on the brain, but the nature of the alterations induced in the developing brain." (17). Hyperphenylalanine. The discovery by Johnson and Shah (12) that the myelin isolated from hyperphenylalaninemic rats had altered levels of long chain and unsaturated fatty acids suggests that the membrane may be unstable and could explain why demyelination occurs in older PKU patients. However, a concise metabolic explanation for these facts is not available despite a generous supply of published hypotheses. One widely accepted concept is that phenylalanine through its 125 inhibition of brain pyruvate kinase and phenylpyruvic acid by its inhibition of brain hexokinase (18) would decrease brain glycolysis and result in a decrease in energy production. I found that L-phenylalanine did inhibit rat brain pyruvate kinase (19) but 2 mM phenylpyruvate had no effect on either chick or rat brain hexokinase. Although Miller g£_§1, (20) were able to verify the inhibition of pyruvate kinase in an in vivo study in which fast frozen tissue was analyzed, they found no differences in ATP or creatine phosphate. The physiological consequences of the pyruvate kinase inhibition, therefore, remain uncertain. In addition to hexokinase, phenylpyruvate has been reported to inhibit at least seven other enzymes (21). The lowest K1 reported was for inhibition of fatty acid synthetase (0.25 mM) (21). These authors suggest that this inhibition could explain the defective myelination characteristic of PKU. There are several reasons why conclusions, like the preceding, must be delayed. Phenylpyruvate has never been detected in brain tissue of PKU patients or of experimental animals. In a study of 39 PKU patients (32), plasma phenylpyruvate averaged only 0.044 mM. Brain phenylpyruvate has been postulated to arise via transamination of phenylalanine with 2-oxoglutarate (23), it could then be enzymatically reduced to phenyllactate by lactic dehydrogenase. In a study in which rats were given intraperitoneal injections of phenylalanine the resultant level of brain phenyllactate, the product of phenylpyruvate reduction, was 2.6 uM (24). Using the kinetic constants (Km and Vmax) reported by Land and Clark (21) for fatty acid synthetase from l4-day—old rat brain and assuming that the 126 concentration of acetyl-CoA is equivalent to the K.In value, 7.6 uM, enzyme velocity was calculated for various concentrations of the competitive inhibitor, phenylpyruvate. Determination of this velocity for a value of phenylpyruvate equal to 25 uM, ten times the level of phenyllactate found in rat brain (24), results in a 5% reduction from that predicted without inhibitor present. The molecular mechanism that explains the hypomyelination in PKU is not available. Although the myelin protein 2',3'-cyclic nucleotide 3'-phosphohydrolase was reduced in rats treated with phenylalanine and p-chlorophenylalanine, two mitochondrial proteins, cytochrome c oxidase and hexokinase, were not affected (6) (see chapt. II). Recently Nordyke and Roach (25) have found that two other mitochondrial proteins, succinate dehydrogenase and glutamate dehydrogenase, were unaffected by this treatment. They also reported that treatment terminated at 21 days of age followed by 3 weeks of recovery still resulted in lower brain weights of experimental animals (26) in agreement with prior studies (6) (see chapt. II). The developing brain seems to be irreversibly damaged during the hyperphenylalaninemic state in agreement with most reported cases of untreated phenylketonuria. 10. 11. 12. 13. 14. 15. l6. 17. 127 REFERENCES SCHEINBERG, H. and STERNLIEB, I. (1967) in Molecular Basis of Some Aspects of Mental Activipy (WALAAS, 0., ed.) Vol. 2, p. 115. Academic Press, New York. NORTON, W.T. and PODUSLO, S.E. (1973) J. Neurochem. 21, 749-757. KURIHARA, T., KAWAKAWI, s., UEKI, K. and TAKAHASHI, Y. (1974) J. Neurochem. 21, 1143-1144. DI PAOLO, R.V., KANFER, J.N. and NEWBERNE, P.M. (1974) J. Neuropath. Exp. Neurol. 32, 226-236. PROHASKA, J.R., LUECKE, R.W. and JASINSKI, R. (1974), J. Nutr. in press. PROHASKA, J.R. and WELLS, W.W. (1974) Proc. Soc. Exp. Biol. Med. in press. GRAVE. G.D., SATTERTHWAITE, 3., KENNEDY, C. and SOKOLOFF, L. (1973) J. Neurochem. g0, 495-501. PROHASKA, J.R. and WELLS, W.W. (1974) J. Neurochem. gg, 91-98. FRENCH, J.H., SHERARD, E.S., LUBELL, H., BROTZ, M. and MOORE, C.L. (1972) Arch. Neurol. 363 229-244. LOU, H.C., HOLMER, G.K., RESKE-NIELSEN, E. and VAGN-HANSEN, P. (1974) J. Neurochem. 2;, 377-381. CROME, L., TYMMS, V. and WOOLF, L.I. (1962) J. Neurol. Neurosurg. Psychiat.‘g§, 143-148. JOHNSON, R.C. and SHAH, S.N. (1973) J. Neurochem. 21, 1225-1240. WOHLRAB, H. and JACOBS, E.E. (1967) Biochem. Biophys. Res. Commun. 28, 998-1002. SCHWAB, A.J. (1973) FEBS Letters éé, 63-66. SCHWAB, A.J. (1974) in The Biogenesis of Mitochondria (KROON, A.M. and SACCONE, C., eds.) pp. 501-504. Academic Press, New York. PORTER, H. and AINSWORTH, S. (1958) Proc. Soc. Exp. Biol. Med. ‘28, 277—280. KNOX, W.E. (1972) in The Metabolic Basis of Inherited Disease (STANBURY, J.B., WYNGAARDEN, J.B. and FREDRICKSON, D.S., eds.) 3d edition, p. 290. McGraw-Hill, New York. 18. 19. 20. 21. 22. 23. 24. 25. 26. 128 WEBER, G. (1969) Proc. Natn. Acad. Sci. 0;, 1365-1369. GRANETT, S.E. and WELLS, W.W. (1972) J. Neurochem. 12, 1089-1098. MILLER, A.L., HAWKINS, R.A. and VEECH, R.L. (1973) Science 179, 904-906. LAND, J.M. and CLARK, J.B. (1973) Biochem. J. 134, 545-555. JERVIS, C.A. (1952) Proc. Soc. Exp. Biol. Med. 81, 715-720. FELLMAN, J.H., BUIST, N.R.M., KENNAWAY, N.G. and SWANSON, R.E. (1972) Clin. Chim. Acta 32, 243-246. EDWARDS, D.J. and BLAU, K. (1972) Biochem. J. 130, 495-503. NORDYKE, E.L. and ROACH, M.K. (1974) Res. Commun. Chem. Path. Pharmacol. 8, 397-400. NORDYKE, E.L. and ROACH, M.K. (1974) Brain Res. 61, 479-488. 129 TABLE 1 ACTIVITY OF 2',3'-CYLIC AMP 3'-PHOSPHOHYDROLASE IN DEVELOPING RAT CEREBELLA FOLLOWING EXPERIMENTAL TREATMENTSa Treatment Body Wt. Brain Wt. Cerebellar Activity (g) (g) (units/g wet wt.) Control 63.2: 4.0* 1.37:0.05* 224:10.5 Pair-Fed 31.4: 1.4* 1.25:0.02* 226: 6.6 Zinc Def. 27.9: 4.6 1.22:0.06 214:19.7 Control 62.2: 6.3* 1.43:0.10* 226:10.l* Copper Def.c 36.7:10.4 1.23:0.04 172: 5.9 Saline Inj. d 59.5: 4.4* 1.45:0.05* 246:14.2 L-Phe DL-p-Clphe Inj. 33.2: 8.5* 1.18:0.11* 234:15.4 Thyroxine Inj. 47.5: 5.4 1.23:0.06 262: 7.1 Control 350 :35.6 --- 402:13.1 Copper Def.e 323 : 7.5 --- 389: 7.3 8Values are means : SD for four animals age 21 days unless noted. Means were tested by Student's E-test (treatments were different from controls * P<0.01). Enzyme activity was determined on thawed 10% (w/v) homogenates after freezing overnight as described in APPENDIX. bData is taken from (5) in which lactational zinc deficiency was carried out and offspring from ad libitum fed (control) and pair-fed dams were compared with zinc deficient offspring. cData is taken from (8) in which gestational-lactational copper deficiency was induced and the offspring compared. dSaline and L-phe DL-p-Clphe injected offspring data is taken from (6). Concurrently additional pups were given 4 subcutaneous injections of 10 pg of thyroxine in saline on days 3, 6, 9 and 12 of age (7); saline injected rats served as controls. eData is from 9 week old male rats who had consumed a copper deficient diet (0.3 ppm) for 6 weeks. APPENDIX 130 ANALYTICAL BIOCHEMISTRY 56, 275-282 (1973) Improved Rapidity and Precision in the Determination of Brain 2’,3'-Cyclic Nucleotide 3’-Phosphohydroluse1 A rapid and precise method for the determination of brain 2’,3’-cyclic nucleotide 3’-phosphohydrolase (CNP) activity has been developed. Total brain homogenates were treated with deoxycholate, and CNP activity was measured as inorganic phosphate (phosphomolybdic acid, 410 nm) released from the product, 2’-AMP, by alkaline phosphatase. Measurements were carried out under optimal conditions of temperature (30°C) and pH (6.2) using the whole brain of the rat, chicken, and quaking mouse. The entire assay was applicable to multiple samples and could be completed in less than 1 hr. Since Drummond et al. (1) reported the presence of a diesterase which hydrolyzes 2’,3’-cyclic nucleotides in the central nervous system (CNS), many reports have appeared further characterizing this activity. The proposition that 2’,3’-cyclic nucleotide 3’-phosphohydrolase (CNP) (EC 3.1.4.1) is associated with the myelin sheath of the CNS was made by Kurihara and Tsukada (2) in 1967. Recent suggestions (3) and evidence (4, 5) that CNP may also be found in the oligiodendroglial cell plasma membrane strengthen the hypothesis for location of CNP in CNS myelin. The activity of CNP has been used to follow CNS myelination in the developing chick brain and spinal cord (6), as well as in the newborn rat brain (7). Because of its association with myelin, CNP has been used as a marker during isolation of CNS myelin or to estimate myelin contamination of other subcellular fractions (8—11). Another application of CNP levels has been the studies involving the neurological mutant mice, jimpy (jp/y) and quaking (qk/qk), both of which have subnormal myelin levels (12). A depression in CNP levels in the brains of jimpy mutants corresponding to reduced myelin levels was shown by several researchers (3, 13—15). Analogous studies on the quaking mutant have also been reported (7, 14). Recent uses of CNP measurement include a study of bovine peripheral nerve myelin (16), drug induced synthesis of CNP in the sciatic nerve of the chick embryo (17), spinal cord CNP in hypothyroid rats (18), and CNP in the chick optic tectum following deafl'erentation ( 19). ‘This work was supported by Grant AM 10209, U. 8. Public Health Service. Michigan Agricultural Experiment Station Journal Article No. 6260. 275 Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved. 131 276 SHORT COMMUNICATIONS This diversity of application led to a variety of analytical techniques which either lack sensitivity, are time-consuming, or both. Because of these technical difficulties, results are often conflicting or noncomparable. The optimization of CNP detection, both for precision and speed of analysis, is the subject of this report. MATERIALS Female quaking mice were purchased from The Jackson Laboratory, Bar Harbor, Maine, and albino rats from Holtzman Co., Madison, Wis- consin. Day-01d male Leghorn chicks were the generous gift of the McPherson Hatchery, Ionia, Michigan. 2’,3’-cAMP, 2’-AMP, and E. coli alkaline phosphatase type III-S were purchased from Sigma Chemical Co., St. Louis, Mo. Triton X-100 was the product of Rohm-Haas, Inc., Philadelphia, Pa. Other chemicals were analytical reagent grade and were used without further purification. Entire reactions were carried out in nitric acid-washed, 10 mm X 75 mm Pyrex test tubes. METHODS Preparation of tissue homogenates. Whole brains, including medulla oblongata to the level of the posterior cerebellum, were rapidly removed, weighed, and homogenized (20 full strokes) in 9 vol of 0.32 M sucrose in a Potter—Elvehjem homogenizer with Teflon pestle. The solubilization procedure was that of Kurihara et al. (13) and consisted of adding 0.1 ml of 0.2 M Tris—HCI, pH 7.5, to 0.2 ml of the 10% (w/v) homogenate, then adding 0.2 ml of a 1% (w/v) sodium deoxycholate solution. After 10 min at 0—4°C, thrice distilled water was added and homogenization was repeated, five strokes, so that protein was of an appropriate dilution (0a., 0.1 mg/ml). Final CN P assay conditions. Velocities were measured which were both linear with respect to time and protein concentration. In 0.2 ml final volume, 5—25 pg of protein were added to 7.5 mM 2’,3’-cAMP, 50 mM Tris—maleate buffer, pH 6.2. The substrate initiated reaction was carried out at 30°C for 10 min. A sample containing substrate and buffer without protein served as a reagent blank. The substrate level which was selected, 7.5 mM, was based on reported procedures ( 1, 2) and the high cost of 2',3’-cAMP. The reproducibility of 2’ ,3’-cAMP concentration was verified spectrophotometrically by the content of adenosine, pH 2.0, and 257 nm, and by estimation of total cyclic phosphate. The reaction was terminated by placing the 10 mm X 75 mm Pyrex tubes in a boiling water bath for 30 sec. The mixture was returned to the 30°C water bath where 0.1 ml of 0.3 M Tris—HCI containing 21 mM MgC12, pH 9.0, was added along with 60 pg (0.72 units) of E. coli alkaline phosphatase (EC 3.1.3.1), re- 132 SHORT COMMUNICATIONS 277 sulting in a final pH of 8.5. It was determined that after boiling and adjusting the pH, no detectable CNP activity remained. The alkaline phosphatase reaction was capable of completely hydrolyzing 1.5 nmoles of 2’—AMP in 20 min at 30°C. Chemical analyses. The inorganic phosphate which was liberated was conveniently determined in the same test tube directly as the phospho- molybdic acid complex (20) and the absorbance measured at 410 nm in a Gilford Model 2000 spectrophotometer. The micromethod of Berenblum and Chain (21) was employed as modified by Martin and Doty (22) using isobutanolzbenzene to extract the yellow chromophore. To the 0.3 ml reaction mixture, 1.2 ml of isobutanolzbenzene (1:1, v/v) and 1.2 ml of 1.5% (w/v) (NIL),Mo7O,,-4 H20 in 0.5}: H280, were added and the solution vigorously shaken (Vortex) for 20 see before centrifugation (Sorvall GLC-l) at 2200 rpm for 5 min. The yellow upper layer was read against isobutanol:benzene. Standard phosphate carried through this pro- cedure generates a linear relation, 0.745 nmoles/absorbance unit. Protein was measured by the method of Lowry et al. (23) using bovine serum albumin as a standard. One unit of enzyme activity is that amount which produces 1 ,dmole of 2’-AMP from 2’,3’-cAMP/min under the conditions described. Specific activity is expressed as units/mg protein. RESULTS Adult rat brain of either sex was used as the source of enzyme in studies attempting to optimize reaction conditions. Pretreatment of the homoge- r-“/ f A 3 r // o / 5 a /' g /, V .1" >- 2 ,, . 1.. > I. O < l .. L a; l A A A A O r 5 6 7 8 p H FIG. 1. Effect of pH on adult rat brain CNP activity. Assay conditions were as described under Methods. The buffer used was 50 mM Tris-maleate. 133 278 SHORT COMMUNICATIONS nate was the first point of study, and although absolute values varied because of animal age, tissue storage, and degree of sonication, the highest and most consistent values were always achieved when using the 1% deoxycholate treatment (13) followed in order of effectiveness by Triton X—100, sonication, and no pretreatment. In order to eliminate bufi'er effects (1) and to determine an accurate pH profile, one buffer, Tris-maleate, was employed over the entire pH range (Fig. 1). An optimum can be seen for the range 6.1—6.7. CNP dis- played a temperature optimum over the range 25—32°C (Fig. 2). Specific activity of GNP at 37°C was 90% of that at 30°C. The K," of 2’,3’-cAMP for crude brain homogenate protein was determined to be 6.0 mM from the slope of the Hofstee plot (24) (Fig. 3). Since the final form of the present CNP assay was related to the method of Kurihara et al. (2, 13), a direct comparison was made by us using both techniques on three adult rat brains. The mean specific activity and standard deviation obtained by our method was 3.52 t 0.193, while the paper chromatographic technique (2, 13) gave 3.29 i 0.126. In order to explore the usefulness of our method to that of published procedures, CNP was determined on the whole brains of adult rats (2 mo of age or older), l4-day old chicks maintained on a commercial mash diet for 2 wk, and 2-mo old female quaking mice with their littermate controls. Table 1 shows the results of our determinations along with re- ported or calculated data from other authors. Since the entire reaction sequence can be performed in the same test T T V T I I 3 { 3.0»- -4 . T: 3 >- y. > 2d" .4 C 0 ‘ L x U |.O>- "‘ .. l. I 1 1 1 i 1 1 ‘ 0 IO 20 30 40 60 TEMPERATURE PC) FIG. 2. Meet of temperature on adult rat brain CNP activity. Assay conditions were as described under Methods. 134 SHORT COMMUNICATIONS 279 '3 E 23» E 3 )- ’: / i\ U 2[ / ‘i \ O _1 , “£3 ./ \\_ I) ’/ Z> \\. ,’ .\ /‘ i ‘x P 073 " . ‘ / Vl‘ Y a— ___l ._ L _ A J 0 2 4 6 8 lO 2', 3'-cAMP(mM) Flo. 3. Effect of 2’,3’-cAMP concentration on the activity of adult rat brain CNP. The inset. shows the results plotted by the Hofstee (24) method. Assay conditions were as described under Methods. tube. our method provides a precise estimate Of CNP activity—«a coef- ficient of variation ts 'f-lOOl of 1.7% for six trials. DISCUSSION Table 2 is a composite of current techniques used for CNP measure- ment. The majority of methods separate 2’,3’-cAMP from 2’-AMP chro- matographically (2, 4, 5). This necessitates an increase in sampling error due to numerous transfers and elution; in addition. hours are required for the development of the chromatograms. Since total reaction time can he as low as 30 min (10 for the reaction and 20 for phosphate cleavage) and no transfer of riaction materials need take place. the speed of the present method can he appreciated when compared to the ascending paper chromatographic techniques more commonly used. ()lafson et al. (7) have developed an assay in which the use of alkaline phosphatase to liberate inorganic phosphate increases the speed of analysis. However. a transfer step after precipitation with TCA intro- duces sampling error. In addition. this method operates at pH 7.5. beyond the optimal range tsee Fig. 11. No detergent pretreatment of homogenates and a substrate concentration of 4 mM further reduce the sensitivity of CNP measurement. A comparison of quaking mice values (% control, Table II shows a lack of agreement with our method (53.8% vs 26.5% 1. Absolute values deviate even further. All current methods for measuring (‘NP Operate at fixed substrate. 135 SHORT COM MUNICATIONS 280 .A is 83 Dom; I an :39; wEen hots vow: 8853.080; 0:35. 0 $3823.. .5 838:: US$55 2: 8.. .5on O5 .3 She Eaves; H 85353. .589? 8a 32¢.» a . _ I a a. s . o assets: 23% Ex m. I an as assesses: :EE 2: E I. a: : .c a Z” we Siege. ens mm. :2 352: 258$ I 5 mod a an we assesses: ”be 8 3:st :2 o l c.. m . e 85825. ass E @320 ELSE 239m l A3 awed H 5; who "Ba—ocohxooc $98 3 :exeib cad min 952 :32 8553.: 3.250 N. Vino E .3 m. . 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EN. 53m 20;? _o :OmrEQEcO _ Egmfir 136 SHORT COMMUNICATIONS 281 TABLE 2 Comparison of Current Procedures for Measuring 2’,3'-Cyc1ic Nucleotide 3’-Phosphohydrolase Tissue 2’,3'- pretreatment‘ cAMP pH T 2’-AMP quantification Reference (mM) (°C) None 7 .5 7 .5 30 Paper electrophoresis, Am 1 None 4 .0 7.5 30 Alkaline phosphatase, Pr 7, 9, l5 Sonication 7 . 5 6.2 37 Paper chromatography, A.» 2, 6, 10, 18 Sonication‘ 7 . 5 6 , 2 37 Paper chromatography, Am 17 Sonication‘ 7 .5 6. 2 37 Paper chromatography, A.” 19 Triton X-100 7.5 6.2 37 Paper chromatography, Am 4, l6 Deoxycholate 7.5 6.2 37 Paper chromatography, Am 11, 13, 14 Deoxycholate‘ 7 .5 6 . 2 37 Paper chromatography, Am 3 Deoxycholate 7.5 6.2 37 Thin-layer chromatography, A.“ 5, ll Deoxycholate 4.0 7 .5 30 Alkaline phosphatase, Pt' 15 ° Unless otherwise stated, the method of sonication was 150 W for 10 min; deoxy- cholate or Triton X-100 pretreatment was 0.4% detergent, 40 mM Tris-HCI, 0°C for 10 min. b No power value listed, 10 min. ‘ 70 W for l min. ‘ 20—25°C for 5 min. ‘ Inorganic phosphate measured as reduced phosphomolybdic acid, Am. rather than saturating substrate. A K... of 6.0 mM agrees well with that of Zanetta et al. who reported a K.,. of 5 mM for various subcellular frac- tions of rat brain (5). Olafson et al. (7) have reported a value of 2 mM for bovine brain. As other workers have noted (1, 7), the high Km of CNP for 2’,3’-cAMP suggests that its activity toward this substrate may not be significant in vivo, and its physiological role remains unknown. From Table 1, it can be seen that our method agrees well with those which pretreat with deoxycholate, operate at pH 6.2, and use 7.5 mM 2’,3’-cAMP. The CNP values of quaking mutant (% control) in our method and that of Kurihara et al. (14) agree well with that reported by Druse and Hogan (25) for the isolated myelin of the quaking mutant (20—26% control). The difierence in specific activities may be due to an activation upon freezing and thawing the 10% homogenates.2 We have found that 30°C gave higher values than 37°C, but temperature was not as critical a parameter as pH (Figs. 1, 2). The precision gained by carry- ing out the reaction in a single vessel and the rapid handling of multiple samples makes this method particularly desirable for measuring CNP ’ Unpublished Observation. 137 282 SHORT COMMUNICATIONS as a myelin marker and for investigations Of myelination under normal and pathological Situations. REFERENCES . DRUMMOND, G. I., IYER, N. T., AND KEITH, J. (1962) J. Biol. Chem. 237, 3535. . KURIHARA, T., AND TSUKADA, Y. (1967) J. Neurochem. 14, 1167. . KURIIIARA, T., NUSSRAUM, J. L., AND MANDEL, P. (1971) Life Sci. 10 (part II), 421. . BANIK, N. L., AND DAVISON, A. N. (1969) Biochem. J. 115, 1051. . ZANE’I‘I‘A, J. P., BENDA, P., GOMsos, G., AND MORGAN, I. G. (1972) J. Neurochem. 19, 881. . KURIHARA, T., AND TSUKADA, Y. (1968) J. Neurochem. 15, 827. .OLAFSON, R. W., DRUMMOND, G. I., AND Lee, J. F. (1969). Can. J. Biochem. 47, 961. . NORTON, W. T. (1971) in Chemistry and Brain Development (R. Paoletti and A. N. Davison, eds.), p. 327. 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B., AND DOTY, D. M. (1949) Anal. Chem. 21, 965. . Lowsv, O. H., RosssROUcH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. . HOFSTEE, B. H. J. (1956) Enzymologia 17, 273. . DRUSE, M., AND HOGAN, E. (1972) Trans. Amer. Soc. Neurochem. 3, 73. JOSEPH R. PROHASKA DOUGLAS A. CLARK WILLIAM W. WELLS Department of Biochemistry Michigan State University East. Lansing, Michigan 48824 Received February 23, 1973; accepted July 3. 1973 lllllllllmll 0 3 9 2 1 I I I I I I I I si.