PERTURBATIONS OF ENERGY METABOLISM IN CHICK BRAIN INDUCED BY HYPERPHENYLALANEMIA AND GALACTOSEMIA Thesis for the Degree of Ph. D. MICHlGAN STATE UNIVERSITY SANDRA SPIEKER GRANETT 197 2 ................. IlllUlllllllIIIHIIIIHIIUUIIIHIIIHllllmllllllllllllllfl ? LIBRARY r 3 1293 10438 2316 ' Michigan State University This is to certify that the thesis entitled Perturbations of Energy Metabolism in Chick Brain Induced by Hyperphenylalanemia and Galactosemia presented by Sandra Elizabeth Granett has been accepted towards fulfillment , of the requirements for ‘ Ph . D . degree in Biochemis try ‘ //./fi 1‘ 1/4" 1/ 2: Major professor Date August ’4, 1972 0-7639 T 111! 800K BlNUERi’ WC. LIGRARY BINDERS --- -~'-‘i-. lumn;-- ABSTRACT PERTURBATIONS OF ENERGY METABOLISM IN CHICK BRAIN INDUCED BY HYPERPHENYLALANEMIA AND GALACTOSEMIA BY Sandra Spieker Granett Cerebral energy metabolism was investigated in the chick treated with L-phenylalanine or D-galactose. Hyperphenylalanemia was produced in the day-old chick by 3 hourly intraperitoneal injections with neutral- ized 0.18 M L-phenylalanine and maintained for 12 - 18 h; controls were injected similarly with 0.154 M saline. Shortly after the last injection the L-phenylalanine-treated animals were observed to be sedated; normal righting re- flexes were impaired and the severely debilitated chicks were prostrated. Levels of phenylalanine in the plasma reached 10 - 15 umol/ml (normal: 0.16): tyrosine increased from 0.10 umol/ml to 0.6 umol/ml. In the brain phenylala— nine and tyrosine were elevated to 2.5 - 3.5 umol/g (normal: 0.05) and 0.15 - 0.30 umol/g (normal: 0.05), respectively. Examination of several glycolytic intermediates and high energy phosphate compounds in the brain at a time Sandra Spieker Granett when levels of phenylalanine were greatest revealed a 15 - 25% increase in phosphocreatine accompanied by a reciprocal decrease in inorganic phOSphate; levels of fructose-1,6—diphosphate decreased by 15 - 25% and levels of lactate and L—a-glycerol phosphate by 25%. Glucose and glucose-G-phosphate were either unchanged or increased, whereas glycogen concentrations did not differ signifi- cantly from those seen in the controls. The levels of the adenine nucleotides were unaffected. The rate of expendi- ture of cerebral high energy phosphates was depressed by 50 - 79% when determined as a function of metabolic rate during ischemia by the "closed~system" technique. A 15% hypoglycemia was consistently observed. Phenylacetic acid accumulated in the brain to levels of 0.01 to 0.06 umol/g. The state of narcosis was reversible within 24 h, although the animals had not regained their appetite. Phenylalanine and tyrosine concentrations in the brain de- creased to normal levels by 18 h, and most of the altered metabolites approached concentrations in control tissue 8 - 12 h after the final injection. The changes observed in the metabolites coincided with cerebral phenylalanine levels of 2 umol/g or more. No in zitrg_inhibition of anaerobic glycolysis by 18 mM L-phenylalanine could be demonstrated with high speed supernatants prepared from chick brain. Commercially Sandra Spieker Granett available creatine kinase activity purified from muscle was unaffected by 9 mM L-phenylalanine. The profile of cerebral amino acids was markedly altered in the phenylalanine-treated chicks. Lysine, histidine, phosphorylethanolamine, taurine, aspartic acid, threonine, serine, glutamic acid, alanine, valine, methionine, isoleucine and leucine were decreased. Gluta- mine, glycine, phenylalanine and tyrosine were elevated. Neither arginine nor ammonia were affected. The summation of free amino acids and ammonia did not differ between the phenylalanine and saline-treated chicks. That the phenylalanine-injected chicks were meta- bolically depressed was supported by the delay in tonic extension observed upon injection of the animals with pentylenetetrazol or picrotoxin. In some cases recovery from the convulsions occurred or convulsive activity was eliminated. Injection of either phenylalanine or saline did not change plasma osmolality or levels of sodium or calcium in comparison to uninjected control animals; however, potassium was increased by 60% regardless of the solute. A survey of other amino acids administered in a similar manner disclosed D-phenylalanine, L-histidine, L-methionine and L-tryptophan to reduce activity in the chick, often resulting in prostration and death. Sandra Spieker Granett The changes produced in cerebral glycolytic meta- bolism in the chick fed a diet 40% in D-galactose for 46 h were compatible with those previously observed: levels of phosphocreatine, ATP, glucose, glycogen, fructose-1,6— diphosphate and lactate were depressed; AMP levels were elevated. Analysis of the utilization of actual and poten- tial high energy phosphate compounds for various intervals of postmorten ischemia (zero time to 10 min) according to the "closed-system" technique indicated a slower rate of glycolysis in the galactose-fed animals in comparison to controls. Galactose and galactitol were not utilized and did not constitute energy sources. ATP expenditure began after 12 sec of ischemia in the galactose-toxic chick in contrast to 24 sec in the chicks fed control diet. Cere- bral glucose levels were initially 0.3 umol/g in the galactose-fed chicks compared with 1.0 - 1.5 umol/g in control animals and decreased to 0.15 umol/g 6 sec after initiation of the ischemia, remaining at this level for the 10 min. Glycogen reserves were depleted sooner and lactate formation was insignificant until after 1 min of ischemia in contrast to the control situation in which accumulation of lactate was immediate. No regulation of glycolysis at the phosphofructokinase point as evidenced by an increase in the level of fructose-1,6-diphosphate during the first 30 sec of ischemia could be demonstrated in chicks fed Sandra Spieker Granett galactose. Citrate levels did not differ between the two groups of animals. Determinations of cerebral glycogen levels by acid hydrolysis were found to be erroneously high due to the presence of a glucose-containing, non-glycogen material. A more specific method for hydrolysis of gly- coqen, employing amyloglucosidase, was proposed. The degradation was accomplished in 50 mM citrate buffer, pH 5, and the glucose released was quantified at pH 8 spectro- photometrically with hexokinase and glucose-6-phosphate dehydrogenase. The procedure was demonstrated as accurate, precise and convenient, and sensitivity could be enhanced by fluorometric analysis. PERTURBATIONS OF ENERGY METABOLISM IN CHICK BRAIN INDUCED BY HYPERPHENYLALANEMIA AND GALACTOSEMIA BY LJ-zflfit‘i SandraASpieker Granett A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1972 This work is dedicated to my husband, Jeff, to my mother, Mildred Spieker and to the spirit of my father, Rudolph Spieker ii GENERAL ACKNOWLEDGMENTS I should like to acknowledge my committee members, Dr. Richard Anderson, Dr. Loran Bieber, Dr. Richard Rech, Dr. John Wilson and my major professor Dr. William Wells, for their conscientious criticism of this work. In particular I wish to recognize Dr. Wells for his encouragement and his confidence in me as a developing scientist. His consistently Optimistic, imaginative, and vigorous approach to research was refreshing. Dr. Leslie Kozak, Dr. Ping Ting-Beall, Dr. Harvey Knull and Dr. James Blosser, whom I had the pleasure of knowing during my graduate career, are thanked for their ideas and comments on aspects of this research, as are many other friends who will remain nameless. The work was conducted in the second and third years of my marriage. For this I acknowledge my husband whose love for me extended to my professional fulfillment, though it necessitated a year's separation. I am indebted to him for my inner satisfaction and contentment and I cherish him more deeply for this. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . ix LIST OF FIGURES . . . . . . . . . . . . xi LIST OF ABBREVIATIONS . . . . . . . . . . xiii I. STATEMENT OF ORGANIZATION . . . . . . l I I 0 SECTION I O O O O O O O O O O O 3 INTRODUCTION 0 O O O I O O O O O 3 LITERATURE REVIEW: Phenylketonuria . . 5 Description of the Disease in Man O I O O O I O O O S Hyperphenylalanemia in Animal Medals 0 I O O O O O O O O 1 0 CHAPTER I. ENERGY METABOLISM IN THE BRAINS OF L-PHENYLALANINE- TREATED CHICKS . . . . . . 15 ABSTRACT O O O O O O O O O O O 1 5 INTRODUCTION I O O O O O O O O O 1 7 MATERIALS AND METHODS . . . . . . . 19 Animals and Materials . . . . . . 19 Production of Hyperphenyl- alanemia. . . . . . . . . . 20 Preparation of Extract . . . 20 Analytical Methods for Glycolytic Metabolites . . . . . . . . 21 iv Page Quantification of Phenylalanine and Tyrosine . . . . . . . 22 Separation and Assay of Pyruvate Kinase . . . . . . . . 23 "Closed— -System" Studies . . . . . 23 Quantification of Phenyl— acetic Acid . . . . . . . . 24 RESULTS . . . . . . . . . . . 26 Brain High-Energy Phosphates and Glycolytic Inter- mediates . . . . . . . 26 "Closed-System" Anoxia . . . . . 29 Studies of the Inhibition of Pyruvate Kinase by Phenyl- alanine . . . . . . . . 34 Levels of Phenylacetic Acid . . . . 36 DISCUSSION . . . . . . . . . . 38 ACKNOWLEDGMENTS . . . . . . . . . 43 CHAPTER II. FURTHER STUDIES ON CEREBRAL METABOLISM IN L-PHENYLALANINE AFFECTED CHICKS . . . . . 44 INTRODUCTION . . . . . . . . . . 44 MATERIALS AND METHODS . . . . . . . 46 Animals and Materials . . . . . . 46 Recovery Studies . . . . . . . 47 Amino Acid Analyses . . . . . . 48 Quantification of Ammonia . . . . 50 Seizure Threshold Studies . . . 51 Study of the Effect of L-Phenylalanine on Anaerobic Glycolysis . . . . 51 Assay of Creatine Kinase in the Presence of L-Phenylalanine . . . 53 Plasma Osmolality Determinations . . . . . . . 53 Plasma Electrolyte Determinations . . . . . . . 54 III. RESULTS . . . . . . . . . . Observations on the Narcotic State Induced in the Chick by L-Phenylalanine . . . Recovery Studies . . . . Amino Acid Analyses . . . Seizure Threshold Studies . Effect of L-Phenylalanine on Anaerobic Glycolysis and Creatine Kinase Activity . . . Studies of the Effects of Other Amino Acids on the Chick . . . Effect of the Injection Procedure on Plasma Osmolality and Electrolyte Concentrations . . DISCUSSION . O O O O O O O 0 ACKNOWLEDGMENTS . . . . . . . SUMMARY . . . . . . . . . . SECTION II . . . . . . . . . . CHAPTER I. STUDIES ON CEREBRAL GLYCOLYTIC FLUX IN GALACTOSE-TOXIC CHICKS . . . . . . . INTRODUCTION . . . . . . . . LITERATURE REVIEW . . . . . . . MATERIALS AND METHODS . . . . . Animals and Materials Substrate Analysis . "Closed-System" Studies Glycogen Analysis . . RESULTS 0 O O O O O O O O 0 DISCUSSION . . . . . . . . . vi Page 55 80 83 96 97 101 101 101 103 106 106 106 107 108 109 128 Page CHAPTER II. DETERMINATION OF GLYCOGEN FROM BRAIN WITH AMYLOGLUCO- SIDASE . . . . . . . . 131 INTRODUCTION . . . . . . . . . . 131 MATERIALS AND METHODS . . . . . . . 134 Materials . . . . . . . . . . 134 Isolation of Glycogen from Brain . . 134 Quantification of Glycogen by Acid Hydrolysis . . . . . . . 135 Quantification of Glucose . . . . 135 Quantification of Glycogen with Amyloglucosidase . . . . . . . 136 Recovery Experiments . . . . 136 Enzymatic Assay for Debranching Activity . . . . . . . . . 137 Purification of Debranching Activity . . . . . 138 Purification of Pullulanase Activity . . . . . . . 138 Assay of Pullulanase Activity on Glycogen . . . . . . . . 139 RESULTS . . . . . . . . . . . 140 Quantification of Standard Glycogen by Acid Hydrolysis and Amyloglucosidase Methods . . . 140 Time Course Study of the Release of Glucose from Glycogen by Amyloglucosidase . . . . . 140 Recovery of Glycogen Added to Tissue . . . . . . . . . 140 Quantification of Glycogen Levels in Brain by Acid Hydrolysis and Amyloglucosidase Methods . . . 146 Assay of Standard Glycogen with Phosphorylase . . . . . 146 Assay of Tissue Glycogen with Phosphorylase and Amyloglu- cosidase . . . . . . . . . 148 vii Page DISCUSSION . . . . . . . . . . 150 ACKNOWLEDGMENTS . . . . . . . . 153 SUMMARY . . . . . . . . . . . 154 REFERENCES . . . . . . . . . . . . . 157 viii Table 1. 10. LIST OF TABLES Plasma and Brain Levels of Phenylalanine and Tyrosine in Chicks Injected Intra- peritoneally with L-Phenylalanine or Saline . . . . . . . . . . . . Effect of Intraperitoneal1y-Injected L—Phenylalanine on Energy-Related Phosphates in Chick Brain . . . . . . Effect of L-Phenylalanine on Inter- mediates of Carbohydrate Metabolism in Chick Brain . . . . . . . . . High-Energy Phosphate Utilization in Brains of Chicks Given L-Phenylalanine Levels of Phenylacetic Acid in the Brains of L-Phenylalanine-Treated Chicks . . . . . . . . . . . Levels of Free Amino Acids and Ammonia in the Brains of Chicks Injected with Saline or L-Phenylalanine . . . Seizure Threshold to Pentylenetetrazol after Intraperitoneal Injections of L-Phenylalanine . . . . . . . Ig_vitro Study of Anaerobic Glycolysis in Cfiick Brain in the Presence of L'PhGflYlalanine o o o o o o o o 0 Survey of the Effects of Several Amino and Imino Acids on the Chick . . . . . . Comparison of Plasma Osmolalities in Phenylalanine and Saline-Injected Chicks . . . . . . . . . . . . ix Page 27 28 30 35 37 7O 74 76 79 81 Table 11. 12. 13. 14. 15. 16. Plasma Concentrations of Sodium, Potas- sium and Calcium in Uninjected Chicks and Chicks Injected with Saline or L-Phenylalanine . . . . . . . Variation of Cerebral Glucose Levels in the Chick During Anoxia . . . Cerebral Levels of Galactose and Galactitol in the Chick after Various Periods of Anoxia . . . . . . Recovery of Standard Glycogen from Tissue Samples . . . . . . . Comparison of Glycogen Levels in Tissue Determined by Acid Hydrolysis and Amyloglucosidase Methods . . . . Degradation of Glycogen Isolated from Tissue by Phosphorylase and Amyloglucosidase . . . . . . . Page 82 122 127 145 147 149 Figure 1. 2. LIST OF FIGURES Enzymatic Metabolism of L-Phenylalanine and L-Tyrosine . . . . . . . Fluxes of Metabolites during Ischemia in the Brains of Chicks Previously Injected with L-Phenylalanine or Saline . . . . . . . . . . Photograph Illustrating the Drowsy State Induced in Chicks by the Intraperi- toneal Injection of L—Phenylalanine Photograph of a Chick Prostrated by Serial Injections of L-Phenylalanine Photograph Demonstrating Loss of Motor Activity in Chicks Injected with L- Phenylalanine . . . . . . . Cerebral Levels of Phenylalanine and Tyrosine in Chicks During a 24 h Period Following Intraperitoneal Injections of L-Phenylalanine . . Cerebral Levels of ATP and Phospho- creatine in the Chick During a 24 h Period Following Injections with L-Phenylalanine . . . . . . . Cerebral Levels of L-a-Glycerol Phos- phate and Fructose-1,6-Diphosphate During a 24 h Period Following L- Phenylalanine Injections in the Chick . . . ._ . . . . . . Levels of Glucose in the Brain and Serum of Chicks During a 24 h Period Following Injections with L-Phenylalanine . . . . . . . xi Page 33 57 57 59 62 64 66 68 Figure 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Seizure Threshold to Picrotoxin in the Chick After Intraperitoneal Injections of L-Phenylalanine . . . . Activity of Creatine Kinase in the Presence of L-Phenylalanine . . . . Change as a Function of the Duration of Postmortem Ischemia in Phospho- creatine in Brains of Chicks Fed Control or Galactose-containing Diets . . . . . . . . . . . . Changes as a Function of the Duration of Postmortem Ischemia in ATP and AMP in the Brains of Control or Galactose-fed Chicks . . . . . . . Change in Cerebral ADP Levels Under Ischemic Conditions . . . . . . . Effect of Postmortem Ischemia on Total Level of Adenine Nucleotides and Energy Charge in the Brains of Control and Galactose-fed Chicks . . . . . . . Changes in Various Cerebral Glycolytic Metabolites and Cerebral Glycogen as Functions of Prolonged Postmortem Ischemia in Control and Galactose- fed Chicks . . . . . . . . . Utilization of Actual and Potential High-Energy Phosphates by Chick Brain During Postmortem Ischemia . . . . . Comparison of Standard Glycogen De- gradation by Acid Hydrolysis and Amyloglucosidase . . . . . . . . Analysis with Time of the Hydrolysis of Glycogen by Amyloglucosidase . . xii Page 73 78 111 113 115 117 119 125 142 144 LI ST OF ABBREVIATIONS ADP Adenosine diphosphate AMP Adenosine monophosphate ATP Adenosine triphosphate CPM Counts per minute EDTA Ethylenediaminetetraacetic acid GABA Gamma-Aminobutyric acid NAD Nicotinamide adenine dinucleotide (oxidized form) NADH Nicotinamide adenine dinucleotide (reduced form) NADP Nicotinamide adenine dinucleotide phosphate (oxidized form) NADPH Nicotinamide adenine dinucleotide phosphate (reduced form) PHE Phenylalanine Tris Tris(hydroxymethyl)aminomethane xiii I. STATEMENT OF ORGANIZATION Galactosemia and phenylketonuria, two genetic diseases of man, are easily distinguished in that the former is a disorder of carbohydrate metabolism and the latter of amino acid metabolism. However, associated with both is mental retardation. For the studies described herein day-old chicks were treated with D-galactose or L-phenylalanine and investigated as model systems for the respective human conditions; galactose proved toxic to the chick, producing convulsions, whereas treatment with phenylalanine was accompanied by sedation. It was of special interest to examine some biochemical parameters of brain function in these animals for two reasons: involve- ment was indicated by the chicks' behavioral signs and impairment of normal mental capacities is an outstanding clinical symptom shared by both galactosemics and phenyl- ketonurics. Perturbations in high energy phosphate com- pounds and glycolytic intermediates had been previously characterized for the galactose-intoxicated chick by Kozak and Wells (1969 and 1971). In Section I evidence is presented for a depressive effect of phenylalanine on energy metabolism in the chick brain. In Section II, further studies on the effects of galactose on glycolytic flux are described. Also a new method for quantification of glyCOgen employed in these investigations is presented. This thesis is divided into two parts, two chapters each, as a matter of convenience; Chapter I of Section I has been published in the form presented here; Chapter I of Section II in conjunction with other pertinent work. II. SECTION I INTRODUCTION Several animals notably the mouse, rat and monkey have received considerable attention as model systems for the study of the human genetic disorder, hyperphenylalanemia or phenylketonuria. The chick, though, had been conspicuously neglected until the late sixties when Tamimie and Pscheidt (1966 a, b and c; 1968) pub- lished a series of papers on the effects of feeding for four weeks a diet 5 or 8% in phenylalanine to the young chick. They found aberrant morphological changes asso- ciated with poor growth and development, but no mortality was reported and post-mortem examination indicated no gross pathology in any internal organs. Symptoms were reversible. Analysis of any biochemical alterations, particularly in the brain, was not extensive. Subsequently, further studies were conducted (Granett, 1970) to eluci- date this latter point. The brain was considered, primar- ily, since irreversible neurologic disturbances are the most outstanding clinical features of phenylketonuria. L-Phenylalanine was administered through the diet (7-1/2 and 10%) to newly hatched chicks for periods of two or three weeks. Abnormally low weight gain was recorded; the animals appeared alert on gross observation. Investi- gation into cerebral high energy phosphate metabolism and glycolysis revealed the phenylalanine chicks to have con- sistently lower lactate levels than controls. PhOSpho- creatine levels were either unchanged or elevated. Changes in concentration of glucose and fructose—1,6-diphosphate were variable; glucose-6-phosphate decreased or did not change. Upon treatment of the chicks with larger amounts of L-phenylalanine by serial intraperitoneal injections, though, they entered a state of narcosis, reversible with time, resembling in some way that produced with barbitur- ates. In this section, then, the cerebral energy metab— olism in the narcotized animals during the highest phenyla- lanine levels and during the recovery period is discussed; the pattern of free amino acids is characterized: and evidence for anti-convulsive activity of phenylalanine is presented. Also, the in 2132 observations are discussed in terms of a direct effect of L—phenylalanine on glycoly— sis. The work presented in Chapter I has been published under the title "Energy Metabolism in the Brains of L- Phenylalanine-Treated Chicks," by Sandra E. Granett and W. W. Wells, Journal of Neurochemistry lg, 1089 (1972). LITERATURE REVIEW Phenylketonuria Description of the disease In man Phenylketonuria is a genetic disorder characterized as autosomal recessive in which the enzyme phenylalanine hydroxylase, responsible in part for the conversion of phenylalanine to tyrosine, is absent (Jervis, 1939 and 1953). The second enzyme required for the hydroxylation, dihydropteridine reductase, is present in normal amounts. Polling first recognized the condition in 1934. Clinical features of the homozygote include microcephaly, hyper- kinesis, electroencephalographic abnormalities, seizures, mental retardation, eczema and deficient pigmentation. L-Phenylalanine accumulates in the plasma of patients to levels as high as 2-6 umol/ml, as compared to a normal range of 0.054-0.12 (Knox, 1972). Alternative metabolites resulting from the diversion of the amino acid through other pathways (Fig. 1) are detected. Plasma concentra- tions of phenylpyruvic acid and o-hydroxyphenylacetic acid measuring 6-64 umol/liter and 7-40 umol/liter, re- spectively, have been reported (Jervis and Drejza, 1966). ocfimoumulq 0cm maficmamamcmnmlq mo Emwaonmume owumE>~cm .H ouzmwm n: 04 034.24) mzaio- «ii i sztzmo- uzifianefifioaszmi mzaxamzfimz .— uzaxamzfimseoz a e 93 0:84.;sz ”125:..sz 92 255.152.... mzaxamzmmmoz 93 0:31:29: :0... 1199a 053%....»sz fl z_z<._m_211|_ H mz_2zwzd>xcma>x_ouv.m allwz_mom>.—.TIIII UZ_ZZNIQ L 904 0.53.;sz :o.H1|I|v\§o_>am>3>zmxa :oa wz_z<.;zfi._>zm:a 064 0:84..»sz Ice 92 oafizmoozo: 0.04 o:.wo HuapH>H0ch .momaaucu 0cm mcoHumummoHQ mamscu mo mHHmuoo How one» cow .:0HuooncH umma ecu Hound : N Nz oanHH oucH voumuHmcoop 0cm oHoHno> ocHHmm nuHs no Ammaoaa n0.H omoc Hmuouv ochoanHSconmrq z ma.0 no as m cuHs mHm>HoucH Samson um oOHHnu maauocouHuomuuucH wouooncH mums mxowno paonampuoco one .aam>HHoommoH .monco m .uqc 0H .mH soum poaoom oommHH co econ 0Ho3 momuancu on» .HHH 0cm HH .H mucmaHHmmxo cH .moamsum 0 co meoHumcHsuouop mo momuuo>u ouu mao>oa ochouau on» «moamsmm m co mGOHumcHauouoc onch no mommuo>u Hammonou mao>oa ochdHuHaaonm on» .vouooHch muons Hmooxm “ouoz >~.0 H HH.N no.0 H 00.0 in: In: 0H.H H H0.HH 00.0 H 0H.0 HHH 0m.0 H 0v.~ u: H0.0 H 00.0 H0.0 H HH.0 «ma.m .mn.0H in HH H0.0 H hm.m In «0.0 H hm.0 H0.0 H 00.0 mH.H H bh.va nu H a a 258.: HmucoEHummxm Houucoo HmucoEHHomxm Houucou HmucofiHuomxm Houusoo ochmHmHmconm ochOHha ochuHuahcosm .mxm chum mammam .ocHHmm Ho ochmHthconmiq squ aaamozouHuommuucH couponcu meHnO cw ucHuouaa 0cm ochuHcamconm mo mao>oq cHuum can mamcamlu.a magma 28 Table 2.--Effect of Intraperitoneally-Injected L—Phenylalanine on Energy-Related Phosphates in Chick Brain. Substrate Exp. Control L-Ph::§::::21ne Cznifiol umol ATP I 1.76 i 0.15 1.81 i 0.06 103 II 1.73 i 0.05 1.83 i 0.12 106 ADP ‘I 0.79 r 0.10 0.66 i 0.03 84 II 0.86 i 0.09 0.75 1 0.06 87 AMP I 0.16 i 0.04 0.11 i 0.03 68+ II 0.18 t 0.02 0.16 i 0.01 90 Total adenine nucleotides I 2.71 r 0.15 2.58 i 0.06 95 II 2.77 t 0.11 2.74 i 0.13 98 Energy charge6 I 0.80 0.83 104 II 0.78 0.81 103 Phosphocreatine I 1.79 i 0.06 2.20 i 0.06 123* II 1.96 1 0.15 2.25 i 0.18 115** Inorganic phosPhate I 3.37 2.74 81* II 3.61 i 0.27 3.25 i 0.10 90++ Sum of phosphates I 12.18 11.81 97 II 12.65 12.64 100 Note: from Atkinson (1968). Each value, except where indicated, represents the average 1 SD of 1 or 2 determinations on 4 or 5 samples from pools described for Table 1. See text section on METHODS for details of sampling and analyses. ++ + *P<0.001. **P<0.01. P<0.05. Pe on» mucomonmun och> 30mm "ouoz 0H0.0 H 00H.H 0nm.0 H 0H0.H 0mn.0 H 0HH.H 00~.0 H 0~0.H somoosnu +00H.0 H 00H.H 00~.0 H can.“ «.0m~.0 H 0HH.~ 0HH.0 H 000.~ «panama .0H0.0 H H00.0 0H0.0 H 00H.0 H.000.0 H 000.0 HH0.0 H HHH.0 mumemmonm Honmosnuisin .000.0 H 00H.0 000.0 H mvH.0 +mH0.0 H 0~H.0 m~0.0 H mmH.0 aumnmmoan0-0.HummoHosnm 000.0 H 0m0.0 m00.0 H 0~0.0 .«m00.0 H 000.0 000.0 H 0m0.0 wumsmmoamu0ummoosH0 0m~.0 H 0H0.H 00H.0 H 0H0.H .0HH.0 H 050.~ 00H.0 H 05H.H «moosHo mgmmmm Hos: wouomncH wouooncH nonooncH wouooncH mchaHHHsaosaun H002 ocHeuanaconauq Houz HH ncwEHnomxm H ucoanomxm «unnumnsm .chnm on50 :H SmHHonduoz onenpanonneo mo moneroanoucH so ocwceauahsonmuq mo noommmli.m manna 31 phosphocreatine, lactate, glucose and glycogen were deter- mined in the brain after 15 and 30 sec of anoxia (Figure 2). In both the control and experimental situations, phOSpho- creatine appeared to be quickly hydrolyzed, while virtually no net utilization of ATP occurred and neither ADP nor AMP accumulated. A significant dissimilarity occurred in the cases of lactate and glucose. In the control group, lac- tate increased considerably within the first 15 sec; how- ever, the increase was delayed in the L-phenylalanine- injected group. Similarly, the control levels of glucose decreased significantly after 15 sec of anoxia, whereas essentially no utilization of glucose was demonstrated in the L-phenylalanine-treated group, even at 30 sec. Exami- nation of the stoichiometry for the control group during the first 15 sec of anoxia revealed that the increase in lactate could not be accounted for by changes in glucose and glycogen. Jongkind and Bruntink (1970) noted a similar imbalance under closed-system conditions in rat brain. They suggested that substrates other than glucose were utilized for lactate production; however, they did not measure depletion of glycogen. In our system, it is un- likely that the lactate carbons originate from alanine since Yoshino and Elliott (1970) have shown that the levels of alanine in rat brain are not altered by a l-min period of anoxia. Conversely, the 30-sec anoxia period in our control group produced a decrease in glucose and glycogen 32 Figure 2.--Fluxes of metabolites during ischemia in the brains of chicks previously injected with L- phenylalanine or saline. The animals were injected as described in METHODS. PHE represents the phenylalanine group. Each value is the average t SD for l or 2 determinations eaCh on 3 samples of brain tissue from a pool of 5 chicks, excepting the zero-time PHE glycogen value which is the average of 2 analyses. After decapitation, the heads were maintained at 41°C for 15- or 30-sec periods of anoxia before being frozen in liquid N , essentially according to the "closed- system" technique 8f Lowry gt_al. (1964). .umolu/g tissue moles I a tissue 33 PHOSPHOCREATINE ATP ADP L5- ._ 0.5 '- LO AMP *——° CONTROL ------ PHE O L l J_ l GLYCOGEN GLUCOSE LACTATE 3.0 ~ - ,I ........ I r _ I”’ +— ’I 1’ i: """""" 1"" l 1 l l l I O '5 30 O '5 30 O '5 3O TIME (SEC) 34 that was greater than the increase in lactate. Glycogen depletion was evident in both groups after 30 sec of anoxia. In the experimental group, this might have been the source of the lactic acid accumulation since glucose was still unchanged. The rate of utilization of high-energy phos- phates (Table 4) was consistently lower in the phenylalanine-treated chick in comparison to that in the control animals at both periods. Studies of the inhibition of pyruvate kinase by phenylalanine Weber (1969) suggested a decline in the production of ATP could occur when the levels of phenylalanine in_vivo approach that of the Ki (6 - 10 mM) for the inhibition of pyruvate kinase by phenylalanine. In our present work with the chick, 2 to 4 mM phenylalanine accumulated in the brain, yet there was no shortage of high-energy metabolites. In- deed, on the contrary, decreased utilization of energy characterized the status of cerebral metabolism. This sit- uation suggested that the pyruvate kinase from chick brain was less sensitive to phenylalanine or that the inhibition was insufficient to affect levels of ATP. Our investigation of pyruvate kinase from chick whole brain has revealed that the enzyme exhibited no inhibition by phenylalanine, even when the concentration of phenylalanine was 400-fold higher than that of phosphoenolpyruvate. As a control, pyruvate kinase from a young rat brain was assayed under identical 35 Table 4.--High-Energy Phosphate Utilization in Brains of Chicks Given L-Phenylalanine. Time Interval L-Phenylalanine After Decapitation Control Injected sec umol/g/min O - 15 6.52 1.36 0 - 30 4.96 2.49 Note: The utilization was calculated from the average values represented in Figure 2, according to the formula (Lowry 22 31., 1964): 2 AATP + AADP + Aphosphocreatine + Alactate, as described in the text section on METHODS. The one-day-old chicks were injected intraperitoneally thrice, at hourly intervals, with 3 ml of 0.18 M L-phenylalanine (total dose 1.62 mmoles) or with saline vehicle and decapitated 2 h after the last injection. The heads were maintained at 41°C for 15- or 30-sec periods of anoxia before being frozen in liquid N2. See text for further details. 36 conditions, and the kinetic parameters were determined. The Vmax was calculated to be 6.7 x 103 umoles of ADP phosphorylated/h per g of tissue; the Km was calculated as 5 M, and the K1 for phenylalanine was 1 x 10"3 M. 3.7 x 10' These values agree with those reported by Schwark et_al. (1971) and Weber 35 31. (1969). Levels of phenylacetic acid The extent to which the metabolite, phenylacetic acid, accumulated in the brains of the chicks treated with L-phenylalanine was determined (Table 5). Considerable variation in the levels of the acid was observed (0.006- 0.065 umol/g), whereas levels of phenylalanine were rela- tively constant (3.04 1 0.39 umol/g). 37 Table 5.--Levels of Phenylacetic Acid in the Brains of L- Phenylalanine-Treated Chicks. Phenylalanine Phenylacetic Acid umol/g Control 0.09 t 0.02* None detected Phenylalanine- + treated 3.04 i 0.39** 0.010, 0.065, 0.0006 Note: The one-day-old chicks were injected intraperi- toneally thrice, at hourly intervals, with 3 m1 of 0.18 M L-phenylalanine or with saline vehicle and decapitated 2 h after the last injection. The brains were immediately removed and frozen in liquid N2. See text for details of the analyses. *An average of single determinations from 3 pools of 3 brains each 1 SD. **An average of duplicate determinations from 3 pools of 3 brains each. +Individual averages for duplicate determinations from each of the 3 pools of brains. DISCUSSION The induction of narcosis in the chick by phenylala- nine has not, to our knowledge, been previously reported. Tamimie and Psheidt (1966) fed diets 5 and 8% (w/w) in L-phenylalanine to day-old chicks for a 4-week period. They observed reduced weight gain, poor feather develop- ment and abnormal develOpment of leg joints with consequent poor motor coordination in the animals. No mortality occurred, and the effects were reversible. In similar ex- periments with a diet 10% (w/w) in L-phenylalanine for 2 weeks, we noted the failure to gain weight (Granett, 1970), and observed at termination of these experiments, a rise in brain phenylalanine levels to 2 umol/g, with plasma levels rising to 4 mM, or a plasma-to-brain ratio lower than in the acute injection experiments described here. The levels of phenylalanine attained in the brain (e.g., 2 umol/g of brain: Granett, 1970) were comparable to those attained in 2 of our 3 injection experiments; yet narcosis was not observed, a finding that suggested that a gradual adjustment to the increased levels of the amino acid had occurred. In the injection studies, tyrosine accumulated 38 39 in the blood to a considerably lesser extent than that observed in the dietary experiments (plasma tyrosine level of 2 mM; Granett, 1970). Thus, the association of the narcosis with the acute studies rather than the chronic does not seem to be related to the absolute level of pheny- lalanine itself, but perhaps to the ratio of phenylalanine to tyrosine or the rapidity of the rise in the levels of phenylalanine or a metabolite thereof. The toxicity of phenylacetic acid, a metabolite of phenylalanine, has already been demonstrated in the hen, monkey, dog, mouse and human (Sherwin and Kennard, 1919; Berthelot and Dieryck, 1939). In the dog, large doses of phenylacetate are accompanied by drowsiness, inability to stand and coma. We have observed similar reactions with cockerels. When fed phenylacetate, the chicks die after the fourth day, and chicks injected intraperitoneally be- come sedated within 30 min. Cerebral levels of phenylace— tate are at least 2 umol/g in these animals. However, it is doubtful that this compound could be the toxic agent in our phenylalanine-injection experiments since phenyl- acetate did not accumulate to levels greater than 0.07 umol/g. In studies on phenobarbital-induced anesthesia in mice, Lowry at El. (1964) observed an increase in phospho— creatine and decreases in inorganic phosphate, fructose-1, 6-diphosphate, L-a-glycerol phosphate and lactate in the 40 brain. Similar changes occurred in the brains of our chicks after injection of phenylalanine. The phenobarbital- induced anesthesia also resulted in increases in glucose, glucose-6-phosphate and glycogen. Elevated levels of brain glucose have been confirmed by Mayman, Gatfield and Breckenridge (1964) and Gatfield gt El' (1966). We ob- served significant increases in glucose and glucose-6— phOSphate and slightly elevated glycogen only in experiment I (Table 3) in which the brain phenylalanine level was highest. Lowry at 31. (1964) found that the barbiturates affected neither the level of the adenine nucleotides nor the energy charge (0.866 for control versus 0.860 for the phenobarbital-treated group), observations with which our findings agree. Recently, Nilsson and Siesj6 (1970) re- ported no effect of various volatile anesthetics and barbiturates on levels of phosphocreatine in rat brain. However, they did substantiate the decrease in lactate associated with barbiturate anesthesia. It is generally accepted that anesthesia lowers the metabolic rate in the brain (Himwich, Homburger, Maresca and Himwich, 1947; Wechsler, Dripps and Kety, 1951). Lowry gt El' (1964) and Gatfield g£_al, (1966) demonstrated the utility of the "closed-system" technique for approxi- mating the reduction in cerebral aerobic energy flux induced by phenobarbital. Likewise, we found that the acute phenylalanine intoxication is associated with a 41 reduction; utilization of actual and potential high-energy phosphates was lowered 50 - 79% at the two time periods examined. The reduced metabolic activity in the brain of the phenylalanine-injected chick is compatible with the increase in phosphocreatine and the decreases in fructose- l,6-diphosphate and inorganic phosphate, which exert glycolytic control of the enzyme phosphofructokinase. Uyeda and Racker (1965a) demonstrated inhibition of rabbit muscle phosphofructokinase at 1 - 5 mM concentrations of phOSphocreatine, and Krzanowski and Matschinsky (1969) showed that the enzyme from sheep brain is more sensitive to the inhibition than the muscle enzyme. The stimulation of phosphofructokinase by fructose diphosphate and in- organic phOSphate is well known (Uyeda and Racker, 1965a and b; Passonneau and Lowry, 1962). In contrast to the pyruvate kinase isolated from rat brain (Weber, 1969 and Schwark gt_al., 1971), pros- tate, seminal vesicles, uterus and skeletal muscle (Vihayvargiya, Schwark and Singhal, 1969), our finding that chick brain pyruvate kinase is not inhibited by L-phenylalanine appears to be a species difference. The inhibition of hexokinase by phenylpyruvate (Ki = 2 - 13 mM in postpartum rat brain) also reported by Weber (1969) was not investigated in the chick. The concentration of phenylpyruvate in the brains of the phenylalanine-injected chicks was not determined; however, it did not accumulate 42 to levels greater than 0.07 - 0.15 mM in plasma of chicks in dietary studies (Granett, 1970). The mechanism of induction of narcosis by pheny- lalanine in the chick is not understood. As a possible explanation, the recent observation that L-phenylalanine can influence the activity of (Na+ + K+)—ATPase from chick microsomes in zi££g_(Ting-Beall and Wells, 1971) is being further investigated. ACKNOWLEDGMENT S We gratefully acknowledge Mr. Joseph Prohaska for the pyruvate kinase study. 43 CHAPTER II FURTHER STUDIES ON CEREBRAL METABOLISM IN L-PHENYLALANINE AFFECTED CHICKS INTRODUCTION This chapter represents a continuation of the previous work, more fully characterizing alterations in cerebral metabolism by L-phenylalanine. A 24-h recovery period following the serial injections of the amino acid is examined for the purpose of relating the duration of the changes in cerebral glycolytic metabolites or high energy phosphates with that of the increased levels of phenylalanine or tyrosine. Changes in cerebral levels of amino acids are described. Seizure threshold is shown to be elevated. Direct inhibition of glycolysis by phenyla- lanine is investigated as a mechanism for the reduced metabolic rate. Also, the effect of the amino acid on creatine kinase is examined. Several other amino acids are tested for narcotic effects similar to phenylalanine's. In light of the extremely large volume of water and 44 45 quantity of sodium chloride or phenylalanine administered, changes in plasma osmolality and cationic composition are determined. MATERIALS AND METHODS Animals and materials Day-old Leghorn cockerels from Rainbow Trail Hatcheries (St. Louis, Mich.) were housed in a brooder at 32°C and fed a commercial chick starter mash received through the Michigan State University Poultry Department. L-Phenylalanine (free form) and picrotoxin were purchased from Sigma Chemical Co. (St. Louis, Mo.). L-d-Glycerol phosphate dehydrogenase (L-glycerol-B-phosphate:NAD oxido- reductase; EC 1.1.1.8) and creatine kinase (ATP:creatine phosphotransferase; EC 2.7.3.2) were obtained from Boeh- ringer Mannheim (New York, N.Y.); glucose oxidase, from WOrthington (Freehold, N.J.). Pentylenetetrazol, a product of the Knoll Pharmaceutical Company (Orange, N.J.) was a gift from Dr. Steve Baskins, Pharmacology Department, Michigan State University. D-(6-14C)-Glucose (Specific radioactivity, 2.9 mCi/mmol) was purchased from Nuclear Chicago (Chicago, Ill.) and DW-14 C)-glucose (Specific radioactivity, 60 mCi/mmol) from Calatomic (Los Angeles, Cal.). All adenine and pyridine nucleotides used in the assays were obtained from either Sigma or Boehringer- Mannheim. 46 47 Recovery,Studies Chicks, 1-day old, were injected intraperitoneally three times at hourly intervals with 3 m1 of neutralized 0.18 M L-phenylalanine or 0.154 M saline. After various periods of time had elapsed (2, 5, 8, 12, 18 or 24 h) following the last injection, the animals were sacrificed. They were deprived of food and water during these times to avoid possible drowning of the sedated chicks, except for the last 60 min before the termination of the 24-h period, at which time the animals' hunger reSponses were checked. Those animals used for quantification of cerebral high- energy phoSphates or glycolytic intermediates were decapi- tated directly into liquid N2, and the brain tissue was later chipped out and powdered over dry ice in the cold room. Three or four individual brains were analyzed at each point. Brains for analysis of phenylalanine and tyrosine levels were removed from the skull and then frozen in liquid nitrogen and pooled, three brains per point. Blood was obtained from these animals by drainage at the neck and was likewise pooled. Serum was separated from the red cells by centrifugation at 3000 g_for 10 min. All tissue samples were stored at -90°C. They were ex- tracted with perchloric acid: ATP, phosphocreatine, L-a-glycerol phosphate and cerebral glucose were quanti- fied as described in METHODS, Chapter I. Free glucose in the serum was measured on zinc sulfate-barium hydroxide 48 filtrates using glucose oxidase as described for Worth- ington's semi-micro method; 0.5 ml of reagent was reacted with 0.5 ml of extract. Amino acid analyses The amino acids were quantified using a Beckman 121 automatic analyzer. Separation of the acidic and neutral amino acids was accomplished at 55°C on resin AA-15 with two 0.2 N sodium citrate buffers (normality with reSpect to sodium) pH 3.25 and 4.25, reSpectively: separation of the basic amino acids was accomplished on resin AA-35 with 0.35 N sodium citrate, pH 5.26. Chicks injected with ~saline or L-phenylalanine according to the usual protocol were decapitated 2-h after the last injection, brains removed, frozen in liquid nitrogen and powdered over dry ice. Weighed tissue was homogenized in 1% picric acid (15 ml/g) using a motor-driven Potter-Elvehjem homogenizer (Stein and Moore, 1954). After centrifugation at 12,000 9 for 10 min, the picrate was removed from the supernatant by batch-wise mixing with Dowex 2-XB resin (chloride form), 4 g of resin per g of original tissue. The resin was filtered and washed with four 2-ml volumes of 0.02 N HCl per g of brain. To remove glutathione which chromatographs in the region between aspartic acid and proline, the filtrate was neutralized to pH 7.2-7.5 with l N NaOH and freshly prepared 0.5 M sodium sulfite was added (80 ul/g 49 of tissue). The mix was allowed to stand open to the air at room temperature for 4 h, at which time the pH was readjusted to 2.0-2.2 with l N HCl. Subsequently, the sample was taken to dryness, redissolved in a known volume of water and aliquots were removed. Typically l-l.5 g of tissue were extracted; the final residue was then dis- solved in 10 m1 of water; two 4-m1 aliquots (equivalent to 0.4-0.6 g of brain) were removed, taken to dryness and redissolved in 0.6 ml of 0.2 N sodium citrate, pH 2, 0.125 mM with reSpect to the internal standard, norleucine or a-amino-B-guanidinopropionic acid for the acid-neutral or basic column, reSpectively. This sized aliquot would allow for quantification of amino acids less than 1 umol/g of brain. For those greater than this, a 0.2-ml aliquot (20-30 mg of tissue) was removed from the original lO-ml volume, taken to dryness on a rotoevaporator and dissolved in 1 ml of citrate-norleucine buffer. Asparagine and N- acetyl aspartic acid and glutamine were quantitated by difference in the aspartate and glutamate levels before and after acid hydrolysis. Also, acid hydrolysis unmasked the serine-threonine peaks beneath glutamine. As an example, a 0.5-ml aliquot (50-75 mg of tissue) from the lO-ml volume was diluted to 2 N HCl with 4 N acid, heated at 100°C for 2 h, taken to dryness, redissolved in water, and again dried to remove HCl. The residue was finally dis- solved in 0.8-1.0 ml of citrate-norleucine buffer. For 50 all analyses, a 0.5-ml aliquot of the final solution was injected on tOp of the column. Standard amino acid solu- tions were chromatographed intermittently throughout the series of samples. Calculations of unknown quantities were based on the area ratios of the compound in question to the internal standard; area was determined by multiplication of peak height by width at half height. When only tyrosine and phenylalanine were quantified, the oxidation of glutathione was omitted as its presence did not interfere with either of these amino acids. Quantification of ammonia Levels of ammonia were determined chemically by a diffusion method described by weil-Malherbe (1969). A small glass rod coated with 0.1 N sulfuric acid was ex- tended through a rubber seal into a 25-ml flask containing 2 ml of saturated potassium carbonate and 0.5 ml of satur- ated potassium bicarbonate. A 0.4 m1 aliquot of a 0.6 M perchlorate extract of brain (0.5 ml/100 mg) was injected into the basic solution, and the flasks were shaken for l h at room temperature. The rods were then immersed into 3 ml of phenol (l%)-sodium nitroferricyanide (5 mg%) re- agent and to this were added 3~ml of an alkaline hypochlor- ite solution (0.34 ml of 4.6% sodium hypochlorite in 100 m1 ' of 0.5% NaOH). Color was allowed to develOp at room temp- erature for 40 min; absorbancy was read at 635 nm. 51 Calculations were based on a standard ammonium chloride solution assayed in the same manner as the samples. Seizure threshold studies The day-old chicks, weighing 40-50 g, were injected 2 or 3 times at hourly intervals with 3 ml of 0.18 M neu- tralized L-phenylalanine or physiological saline. Approxi- mately 2 h after the last injection when narcotic effects were visible in the phenylalanine-treated group, the ani- mals were injected intraperitoneally with 1 ml or less of picrotoxin or pentylenetetrazol solutions. The picrotoxin was dissolved either in water (250 mg/100 ml) or 95% ethanol (250 mg/lO ml); the pentylenetetrazol was used full strength (100 mg/ml) or diluted 1 to 10 with water. The interval between injection of the convulsant agent and the first tonic extension was measured. Study of the effect of L-phenylalanine on anaerobicgglyCOIysis Day-old chicks were decapitated, brains removed and homogenized in 1.15% neutralized KCl (9 ml/g tissue). After an initial 10-min centrifugation at 12,000 g, the fraction containing the soluble glycolytic enzymes was pre- pared by centrifugation at 100,000 g_for 30 min in a No. 40 rotor using a Beckman L2 ultracentrifuge. The glycolytic mixture (Weber, Glazer and Ross, 1970) contained the follow- ing components given as final concentration: ATP, 1 mM: MgSO 5 mM; NAD+, 1 mM: nicotinamide, 30 mM; 2 mM; KH PO 4’ 2 4' 52 glycylglycine, pH 7.4, 50 mM; glucose, 1 mM; a minimum of 20,000 cpm of D-(14C)-g1ucose per ml; 0.2 m1 of high-Speed supernatant per m1. Neutralized L-phenylalanine was added at the expense of water to a final concentration of 9 or 18 mM. The samples were incubated for periods of 0 to 60 min at 37°C in a Dubnoff shaker bath, after which the reaction was stopped by the addition of 10% trichloroacetic acid (1 ml per m1 of incubate). Radioactive lactate was recovered as the acetaldehyde-dimedone derivative according to the method of Long, Mashimo and Gump (1971). Into a wide-mouthed jar were placed 15 ml of a 0.4% aqueous dime- done solution, pH 6. An aliquot of the deproteinized sample (0.5 or 1.0 ml) was combined with 1 m1 of a lactate solution (10 mg/ml) in a 15-ml beaker; to this was added 1 ml of 20% cerric sulfate in 2 N H SO The beaker 2 4‘ attached to a rubber stopper by tape was immediately sus- pended in the jar; the jar was stoppered and shaken for 3 h at room temperature, during which time the lactate was chemically converted to acetaldehyde which, upon diffusion, reacted quantitatively with the dimedone. The product was precipitated upon acidification to pH 4 with 2 N acetic acid and recovered by filtration on preweighed Whatman No. 540 filter papers (2.4 cm). The air-dried precipitate was then weighed and counted on a Beckman CPM-100 scintil- lation spectrometer in 10 m1 of scintillation fluid (10 g 53 of 2,5-diphenyloxazole, 100 g of naphthalene and l leter of dioxane). No quenching was observed. Assay of creatine kinase in the presence of L-phenylalanine Commercial creatine kinase from muscle was assayed at 25°C by a Gilford recording Spectrophotometer with and without L-phenylalanine according to the procedure of Dawson (1970). The assay medium consisted of the follow— ing expressed as final concentrations: Tris-HCl, pH 7, 65 mM; glucose, 3 mM; NADP+, 0.125 mM: ADP, 2 mM: MgC12, 6.25 mM: 3.33 ug each of hexokinase and glucose-6-phosphate dehydrogenase per ml; 10 pg of creatine kinase per m1. L-Phenylalanine was added to yield either 3.6 or 9 mM. A reaction attributed to ATP contamination of the ADP was encountered and allowed to go to completion before addition of creatine kinase and phosphocreatine. The levels of phosphocreatine assayed ranged from 20 to 80 nmol/ml. Plasma osmolality determinations Hyperphenylalanemia was produced in the chicks by intraperitoneal injection of L-phenylalanine as previously described. Saline-injected animals served as controls. Osmolality was measured on plasma obtained by heart punc- ture, employing a Fiske G-66 osmometer. 54 Plasma electrolyte determinations The levels of potassium, sodium and calcium were measured on a Coleman flame photometer for the same samples used in the osmolality studies. As an additional control, the plasma from uninjected chicks fed a mash diet was included. A 1/100 dilution of the plasma in 0.02% Sterox was made for the potassium determination; l/200 for sodium; 1/25 for calcium. RESULTS Observations on the narcotic state induced in the chiCk by L:phenyl§1anine Photographs were taken of the animals 2 h after the third injection of the amino acid. The drowsy state is evident in Figure 3; the more alert, wide-eyed chick is representative of the saline-control group. Some of the phenylalanine-treated animals were more severely incapaci- tated (Figure 4). A loss of motor activity could easily be demonstrated by challenging the animal's normal right- ing reflexes. If a control chick were placed on its back, it would immediately maneuver to its feet again; however, the chick injected with L-phenylalanine had great diffi- culty in accomplishing this and would remain on its back for 5 sec or more (Figure 5). The legs of the chicks in this position could easily be flexed. Recovery studies When chicks were injected intraperitoneally with a total of 1.62 mmole of L-phenylalanine, levels of the amino acid greater than 2 umol/g of tissue were maintained in the brain for a period of 12 h after treatment; the increase in tyrosine followed that of phenylalanine, 55 56 Figure 3.--Photograph illustrating the drowsy state induced in chicks by the intraperitoneal injection of L-phenylalanine. The chicks were injected intraperitoneally 3 times at hourly intervals with 3 m1 of 0.18 M L-phenylalanine or 0.154 M saline and observed 2 h after the final injection. The more alert animal is representative of controls. Figure 4.--Photograph of a chick prostrated by serial injections of L-phenylalanine. The animals were treated as described for Figure 2. The animal standing is representative of the control group. 57 Figure 3. l” h‘unnma Figure 4. 58 Figure 5.--Photograph demonstrating loss of motor activity in chicks injected with L-phenylalanine. The injection procedure was identical to that described for the animals of Figure 2; 2 h following the last injection the chicks were placed on their backs and their righting reflexes were observed. The animals were capable of standing. 59 Figure 5. 60 peaking at 12 h (Figure 6). Both amino acids appeared to be rather rapidly metabolized and levels returned to normal by 18 h. Maximally, phenylalanine increased about 60 times the control value, whereas tyrosine was elevated 5 fold. Levels of ATP in the brain were not observed to change in the phenylalanine-injected group throughout the 24 h period; however, phosphocreatine increased by 15 - 20% after 2 h and the rise was maintained for about 12 h (5 h, p<0.02; 8 h, p<0.1) (Figure 7). L-a-Glycerol phos- phate (Figure 8) was affected earliest; levels remained depressed by 30 - 45% for 12 h compared to the saline- control values (2 h, p<0.02; 5 h, p<0.02; 8 h, p<0.10). The levels of fructose-1,6-diph03phate fell 30 - 40% after 2 h had elapsed (5 h, p<0.05; 8 h, p<0.10) and appeared to return to near normal levels after 8 h (Figure 8). Cere- bral levels of glucose in the control chicks decreased with time, rising slightly between 18 and 24 h (Figure 9); the rise correlated well with an increase in glucose in the plasma and is probably related to the intake of mash 60 min before the sacrifice. This was not observed in the phenylalanine group, but these animals did not eat when given the food. A very slight hypoglycemia (15%) was detected in the phenylalanine group at the early times studied (Figure 9). It appears that the phenylalanine- treated chicks maintained higher cerebral levels of glucose than the controls. 61 Figure 6.--Cerebral levels of phenylalanine and tyrosine in chicks during a 24 h period following intra- peritoneal injections of L-phenylalanine. Day-old chicks were injected intraperitoneally 3 times at hourly intervals with 3 m1 of 0.18 M L-phenyla- lanine or 0.154 M saline. The tissue was obtained as described in METHODS. Each value represents one deter- mination on tissue pooled from 3 animals. Analyses were done on an amino acid analyzer, utilizing 400 mg of brain. 62 5:00.00 :30. 00. 0 0* 2800 --.o..:...:.- 3 2 o. _ o. o. _ . . o . . . _ \L - p n 3 2 .l Incl... 00mm: .6 a .00 BE: 0502030095 4 24 l8 l2 Time in hours 2 1T7 Phe inj 63 Figure 7.--Cerebral levels of ATP and phospho- creatine in the chick during a 24 h period following injections with L-phenylalanine. Day-old chicks were injected intraperitoneally 3 times at hourly intervals with 3 ml of 0.18 M L- phenylalanine or 0.154 M saline. Values represent the average of Single determinations 1 standard deviation on 4 animals with the exception of the control values at 12 and 24 h for which n is equal to 3. Tissue was obtained and assays performed as described in METHODS. ' umol per g of tissue 64 2.5_ ATP ILL Ph Iolon’ 2.0- l/’ ~ any me “‘11 I 0} J I 1 1 l L 3'5 ' Phosphocreotine 3 o - L ‘ Phenylalanine Control L 1 Ht 2 5 8 I2 24 PM”! Time in hours 65 Figure 8.--Cerebral levels of L-d-glycerol phosphate and fructose-1,6-diphosphate during a 24 h period following L-phenylalanine injections in the chick. Refer to the description of Figure 4 for experimental details. 0.20 O.IO umol per g of tissue O.|O 66 L- d-glycerol phosphate L Phenyfilghine _ ._ _ _ _____I I Oftfit'rol 1 1’ . ixliT/z H..,........ l l 1112 5 e ['2 24 Time in hours 67 Figure 9.--Levels of glucose in the brain and serum of chicks during a 24 h period following injections with L-phenylalanine. Experimental details are identical to those for Figure 4. The serum glucose levels represent single determinations on blood pooled from 3 animals. 68 Cerebral glucose 4. 2 L 'r 3.4 ~ Phenylalanine j c» 1 fl ,,,,, g 2 6 - — -"" " ‘- '5 E l. 8 - :1 . I. OJ; COMI’O' 4L / . l . 1 . I e L Serum glucose __ l 6l' Control E \ as I 4+ a. '5 ~~- E I 2 '- // ““s~ =1 0” ’k‘ "’ Phenylalanine I O- I, ‘L 1 J l L 1 1n 2 5 3 l2 24 PM “'i Time in hours 69 Amino acid analyses The large dose of L-phenylalanine administered to the chicks produced very drastic changes in the profile of cerebral amino acids at two hours after the final injec- tion (Table 6). Lysine, histidine, phosphorylethanolamine, taurine, aspartic acid, threonine, serine, glutamic acid, alanine, valine, methionine, isoleucine and leucine were decreased. Neither arginine nor ammonia were affected. Only glutamine, glycine, phenylalanine and tyrosine were elevated. The level of histidine as calculated represents a minimum value in the saline-injected animals. A second component that chromatographed beneath the histidine peak may possibly have contained some histidine but was not included in the calculations. In the phenylalanine group the histidine level was below limits for accurate quanti- fication (less than 0.015 umol/g of tissue). The summation of free amino acids and ammonia in the control and phenylalanine-treated groups was 36.944 and 36.787 umol/g of brain, reSpectively (Table 6). Seizure threshold studies Convulsions produced in the chicks with either picrotoxin or pentylenetetrazol appeared similar; the animals underwent tonic-clonic movements followed by a tonic extension of limbs. In the experiments in which the chicks were injected with picrotoxin, the animals pre- treated with either l.08 or 1.62 mmol of L-phenylalanine Table 6.--Levels of Free Amino Chicks Injected with 70 Acids and Ammonia in the Brains of Saline or L-Phenylalanine. mom “32:23:?" (3.23:1! Umol/g of wet wt. Lysine 0.593 i 0.022* 0.414 t 0.051 70 Histidine 0.040 i 0.004* Not measurable Arginine 0.067 t 0.001* 0.073 t 0.002 109 Phosphorylethanolamine 1.910 t 0.100* 1.610 1 0.056 84 Taurine 8.750 t 0.310* 7.510 t 0.182 86 Aspartic Acid 2.450 t 0.136 1.930 t 0.136 79 (p<0.005) Asparagine and N-Acetyl ASpartic Acid 4.130 t 0.897 4.220 t 0.375 102 (NS) Threonine 0.383 t 0.041 0.240 t 0.034 63 (p<0.010) Serine 0.576 t 0.043 0.441 i 0.006 77 (p<0.025) Glutamic Acid 8.820 t 0.342 5.480 t 0.371 62 (p<0.001) Glutamine 6.880 t 1.340 9.480 t 0.729 138 (p<0.050) Glycine‘ 1.160 t 0.045 1.290 t 0.042 111 (p<0.025) Alanine 0.451 t 0.028 0.318 t 0.015 71 (p<0.005) valine 0.097 t 0.004 0.044 t 0.004 45 (p<0.001) Methionine 0.029 1 0.008 0.014 t 0.003 48 (p<0.050) Isoleucine 0.060 t 0.009 0.041 t 0.005 68 (p<0.050) Leucine 0.083 t 0.011 0.042 t 0.005 51 (p<0.005) Tyrosine 0.049 t 0.006 0.160 t 0.022 327 (p<0.001) Phenylalanine 0.048 t 0.010 3.140 t 0.414 6542 (p<0.001) Ammonia 0.368 t 0.127+ 0.340 t 0.103 92 (us) Total 36.944 36.787 100 Note: The 1-day-old chicks were injected intraperitoneally 3 times at hourly intervals with 3 m1 of 0.18 M L-phenylalanine or with 0.154 M saline, and decapitated 2 h after the last injection. The brains were immediately removed and frozen in liquid N 2. *These values represent the average of single or duplicate analyses i range from 2 pools of 5 brains each. averages of single or duplicate determinations from 3 pools of tissue, 5 brains each. the student's t-test. All other values are Statistical significance was determined according to +Quantified spectrophotometrically (Weil-Malherbe, 1969). 71 (Figure 10) underwent tonic extension at a later time than controls. This delay was more apparent with the higher amino acid treatment. The dose response curve for the convulsant agent was straight over a 50 fold concentration range in the controls. Some variation in control values was observed between experiments at the lower doses of picrotoxin. The survival rates of the saline and phenyla- lanine (1.08 mmol) injected groups differed markedly at 5 mg/kg picrotoxin. Only 1/5 controls recovered from the convulsions; the average time of death of the remaining four was 44 i 8 min; whereas 4/8 of the phenylalanine- treated animals did not undergo convulsions; 2/8 underwent convulsions but recovered completely and the other two chicks died after 45 and 60 min. No such protective ef- fect could be demonstrated at any other doses of picrotoxin. Levels of phenylalanine and tyrosine determined from a pool of brains from animals treated with 1.08 mmol of amino acid were 0.68 and 0.46 umol/g of tissue, respectively; phenylalanine measured in 3 pools of brains from animals treated with 1.62 mmol was 2.96 t 0.30 and tyrosine was 0.20 2 0.05 umol/g. This anti-convulsive effect of L- phenylalanine was also shown with pentylenetetrazol (Table 7). At a pentylenetetrazol dose of 200 mg/kg, the tonic phase was delayed by 20 min over the control (p<0.001). The brains from these animals were selectively pooled, 3 - 5 per pool and phenylalanine and tyrosine 72 Figure lO.--Seizure threshold to picrotoxin in the chick after intraperitoneal injections of L-phenylalanine. Day-old chicks were injected intraperitoneally 2 or 3 times at hourly intervals with 3 m1 of 0.18 M L- phenylalanine or 0.154 M saline; 2&1/2—3 h after the final injection, the animals were injected with picrotoxin as described in METHODS and the interval before the onset of tonic extension was noted. The values 2 standard devia- tion represent averages from 5 animals excepting one phenylalanine group mentioned in RESULTS. N 01 uh O O O 0 Time before tonic extension (min) 73 I.08 mmol injected 3.56 g per kg body weight Phenylalanine ‘ ‘ c—C ontrol l.62 mmol injected 5.34 g per kg body weight § | ~. .~ rut .§ 4 5. _’ + Phenylalanine Control \ #f/g L I 11 l IOO ISCT r 500 Picrotoxin mg per kg 74 Table 7.--Seizure Threshold to Pentylenetetrazol after Intraperitoneal Injections of L-Phenylalanine. Interval Before Tonic Extension Pentylenetetrazol Saline Phenylalanine 200 mg/Kg 8.5 i 4.2 min (n = 15) 28.8 i 21.2* min (n = 16) 400 tug/Kg 3.0 i 2.7 min (11 - 6) 3.1 t 1.61 min (n = 6) Note: Chicks were injected intraperitoneally 2 times at hourly intervals with 3 m1 of 0.18 M L-phenylalanine or 0.154 M saline; 2&1/2 - 3 h after the last injection, the pentylene- tetrazol was administered. The amino acids were determined on representative tissue samples using an automated analyzer. *p<0.001; cerebral level of phenylalanine is 1.52 i 0.69 umol/g; level of tyrosine, 0.21 i 0.07 umol/g. +Cerebra1 level of phenylalanine is 1.01 and tyrosine, 0.26 mmol/g- 75 were determined for 4 such pools. Phenylalanine was pre- sent at levels of 1.52 i 0.69 umol/g and tyrosine at 0.21 t 0.07 umol/g. Effect of Ljphenylalanine on anaerobic glycolysis and creatine kinase activity It was of interest to determine if any of the in zizg_observations could be demonstrated in simple i2_!iE£g_ systems. Production of lactate was studied for the 100,000 g soluble fraction of chick brain with and without L- phenylalanine (Table 8). Concentrations of the amino acid as high as 18 mM exerted no inhibition. Also 9 mM L-phenylalanine had no effect on creatine kinase cata- lyzed phosphorylation of ADP (Figure 11). Studies of the effects of other amino acids on the Ehick Several amino acids were injected into the chick according to the same procedure used with L-phenylalanine, and the animals were observed for a 24 h period after the third injection (Table 9). The animals treated with L-histidine, L-methionine, D- or L-phenylalanine and L- tryptophan were most severely debilitated; they were most inactive and in a majority of instances prostrated. L-Leucine, L-lysine, L-threonine, L-valine or hydroxy- proline had slight or no effect. The mortality rates correlated with the severity of the conditions. 76 Table 8.-—In vitro Study of Anaerobic Glycolysis in Chick Brain in the Presence of L-Phenylalanine. Conversion of [14C] Glucose to [14C] Lactate Control 9 mM % of 18 mM % of L-Phenylalanine Control L-Phenylalanine Control 3M3" sari/22* Exp . I+ 15 min 35 36 103 36 103 30 min 134 132 99 148 110 45 min 250 291 116 297 119 60 min 391 420 107 496 126 Exp. 11* 15 min 1.4 1.8 129 1.4 100 30 min 7.8 6.8 87 6.8 87 45 min 17.0 20.0 118 19.4 114 *[14C]lactate was isolated as the acetaldehyde-dimedone derivative. See METHODS. cpm/mg indicates the total radioactivity in this final product. +290,000 cpm of [6-14C]glucose/l ml incubation. +18,750 cpm of [U-14C1glucose/l ml incubation. 77 .mcficmamaxcmnmlq mo mocmmmum 03» CH mmmcfix mCHuomHo mo mafi>wuo¢ll.aa ousmflm 78 _E be oczootoocamoca 8 BE: oo. 8 8 oe om - u u q - use—c.2325 LEWM ” \. .2200 o .. ID (5 ugul-|ul Jed peuuo; d iv 10 |OUJU L 10 79 Table 9.--Survey of the Effects of Several Amino and Imino Acids on the Chick. Compound Physggglcicigvity degtgzrigd L-Leucine Slight Reduction 0/5 L-Lysine Slight Reduction 0/5 L-Histidine Severe Reduction 1/4 L-Methionine Severe Reduction 3/5 L-Phenylalanine Severe Reduction 2/5 D-Phenylalanine Severe Reduction --- L-Tryptophan Severe Reduction 4/5 L-Threonine No Change 0/5 L-Valine No Change 0/5 Hydroxyproline No Change l/S Note: The chicks were injected intraperitoneally 3 times with 3 ml of a 0.18 M neutralized amino acid solution, except for tryptophan which was 0.09 M. The animals were observed for a 24-h period. 80 Effect of the injection procedure on plasma osmolality and Electrolyte concentrations A considerable volume of water (9 m1) and quantity of sodium chloride or L-phenylalanine was injected into the chicks. Previously the level of phenylalanine in the plasma of the chicks was observed to increase from 0.16 to 10 - 15 umol/ml (Granett and Wells, 1972). The extent to which the treatment alters blood electrolytes was investigated by determining plasma osmolality and the levels of several cations. The absorption of the water, NaCl or amino acid by the chick did not modify the osmolality (Table 10) compared to a value of 309 i 7 milliosmol/kg obtained by Knull, Wells and Kozak (1972) for uninjected chicks. The levels of potassium, sodium and calcium did not differ in the two injected groups; but when compared to the uninjected animals, potassium was 160% higher regardless of the solute injected (Table 11). 81 Table 10.--Comparison of Plasma Osmolalities in Phenyla- lanine and Saline-Injected Chicks. Osmolality milliosmol/Kg Saline Group 306 i 7 Phenylalanine Group 307 i 4 Note: Chicks were injected intraperitoneally 3 times at hourly intervals with 3 m1 of 0.18 M L-phenylalanine or 0.154 M saline. Blood was obtained by heart puncture 4 h after the last injection. Each value represents the average of duplicate determinations on 3 separate pools of plasma, 5 animals per pool. 82 Table 11.--Plasma Concentrations of Sodium, Potassium and Calcium in Uninjected Chicks and Chicks In- jected with Saline or L-Phenylalanine. Injection K+ Na+ Ca++ milliequivalents/liter None* 5.03 t 0.36 136 H- l 5.80 t 0.06 Saline+ 7.98 I+ 0.80 139 t 2 6.56 i 0.08 L-Phenylalanine+ 8.65 i 0.35 135 i 2 6.16 H- 0.08 Note: The values represent averages of duplicate deter- minations on 3 different samples, except for the value for potassium in the uninjected group for which n = 6. *The chicks were mash-fed, uninjected. +These same samples were used in the osmolality studies. DISCUSSION The recovery studies confirm many of the earlier observations (Granett and Wells, 1972); that is, the level of phosphocreatine is elevated and the concentra- tions of fructose diphosphate and L-a-glycerol phosphate are depressed. Standard deviations are larger than pre- viously recorded since analyses were performed on indi- vidual brains rather than on pooled tissue and thus varia- bility amongst animals is quite prominent. A very slight hypoglycemia was evident throughout the time course, which is compatible with the report of Fajans, Floyd, Knopf and Conn (1967) that many essential amino acids including phenylalanine are capable of invoking an in- sulin release and thus lowering plasma levels of glucose in humans and rats. The abrupt rise in glucose concentra- tions in the control plasma, also reflected in the brain, correSponds to the intake of food 60 min before the 24 h sacrifice. Up to this time the animals had been deprived of food and water. The phenylalanine-treated animals refused the food and were abnormal in this respect, al- though they did appear more alert at the end of the 83 84 experiment. No decline was observed in the glucose levels in the brains of the phenylalanine-treated chicks as in controls. Evidence has been presented (Lowry, Passonneau, Hasselberger and Schulz, 1964; Mayman, Gatfield and Breckenridge, 1964; Brunner, Passonneau and Molstad, 1971) that barbiturate or volatile anesthetics increase serum or cerebral levels of glucose and the brain/serum glucose ratio. The levels of glucose-G-phosphate also increase in the brain. It has been difficult with our system to consistently demonstrate an increase in glucose or glucose-G-phosphate (Granett and Wells, 1972), but in these recovery studies it would appear that the brain/ serum glucose ratio is increased in the phenylalanine- treated group and that the glucose is not being depleted from the brain as in control animals, perhaps because of a postulated reduced utilization. Of the metabolites examined L-a-glycerol phos- phate was affected earliest. The most significant changes in this compound, fructose diphosphate and phosphocreatine were associated with the highest concentrations of phenyl- alanine in the brain and their levels appeared to return to those observed in control animals as the amino acid concentration fell. The data implies that phenylalanine may be the active compound, directly or indirectly, causing the altered state of energy metabolism; however, 85 the participation of a metabolite of phenylalanine follow- ing a similar concentration profile cannot be ruled out. Evidence has been presented from the rat and human brain systems that phenylalanine directly inhibits glycoly- sis at pyruvate kinase (Weber, 1969). This inhibition was not demonstrable in the chick (Granett and Wells, 1972). Furthermore, L—phenylalanine did not inhibit lactate forma- tion in a fortified 100,000 g_supernatant fraction prepared from chick brain. Inhibition was reported by Weber, Glazer and Ross (1970) for such a system purified from human brain and in human and rat brain slices (Ki of 2 and 17 mM for 2 and 7 day-old rats, respectively). In some preliminary work with brain slices from the chick, we found L- phenylalanine (3.6 mM) to inhibit lactate formation by 10 - 30%; in one experiment with 10 mM L-phenylalanine both stimulation and inhibition were observed. More work is needed before any significance can be attached to these findings in slices, but it would appear that L-phenylalanine in_gi££2_does not affect glycolysis in chick brain as it does in rat brain. Any direct interaction of phenylalanine with a glycolytic component in chick brain remains unproven. Recently levels have been reported for cerebral amino acids in the young chick (Levi and Morisi, 1971; Blosser and Wells, 1972). In general our analyses are in good agreement with their work. Discrepancies may be due 86 to differences in strain (White Leghorn versus White Livorno). Also the validity of equating our saline-treated control animals with uninjected controls is questionable. It is known that brain slices under certain conditions will swell in saline with sodium being taken up and potas- sium being extruded from the tissue (Franck and Schoffeniels, 1972). If such were occurring in our ani- mals in which elevated plasma levels of potassium were observed, it is reasonable that the concentrations of other cellular constituents would be altered. Dobkin (1972) demonstrated that cerebral levels of glutamine were altered by intraperitoneal injection of 0.9% NaCl in the rat. The differences between the amino acid levels in the control and phenylalanine-treated animals suggest in- hibition at transport sites. In yitrg L-phenylalanine (2 - 10 mM) has been shown to reduce the initial rate of influx or the steady-state accumulation of glycine, ala- nine, leucine, arginine, lysine, methionine (Blasberg and Lajtha, 1965 and 1966), histidine (Neame, 1964) and serine (Abadom and Scholefield, 1962) in cerebral slices. On the basis of their studies Cohen and Lajtha (1972) distin- guished separate transport sites for at least eight classes of amino acids; namely, small neutral, large neutral, small basic, large basic, acidic, GABA, amido and imino acids. L-Phenylalanine was described as having a significant affinity for the transport systems of other neutral amino 87 acids and basic amino acids, but as not affecting the influx of the dicarboxylic group. This corresponds well to our finding except for arginine and glycine which were not decreased in concentration and aspartic and glutamic acids which were decreased. Since the decrease in gluta- mate balances the increase in glutamine, the formation of glutamine may represent an attempt of the cell to alle- viate an accumulation of ammonia resulting from metabolism of phenylalanine in the liver. However, plasma levels of amonia were not quantified and therefore cannot be assumed to be elevated. Should the detoxification mecha- nism be active, it would appear to be adequate as ammonia levels are essentially equal in the brains of both saline and phenylalanine-treated groups. Since aspartate and alanine are synthesized by transamination of oxaloacetate and pyruvate, respectively, by glutamate, a reduction in their levels could be due to the decrease in amine donor. An alternative explanation for the decreased cerebral levels of aspartic and glutamic acids and alanine and serine as well is related to their metabolic proximity to glycolysis and the tricarboxylic acid cycle; the levels may reflect slower carbon flow. The rate of glycolysis has been shown to be lower in the phenylalanine-injected group (Granett and Wells, 1972) (see Chapter I, this section), although the rate of the tricarboxylic acid cycle was not examined. The synthesis of these amino 88 acids appears to be rapid enough to be affected within the few hours of the acute study as Yoshino and Elliott (1970) demonstrated considerable radioactive labeling in the amino acids in rat brain 15 min after injection of [14C] glucose in the tail vein. In addition to influx and metabolic alterations, another dimension to the steady-state levels of cerebral amino acids possibly disturbed under our conditions is the efflux of amino acids from the central nervous system. Active transport of amino acids between the blood and cerebrospinal fluid has been demonstrated ig_vitro with choroid-plexus tissue (Lorenzo and Cutler, 1969) and in giyg with the ventricular perfusion technique (Cutler and Lorenzo, 1968). The effects of excess phenylalanine on cerebral amino acids have been described most frequently for the rat. Significant reductions in cerebral conCentrations of threonine, valine, methionine, isoleucine, histidine and leucine were observed by McKean, Boggs and Peterson (1968) in acute injection studies with both immature and mature rats. No change was observed in the level of arginine. Most of these observations were confirmed by Castells, Zischka and Addo (1971). Lowden and LaRamée (1969) examined non-essential amino acids and found ala- nine, serine, glutamic acid and aspartic acid decreased in the brains of 10 day-old rats. Analyses of the brain 89 tissue from untreated phenylketonuric subjects (McKean and Peterson, 1970) demonstrated a reduction in threonine and an elevation in glutamine, but the paucity of data available disallows any extensive comparison with the chick. The depressed physical response of the chick to extremely high levels of L-phenylalanine is unlike that of the rat. Rats subjected to a rigorous injection series with the amino acid reacted in a manner characteristically sympathomimetic (Goldstein, 1961). Heart and reSpiratory rate were increased; surface blood vessels were constricted; gastrointestinal tone was reduced; and analgesia was dis- played. Most severely affected animals demonstrated tremors. Injection of the rats with the amino acid and a monoamine oxidase inhibitor, iproniazid (also an anti- depressant), produced similar and more potentiated effects. In contrast the phenylalanine-treated chicks at no time were hyperactive. Noteworthy is the fact that high phenyl— alanine concentrations in the brain greater than 3 umol/g of tissue were necessary to produce either of the two behavioral states; these different responses would indicate separate mechanisms for toleration of this amino acid in the rat and chick. More compatible with our observations is a deple- tion in brain catecholamines. It has been reported that rats treated with non-toxic doses of a-methyl tyrosine, an 90 inhibitor of tyrosine hydroxylase, become depressed as quantified in behavioral studies, and the cerebral catecho- lamines are reduced (Rech, Borys and Moore, 1966; Rech, Carr and Moore, 1968). This particular aspect has not as yet been examined in the phenylalanine-treated chick, but it is possible that the extremely high concentrations of the amino acid could alter synthesis or storage of nore- pinephrine. Phenylalanine has been reported to competi- tively inhibit the formation of 3,4-dihydroxyphenylalanine from tyrosine with tyrosine hydroxylase from adrenal gland (Xi = 1 - 3 x 10“ M), heart and caudate nucleus (Ikeda, Levitt and Udenfriend, 1967). Phenylalanine could also inhibit synthesis by chelating copper (Albert, 1950) at the dopamine B-hydroxylase step. Cu (++) chelators have been found to inhibit this enzyme (Goldstein and Nakajima, 1967). The studies with picrotoxin and pentylenetetrazol confirm the hypoactive state produced in the chick by phenylalanine. Decreased cerebral excitability was evi- denced by the delayed tonic phase of the convulsion and occasional total elimination of clinical signs; a rather large dose of L-phenylalanine was required. In this re- spect the chick also differs from the rat, for using the convulsant, hexafluorodiethyl ether, Gallagher (1970) demonstrated that phenylalanine reduced seizure threshold in immature rats. The effect appeared to be independent 91 of derived tyrosine or phenylpyruvate (Gallagher, 1971). However, his experiments differed from ours in that DL- phenylalanine was utilized and the levels of phenylalanine did not exceed 1 umol/g of brain. In agreement more with phenylalanine's effect in the chick than in the rat is Nigam, Watson and Marcus' (1968) study of electroencephalo- graphic effects of L-phenylalanine in the cat. Generalized slowing was observed when plasma phenylalanine levels were elevated beyond 1.1 umol/ml. Utilization of varied methods for production of convulsions should aid in elucidating the mechanism of phenylalanine's effect. However, the studies with picro- toxin and pentylenetetrazol thought to induce cerebral excitability by two distinct pharmacological actions (namely, picrotoxin blockage of presynaptic inhibition and pentylenetetrazol shortening neuronal recovery; ESplin and Lablicka, 1967) would indicate a general rather than specific effect since phenylalanine suppressed convulsive activity induced by either agent. Attempts to measure phenylalanine's protection against a third seizure inducer, electroshock, were unsuccessful due to technical diffi- cultires involving the eyelid closure reflex of the chick displacing the corneal electrodes. Several substances including GABA and alanine injected into mice as hyperosmolar solutions (1 M) have offered protection against convulsions. Mild dehydration 92 of the brain with increased plasma osmolarity followed the treatment (DeFeudis and Elliott, 1967; DeFeudis, 1971), and it was suggested that the anti-convulsive activity was a consequence of the loss of water from the brain and unre- lated to the particular compound injected. This theory is not new. In 1929 Fay used fluid restriction as therapy against convulsions and McQuarrie, Anderson and Ziegler (1942) introduced as a diagnostic tool a hydration test for inducing seizures. Toman and Goodman (1948) emphasized the concommitant changes in electrolyte balance. Our ex- perimental conditions differ from those of DeFeudis in that our solutions of L-phenylalanine were slightly hypo- osmolar and regardless of the increased plasma levels of phenylalanine to 10 - 15 umol/ml (Granett and Wells, 1972) the plasma was iso-osmolar. Also the elevated levels of potassium in the plasma were compatible with water uptake by various tissues, not dehydration (Franck and Schoffe- niels, 1972). Since the saline-injected controls, which were alert, exhibited the same elevated potassium levels, it is unlikely that water exchange is instrumental in pro- ducing the sedated state in the animals treated with L- phenylalanine. Another factor worth consideration is the role of ketone bodies in our system. Phenylalanine is catabolized eventually to acetoacetate and fumarate. Ketosis induced by fasting has been a classic treatment for seizures since 93 ancient times in lieu of SOphisticated therapy. Currently high fat and low carbohydrate diets are being used with children with seizure disorders who haven't responded positively to normal anticonvulsive drugs. Uhlemann and Neims (1972) demonstrated significant protection against maximal electroshock using ketotic mice, but they were unable to quantitatively relate blood ketones and the posi- tive or negative response. Levels of B-hydroxybutyrate presently being determined in the plasma of the phenylalanine-treated chicks have been found to be about 100% higher than in controls. Acetoacetate hasn't been quantified as yet. Mechanisms by which ketosis exerts anti-convulsive activity are not well defined. Various laboratories have emphasized the ketone bodies, per se, utilization of ketone bodies by the brain for energy instead of glucose, dehydration, electrolyte imbalance, metabolic acidosis, or hyperlipemia in one way or another (refer to Uhlemann and Neims, 1972, for specific refer- ences). Fischer (1951) concluded from studies of ketosis in rabbits and humans that ketone bodies probably are not the direct cause of the depressed state in diabetic coma. It was reported that fatty acids and not B-hydroxybutyrate reduced muscular tone and activity, producing unconscious- ness in the rat (Samson, Dahl and Dahl, 1956) and altered electroencephalographic patterns in the rabbit, suggestive of sleep (White and Samson, 1956); of carbon chains ranging 94 in length from acetate to decanoate, the longer chain fatty acids were demonstrated more effective. Acetoacetate was not examined. Thus at this point it is very difficult to definitively relate the presence of the ketone bodies in the chick to the sedation associated with phenylalanine, but it is a factor deserving additional experimentation. At present nothing is known about the steady-state levels of lipids in the phenylalanine-treated chick; if insulin were being secreted, a lowering in fatty acids could be expected; but it is an interesting area in view of the narcotic effect reported with fatty acids. Already a difference is apparent between that system and ours. Dahl (1968) correlated the effective narcotic concentra- tion of the various fatty acids ig_ziyg_with in yitgg in- hibition of the (Na+ + K+)-ATPase, postulating that the two phenomena rather than being causally related were linked through a common action of the fatty acid on the membrane lipids. This is in contrast to the observation of Ting- Beall and Wells (1971) with L-phenylalanine, which stimu- lates (Na+ + K+)-ATPase, in vitgg, probably through a mechanism involving chelation of divalent cations that would otherwise inhibit the enzyme. Reversible blockage of the action potential in vagus nerve preparations was demonstrated with sodium octanoate (Dahl, Shirer and Balfour, 1966). Now in progress in this laboratory are experiments by Dr. Ting-Beall to determine the effect of 95 L-phenylalanine on the action potential in peripheral nerve. It has been demonstrated that L-phenylalanine complexes with calcium which suggests that the amino acid could dis- place the calcium from the axonal membrane surface (Ting- Beall and Wells, 1971). Tasaki, Watanabe and Lerman (1967) have shown that calcium is necessary for axonal membrane excitability. ACKNOWLEDGMENTS Dr. Hyram Kitchen is acknowledged for the use of his amino acid analyzer and Miss Irene Brett for her very fine technical assistance. I am indebted to Mrs. Betty Schoepke for the electrolyte analyses. Also I would like to thank Mr. Willie Taylor for his energies on the anti- convulsion studies. 96 SUMMARY The chick was investigated as a model system for the study of phenylketonuria, the genetic disease in which the hydroxylation of phenylalanine to tyrosine is blocked. Hyperphenylalanemia was produced by hourly intraperitoneal injections of 3 ml of 0.18 M L-phenylalanine over a 3 h period, resulting in the administration of 1.62 mmoles of the amino acid. Shortly following the final injection, the animals were observed to enter a state of narcosis which was reversible within 24 h. The animals lost normal righting reflexes. Levels of phenylalanine reached 10 - 15 umol/ml of plasma (control value, 0.16) and 2.5 - 3.5 umol/g of brain tissue (control value, 0.05). Examination of several cerebral glycolytic intermediates and high energy phosphate compounds at a time when the concentra- tion of phenylalanine in the brain was highest disclosed elevated levels of phosphocreatine and lowered levels of fructose-l,6-dipho5phate, lactate and L-a-glycerol phos— phate. Glucose and glucose-6-phosphate were either un- changed or elevated; glycogen remained unchanged and adenine nucleotide energy charge was not affected. Some 97 98 of these data are compatible with those observed for mouse brain under conditions of barbiturate anesthesia. The ”closed-system" technique revealed that the rate of utili- zation of high energy phoSphates was depressed as is also characteristic of the anesthetized state. Attempts to demonstrate ig_yi£§2_an effect of phenylalanine on anae- robic glycolysis with high-speed supernatant fractions prepared from chick brain or on creatine kinase were nega- tive. In recovery studies the changes in the metabolites indicated previously coincided quite well with cerebral phenylalanine levels of 2 umol/g or more. A very slight hypoglycemia was associated with the phenylalanine treat— ment . The large increase in phenylalanine in brain was shown to significantly reduce the levels of all the essen- tial amino acids, with the exception of tryptophan which was not quantified. A probable explanation is that phenylalanine inhibited entry or exit of the amino acids at translocation sites on the membrane. Some of the non- essential amino acids were also decreased which may be attributed to the reduction in carbon flow through gly- colysis. Only phenylalanine, tyrosine, glutamine and glycine were elevated. Ammonia levels were unaltered. In the animals treated with L-phenylalanine the tonic phase of pentylenetetrazol and picrotoxin induced 99 convulsions was delayed and in some cases eliminated which is compatible with barbiturate actions. When the injected animals were compared with uninjected ones, the injection procedure was found not to alter plasma osmolality; however, potassium was ele- vated in the plasma of both the saline control and phenylalanine-injected groups, while sodium and calcium were unchanged. It was concluded that the technique alone was not a factor in the response observed with the chicks injected with phenylalanine. This investigation has revealed two major dif- ferences between the chick and the rat as model systems for phenylketonuria. Firstly, the two species react to the amino acid loads in different ways. The rat does not become sedated as does the chick. Phenylketonurics do not display hypoactivity, but more often hyperkinesis, agitated behavior, tremors and seizures. Secondly, phenylalanine appears to inhibit brain pyruvate kinase in both rats and humans (Weber, 1969) but is ineffective in the chick. Thus it would appear that studies on the effects of phenylalanine in the chick may be less rele- vant to hyperphenylalanemia in humans and more significant for the demonstration of sedation, a previously unknown action of the amino acid. Indications are that studies with histidine, tryptOphan and methionine would be useful from a comparative point of view as these amino acids also 100 showed on gross observation similar toxicity in the chick. In conclusion, from indirect evidence the state of narcosis induced in the chick may be mediated by an activity of L-phenylalanine on transmission of nerve impulses either by interference with the synthesis of the transmitter species, norepinephrine, or by impair- ment of the propagation of the impulse along the nerve. The alterations in the glycolytic intermediates that were observed may reflect a depressed electrical activity in the brain and a decreased demand for energy. III. SECTION II CHAPTER I STUDIES ON CEREBRAL GLYCOLYTIC FLUX IN GALACTOSE-TOXIC CHICKS INTRODUCTION A study was made of certain aSpects of the cerebral energy metabolism in chicks intoxicated with galactose. Previously, the levels of various glycolytic intermediates had been found to be lower in the brains of cockerels fed a diet containing galactose (40% w/w), and it was of interest to know to what extent the rate of glycolysis was affected. The "closed—system" technique described in the previous section was used to estimate the flux. In combi- nation with other relevant work, the data presented here is currently in press under the title, "Studies on Cerebral Energy Metabolism during the Course of Galactose Neuro— toxicity in Chicks," by S. E. Granett, L. P. Kozak, J. P. McIntyre, and W. W. Wells, in Journal of Neuro- chemistry. Striving to provide the reader with a complete dimension of the problem of galactose-toxicity in the 101 102 chick, in the following literature review I have summarized various efforts in the area, including those perhaps only indirectly related to cerebral energy metabolism. LITERATURE REVI EW Rutter, Krichevsky, Scott and Hanson (1953) re- ported that chicks fed a diet greater than 10% in galac- tose displayed ataxia, tremors and convulsions, followed by death. Degeneration of neurons was associated with the toxicity (Rigdon, Couch, Creger and Ferguson, 1963). Galactose and its metabolites, galactitol, UDP galactose and galactose-l-phosphate, accumulated in the brain as well as in other tissue (Wells and Segal, 1969; Kozak and Wells, 1971). It has been established that the female is more sensitive to the high dietary levels of galactose than the male (Nordin, Wilken, Bretthauer, Hansen and Scott, 1960). Mayes, Miller and Myers (1970) demonstrated that the levels of galactose-l-phosphate were higher and the activity of galactose-l-phosphate uridyl transferase lower in females than males and suggested these differences might account for the higher mortality rate in female chicks. The capacity of chick brain to oxidize galactose was found to be low in comparison to that for glucose, and the activity was 5 to 10% of that observed for the kidney (Wells, Gordon and Segal, 1970). 103 104 Kozak and Wells (1969) reported a perturbation in high energy phosphate metabolism (fall in adenylate energy charge) and glycolysis in galactose-toxic chicks. The cerebral levels of phosphocreatine, ATP and many glycolytic intermediates were depressed, while ADP, AMP and Pi were elevated. The incorporation of 32P into galactose-l- 32 pho3phate was rapid and very similar to that for P into -32P]-ATP while little galactose was being incorporated [Y into glycogen, lipid, or glutamate. To explain this enigma and account for hydrolysis of ATP, the following mechanism of a futile ATPase was proposed: Galactokinase Galactose + ATP : Galactose-l-Phosphate -+ ADP Phosphatase Galactose-l-Phosphate + Galactose + Pi 4. A time course study of the dietary period revealed that ATP and g1ucose-6-phosphate decreased early in the feeding period (9 h), whereas reductions in other glycolytic metabolites were not observed until 18 or more hours had elapsed. The cerebral level of glucose fell from 1 - 2 umol/g of tissue to as low as 0.2 - 0.3 (Granett, Kozak, McIntyre and Wells, in press). No hypoglycemia has been observed (Rutter EE.§l:' 1953). Chicks administered galactose via drinking water as Opposed to diet display hyperosmolality (160% of control), and dehydration of nervous tissue has been suggested as a 105 mechanism for the convulsions (Malone, Wells and Segal, 1972). However, Knull, Wells and Kozak (1972) have pro- duced seizures under more mild hyperosmolar conditions (110% of control) using the synthetic diet 40% by weight in galactose and shown that symptoms of excited motor activity and alterations in the cerebral energy metabolites can be reversed by treatment with glucose without simultaneously decreasing the slight hyperosmolarity. Rates of protein synthesis and degradation were examined in the brains of the galactose-toxic chick and found to be similar to those in the control animal. Analy- sis of the free amino acids revealed alanine and leucine most significantly reduced and aSpartate elevated. Poly— riboSomal profiles were unaffected by galactose diet (Blosser and Wells, 1972), Glycoprotein metabolism, how- ever, appeared to be altered as judged by a faster rate of incorporation of [3Hl-glucosamine which could not be attri— buted to a difference in Specific activity of precursor pools (Knull, Blosser and Wells, 1971; Blosser and Wells, 1972). Lysosomes from galactose-toxic chicks were found to be more labile to hypo-osmotic shock and temperature in contrast to those isolated from control chicks, and the fragility was associated with the accumulation of galactose and galactitol (Blosser and Wells, in press). MATERIALS AND METHODS Animals and materials Male White Leghorn chicks (1 to 2 days old) were purchased from Cobbs, Inc., Goshen, Indiana, and housed in a brooder at 32° for the experimental period. As previously described (Kozak and Wells, 1969), the diets were either free of galactose or contained 40% (w/w) of D-galactose (General Biochemicals, Chagrin Falls, Ohio) in place of an equal amount of cerelose (D-glucose monohydrate). Enzymes and coenzymes were purchased from Sigma Chemical Co. (St. Louis, Mo.) or Boehringer Mannheim (New York, N.Y.). Substrate analysis Perchlorate extracts of tissue were prepared as described by Kozak and Wells (1969). The acid-soluble phosphates and glycolytic intermediates were quantified enzymatically from neutralized extracts according to methods previously described (Granett, 1970; Kozak and Wells, 1969). Citrate concentrations were determined by the fluorometric procedure of Williamson and Corkey (1969) utilizing citrate lyase (EC 4.1.3.6) and malate dehydrogenase (EC 1.1.1.37). 106 107 The gas liquid chromatographic procedure of Sweeley, Bentley, Makita and Wells (1963) was employed to measure galactose and galactitol levels on perchlorate extracts desalted with MB-3 resin (Rohm and Haas Co., Philadelphia, Pa.). "Closed-system" studies The chicks, 3 to 4 days of age, were fed their re- spective diets for 46 hours. Anoxic conditions were pro- duced by decapitation, and the heads were frozen in liquid N2 after periods of up to 10 minutes. For intervals beyond 18 sec, the heads were incubated at 40°C; otherwise, they were maintained at room temperature. For each period of anoxia, there were four control and four experimental chicks and individual brains were chipped out and pooled. Metabolic rate, defined in these studies as the rate of utilization of actual and potential high energy phosphates, was calculated according to the formula (Lowry, Passonneau, Hasselberger and Schulz, 1964): 2 AATP + AADP + Aphosphocreatine + Alactate where Aexpresses the change in the level of the metabolite during the period of anoxia. The term, Alactate, was used instead of 2 Aglucose + 1.45 (Alactate - 2 Aglucose) since glucose exhibited considerable variation early in the time course (see RESULTS). 108 Glycogen analysis Brain glycogen was purified and hydrolyzed according to the method of Walaas and Walaas (1950), which involves base digestion of the perchlorate pellet, ethanol precipi— tation of the glycogen, followed by acid hydrolysis. The glucose released was then quantified according to the fluorometric procedure of Lowry eE_§l. (1964). After 10 min of anoxia, glucose-yielding material was still isolated in amounts (0.5 umol glucose/g of brain) similar to those observed at 2 and 5 min of anoxia. This value was observed for both dietary groups. The possibility that the basal level of 0.5 umol released glucose/g of brain may be due to sources other than glycogen was investigated since glycogen should have been depleted under our conditions of anoxia. An attempt was made to demonstrate that the material iso- lated at the 10 min period was not glycogen using highly purified a-l,4-glucan glucohydrolase (EC 3.2.1.3) (Pazur, Simpson and Knull, 1969), an enzyme previously shown to hydrolyze a-1,4— and a-l,6-glucosidic linkages (Pazur and Kleppe, 1962). The 10 min sample proved to be completely resistant to treatment with the hydrolase, whereas material representing the zero time was not. Thus, all the glycogen data presented here has been corrected for contamination from non-glyCOgen, glucose-containing polymers. Refer to the following chapter for a more complete report on the analysis of glycogen with the glucohydrolase. RESULTS Employing the "closed-system" technique of Lowry gt El' (1964), the levels of the adenine nucleotides, phoSphocreatine, glycogen, glucose, fructose-1,6-diphoSphate and lactate were determined in the brains of chicks fed, reSpectively, control or galactose-containing diets for 46 hours at various intervals of anoxia (Figures 12-16). As expected, utilization of phosphocreatine occurred promptly in both the control and galactose-fed groups, the phosphocreatine being lower with the onset of anoxia in the brains of chicks fed galactose (Figure 12). The ATP expenditure was initially delayed in both groups, the ATP presumably being replenished by the action of creatine phosphokinase (EC 2.7.3.2), but utilization was apparent as phosphocreatine became limiting. This occurred at an earlier period (12 sec) in the brains of chicks fed galac— tose than in those fed the control diet (24 sec) (Figure 13). One minute after decapitation, the level of ATP was only 10% of the initial in chicks fed galactose, whereas 57% remained in brains of animals fed the control diet. AMP levels were elevated in animals fed galactose at the onset of ischemia as previously noted (Kozak and Wells, 1969) 109 110 .mafimuoo HmucoEHummxw How moomamz mom .mcoHumcflfinwumo 0808 no m now am a ucommumou when HMOfluum> one .mcflmun v mo doom a 800m moHdEmm wommfiu Hmsofl>fiocw so 050 oowuumo mums mcoflumcflEumumo one .n me How muoflo o>Huommmmu neon» pom mumz mxofino one .mumao mcflcfimucoolomouomHmm no Houucoo new mxoflno mo mcflmun ca mafiummuoonmmocm ca mHEmsomH Saunas numom mo cofiumuso on» no coHuocsm a mo omcanI|.~H musmflm wholes per g of tissue 2.0 _Lf IS 30 111 PHOSPHOCREATINE galactose-fed ' 45 6° Time in Seconds 112 .NH ousmfim How we oEmm ecu mum3 mcoHuwocoo HmucmEHummxm .owuuoam OHM mCOHHMGflfiuwnwa OHOE .HO m MOM 0m + mGMOE $39 .meHno cowlomouomamm no Houucoo mo mcflmun as» aw mz¢ can ma< CH seawnomfl EmuHOEumom mo coflumuso 0:» mo cofiuoc5m a mo momcmnunn.ma musmflm 113 25... 3352 3:33 o. n u r. 9 on o. E q d 1 m \ I. \O/ \\MIIIIW\\~ /H\ I \ \ xx than.” \N. \\ a 1 «at: t \ one-8206.3 :<\\n\Hr-N .. -x-x-H- a l-xa \n \ I, s \ I l I / K II I - I . — . l 68.08.0269 a .p < 0.0 0.. m.. on»); )0 6 100 «noun 114 .maflmuoo HmucoEwuomxo 00w NH musmfim mom .mcofluflocoo UHEonomfl Moos: mao>oa mod Hmunoumo CH omc0501|.va musmflm 115 m 8 fi 3352 o. n F. nc - _ 68-82023 moeooom on q an: enssu )0 0 10d se|oum 116 .<.< no? mmumno mmuwcm use 0.0 spas ooumoflocw mum moofluomaosc ocwcood _mz<_ + "mada + _me¢_ Haas“ m.o + "meal ”masauom mcwsoaaom can an oocwmoo ma Amoma .comcfixumv omumno monocm .va one ma mousmfih ca ooucmmoum mommnm>m mnu no woman ouo3 mcofiumasoamo .mxowco coulomouomHmm one Houucoo mo mcfimun ecu ca omumno amumco one moofluooaosc ocflcoom mo Hm>oa Hmuou co masonomfl EouuoEumom mo uoommmur.ma ousmwm 117 as: 0.0! o 9 r a h c V. W 00...: n E 0.1 33...: 8:38 o. 0 NF. nc on n. u - q 4. dl j q u j l 1 IA. . 68.32023 82.3.8.8 \ [4“‘IQ"‘I’ I a, (x .41 o ' ' o N .' onset; 10 6 10d soloum ug ewuiuepv "no; 0. l0 118 .NH ousmfim now no 080m on» ouoz mcofluwocoo Hmucmefluomxm .moHQEmm momma» once so m now am a mamas ucomoumwu mucflom owuuon .mx0flno cowlmmouomamm can Houucoo CH wHEoEOmfl Emuuoeumom oomcoaoum mo mcofluocsm mm comoomam Hounmumo can mmpflaopmuoe cephaoowam Hangouoo msoHHo> ca mmmcmnolt.mH musmflm 119 Egan?! use»? .. . h n..o 88.... 8 238.56 F W m “.255 of 0.. mmogdo " 'v n _ w n . p a S m. Ru 0.. I... 0.0 9. S n a 0.. r... 120 and increased in proportion to the decrease in ATP levels observed in either group of chicks. In contrast, although fluctuations were apparent, the levels of ADP (Figure 14) were generally unchanged up to one minute after decapita- tion; then they gradually decreased until at 10 minutes, low but detectable amounts remained. The ratio [ATP][AMP]/ [ADP]2 was calculated from data for adenine nucleotides at the various periods after decapitation, and gave an average of 0.60 i 0.25 and 0.58 t 0.19 for chicks fed galactose and control diets, respectively. These are in good agreement with values calculated for mouse brain by Lowry EE.E£’ (1964). As seen in Figure 15, the total adenine nucleotide content remained stable. The differences in the onset of expenditure of high energy phosphate between the dietary groups can be seen by the more rapid decline of whole brain energy charge in chicks fed galactose as compared with those fed control diets. Analysis of brain glucose and glycogen revealed the low supply of these vital carbohydrate reserves in brains of chicks fed galactose (Figure 16). Immediately after onset of ischemia, the levels of glucose and glycogen in the brains of control chicks decreased synchronously in sawtooth-like patterns which were strikingly similar. A study was made of the variability of the glucose levels in brains of chicks fed the control diet during ischemia. Analyses of 4-5 animals per time period up to and including 121 18 sec revealed considerable standard deviation; e.g., 2.14 i 0.72 umol glucose/g of fresh tissue at zero time, 3.66 i 1.31 at 3 sec after decapitation, and 1.13 t 0.31 at 18 sec (Table 12). The wide standard deviation would indicate that the patterns for the levels of glucose and glycogen (Figure 16) are due to animal variability and do not suggest synthesis of either of the two molecules during ischemia. Large variation in cerebral levels of glucose (2.5 i 1.2 umol/g) among individual chicks has been pre- viously noted, while the variations in ATP (1.9 i 0.1 umol/g) and lactate (3.0 i 0.4 umol/g) were less (Kozak and wells, 1969). The large percentage of carbohydrate in the diet (60%) may also cause the individual variability to be more striking. Glycogen in the brains from chicks fed galactose was completely exhausted after one min of anoxia, while minor amounts of glucose remained (0.15 umol/g of tissue). This level of glucose had been main- tained in the animals fed galactose since 6 sec of anoxia had elapsed and persisted for the remaining experimental times. The fact that this concentration approximates 20 times the Km of hexokinase for glucose (Crane and Sols, 1955) suggests that this residual glucose may be compart- mentalized, e.g., due to trapped blood in the cerebrum, and unavailable to the enzyme. In fact, Dr. H. R. Knull (unpublished results) has demonstrated using [14c]-inulin that there is contamination of powdered brain from blood. 122 Table 12.--Variation of Cerebral Glucose Levels in the Chick During Anoxia. Period of Anoxia Glucose ESE umol/g tissue Zero time 2.14 t 0.72 3 3.66 t 1.31 6 2.21 t 0.41 9 1.92 i 0.84 12 1.60 i 0.73 18 1.13 i 0.31 Note: The chicks were fed the control diet for 46 h, at which time they were sacrificed according to the "closed-system" techniques. The values represent the average t standard deviation of determinations on 4 or 5 individual brains. 123 If the cerebral glucose is then corrected for blood glu- cose content, the zero time concentration in brain would be 0.1 umol/g and the levels after 6 sec closer to the Km for hexokinase than previously estimated. That less energy could be derived from glucose in the animals fed galactose than in the controls after de- capitation was supported by analyses of lactate. The level of lactate initially was depressed in brains from galactose— fed chicks and remained relatively constant throughout the first minute of anoxia, increasing significantly only be- tween 2 and 10 minutes. The increase cannot be accounted for by corresponding decreases in glucose, glycogen, or glycolytic intermediates. In control tissue, a rapid in- crease in lactate, inversely proportional to the rate of utilization of glucose and glycogen, was observed follow- ing decapitation, in agreement with the results of Lowry EE.E£' (1964) for normal mouse brain. Similarly, the con- centration of fructose-1,6-diphosphate increased initially, then declined as carbon sources were depleted. No similar increase in the levels of fructose-1,6-diphosphate immed- iately after decapitation was observed in the brains of chicks fed galactose, presumably because of the paucity of carbohydrate reserves prior to decapitation. InsPection of the actual plus potential high energy phosphate util- ization (Figure 17) reveals a generally depressed rate of energy expenditure in brains of chicks fed galactose. 124 .moomamz co cofluoom uxou on» Ca ooflm lflommm manfiuom may on mcwouooom .ma new va .mH .NH mwusmwm cw concomoum sumo on» no women ouw3 mGOADMHSUHmu .maEmzomw couuoaumom msfluso gamma xoano an monogamonm hmuwco inmfln Hmwucmuom can Hmsuoo mo coHDMNHHHuDII.nH ousmwm Actual and Potential mP Used umoles/ g of tissue 125 I l l l A: 1 I5 30 45 60 'fi l2 Time in seconds 126 During the entire "closed-system" study period, cerebral levels of galactose and galactitol (Table 13) were virtually unchanged at 9 periods of anoxia between "zero" time and 10 min, verifying the inability of either carbohydrate to act as a significant energy source (Kozak and Wells, 1971). However, variability among the various pools was again evident as previously noted for glucose. Citrate, known to act as a modulator of phOSpho- fructokinase (EC 2.7.1.11) activity, was quantified in brains of chicks of both dietary groups. At "zero" time, brain citrate levels were 0.183 t 0.012 and 0.170 t 0.012 umol/g fresh tissue for control and galactose-fed groups, reSpectively. 127 Table l3.--Cerebral Levels of Galactose and Galactitol in the Chick after Various Periods of Anoxia. Period of Summation of Anoxia Galactose Galactitol Ggiigziiioind umol/g tissue Zero time 4.90 5.14 10.04 3 sec 2.87 2.92 5.79 6 sec 1.14 2.77 3.91 9 sec 2.26 2.61 4.87 18 sec 4.93 t 0.29 6.71 i 0.32 11.64 1 min 3.37 2.28 5.65 2 min 7.11 t 0.18 4.19 t 0.26 11.30 5 min 1.65 i 0.23 4.61 i 0.35 6.26 10 min 4.15 4.94 9.09 Note: The tissue was the same as used for the analyses in Figures 12-17. Where standard deviations are given, the values represent averages of 3 deter- minations on the pooled tissue; otherwise, 2 values were averaged. DISCUSSION The "closed-system" represents an attempt to eval- uate the ability of the brain in the galactose-fed animal to carry out glycolysis sufficiently well to meet the re- quirements for energy production. Consideration of the expenditure of potential ~P points out that animals treated with galactose are unable to replenish significantly the energy expended because of the limiting amount of sub- strate available. This depletion of substrate effectively slows glycolysis down. In the control animals, 0.5 umol glucose/g tissue (Figure 16) appears to be the critical level at which the brain can no longer maintain near-normal concentrations of ATP (Figure 13). During ischemia, in the animal treated with galactose, lactate does not accu- mulate until after 48 sec of anoxia have elapsed. Further- more, fructose-l,6-diphosphate did not increase throughout the period of ischemia. Possibly the intrinsic ability of the glycolytic pathway to oxidize glucose in the chicks fed galactose is not impaired, and if more substrate were provided, metabolic control of phosphofructokinase and increased formation of lactate could be demonstrated. 128 129 The very severe lowering of the cerebral level of glucose simultaneous with normal plasma levels suggests that the translocation of glucose across the blood-brain barrier is limited. Additional evidence is provided by experiments in which the Specific activity of brain glu- cose was monitored after intraperitoneal injection of [14C1-glucose. A nearly 10-fold higher amount of [14C]- glucose from plasma pools virtually equivalent in glucose concentrations and specific radioactivity was found in the brains of control-fed chicks compared with those of galactose-fed animals 5 min after administration of the tracer (Granett 35.31., in press). Bidder (1968) pre- viously presented evidence suggesting a common transport mechanism for glucose and galactose based on their inhi- bition of 3-0-methylglucose transport across the blood- brain interface. In our studies, plasma galactose may reach values of greater than 22 mM in the galactose-toxic animals, whereas in the control animal, the free sugar is absent from the blood. The observation that treatment of the galactose-fed chicks with glucose, which doubles the plasma level of glucose, increases cerebral fructose di- phosphate, lactate, glycogen and glucose to near normal levels also supports the concept that the intracellular concentration of glucose is limited by the competition of galactose with glucose at transport sites (Knull, Wells and Kozak, 1972). 130 Although the etiology for the galactose-induced neurotoxicity in the chick is undoubtedly complex, it is reasonable that the exhausted metabolic state of the brain described here is intimately involved in the abnormal motor activity and neuronal degeneration associated with the toxicity. The brain is unique in that it depends almost completely on carbohydrate furnished it by the blood for its energy sources. Its utilization represents 25% of the total bodily consumption of glucose, and the metabolic rate may be 20 times the average for the body as a whole (Bachelard, 1970). Thus impairment of glucose uptake by the brain which appears to occur in the galactose-toxic chick would be of major significance. Comparison of the changes induced in energy metabolism in chick brain by galactose versus phenylalanine reveals an interesting similarity. Both agents produced a depressed cerebral metabolic state in the animals, galactose probably limiting the substrate available for glycolysis; however, no carbohydrate paucity in the brain was associated with the phenylalanine treatment and no evidence was obtained for a direct interaction of the amino acid with glycolysis. Thus the two compounds, galac- tose and phenylalanine, appear to exert primary and secondary effects on carbohydrate metabolism. CHAPTER II DETERMINATION OF GLYCOGEN FROM BRAIN WITH AMYLOGLUCOSIDASE INTRODUCTION After various periods of ischemia the concentration of glycogen in the brains of normal chicks decreased from 1.5 to 0.5 umol of glucosyl units/g after 1 min and re- mained at this level after 10 min had elapsed (Granett, Kozak, McIntyre and Wells, in press). Since simultaneously glucose and high energy phosphate compounds were exhausted, it was logical that glycogen should have been utilized more completely. For these studies the glycogen had been isolated from perchlorate insoluble material, digested in base, precipitated in ethanol and quantified by enzymatic determination of the glucose released upon acid hydrolysis. Employing similar analytical methods Lowry, Passonneau, Hasselberger and Schulz (1964) also observed a glucose- containing material stable to ischemia. An attempt was made to demonstrate that the material isolated at 10 min was not glycoqen using amyloglucosidase (a-l,4-glucan glucohydrolase), an enzyme shown to hydrolyze 131 132 a-1,4- and a-l,6-glucosidic linkages (Pazur and Kleppe, 1962). Incubation of partially purified glycogen with the enzyme revealed that glucose was released from the zero time sample and not from the 10 min sample, indicating that a non-glycogen substance containing glucose was present. Of the methods available for glycogen quantifica- tion, the procedure employing phosphorylase degradation coupled with conversion of the glucose-l-phosphate to 6-phosphogluconate with phosphoglucomutase and glucose-6— phosphate dehydrogenase is most specific (Passonneau, Gatfield, Schulz and Lowry, 1967). The non-Specific methods are based on a Pflfiger type procedure (Pflfiger, 1909; Good, Kramer and Somogyi, 1933) for isolation of the glycogen by a KOH digestion of the tissue and ethanol precipitation of the polysaccharide. This is followed by measurement directly with anthrone reagent (VanHandel, 1965) or indirectly by acid hydrolysis and enzymatic determination of the glucose with hexokinase and glucose- 6-phosphate dehydrogenase (Slein, 1963) or glucose oxidase (Hugget and Nixon, 1957). In the following chapter a method for quantifica- tion of glycogen utilizing amyloglucosidase is described. The assay is Specific for glycogen in the brain and is equally as sensitive as the phosphorylase assay. Also it is very convenient; only one enzyme iS required for the 133 degradation of the glycogen and no activation is necessary as with the phosphorylase method. While this work was in prOgress Jongkind, Corner and Bruntink (1972) published an abbreviated method for glycogen determination employ- ing amyloglucosidase. MATERIALS AND METHODS Materials Rabbit liver glycogen (Type III) and amylogluco- sidase from Rhiszopus and ASpergillus were obtained from Sigma Chemical Co. (St. Louis, Mo.). The purified amylo- glucosidase (a-l,4-g1ucan glucohydrolase; EC 3.2.1.3) (Pazure, Simpson and Knull, 1969) was a gift from Dr. Harvey Knull. G1ucose-6-phoSphate dehydrogenase (D- glucose-G-phosphate : NADP oxidoreductase; EC 1.1.1.49) hexokinase (ATP : D-hexose-6-phosphotransferase; EC 2.7.1.1), phosPhoglucomutase (a-D-glucose-l,6-diphosphate : a-D-glucose-l-phosphate phosphotransferase; EC 2.7.5.1), phosphorylase a (a-1,4-g1ucan : orthophosphate glucosyl- transferase; EC 2.4.1.1) and nucleotides were obtained from Boehringer Mannheim (New York, N.Y.) or Sigma. Isolation of glycogen EEOm brain Chicks were decapitated directly into liquid N2; brain tissue was chipped out and powdered over dry ice in a cold room and later stored at -90°C. The tissue was homogenized in 0.6 M perchlorate-1 mM EDTA (0.5 ml/100 mg) 134 135 and the homogenate centrifuged for 10 min at 12,000 g. The pellet was digested in 5 N KOH (0.4 ml/lOO mg original tissue) for 30 min at 100°C, whereupon 3% Na2803 (0.1 ml/ 100 mg) and absolute ethanol (1 ml/100 mg) were added. Glyc0gen precipitated upon cooling of the samples to -20°C for l h or overnight and was recovered by centrifugation and washed in 70% ethanol (0.5 m1/100 mg) (Walaas and Walaas, 1950). The pellet was then dried with warming under a N2 stream. Quantification of glycogen By acid hydrolysis A standard solution of glycogen (8mg/50 ml) was diluted 1:1 with 2 N H2804 and hydrolysed in a sealed ampule for 3 h at 100°C. Partially purified glycogen from brain was dissolved in 1 N H2804 (0.6 ml/100 mg) in an ampule and hydrolysed similarly. The hydrolysates were neutralized with NaOH and the glucose content was deter- mined enzymatically. Quantification of glucose Free glucose was determined enzymatically accord- ing to a modification of the method of Slein (1963). The reaction cuvette contained the following components expressed as final concentration in a 0.25 ml volume: 320 mM Tris-HCl, pH 8; 6 mM MgCl 1 mM ATP; 0.32 mM NADP+; 23 10 ug/ml g1ucose-6-phoSphate dehydrogenase; 20 ug/ml 136 hexokinase. The production of NADPH was monitored on a Gilford 2400 recording spectrophotometer at 25°C at 340 nm. Quantification of glycogen with amyloglucosidase A standard glycogen solution (8 mg/SO ml) was diluted 1:1 with 0.1 M citrate buffer, pH 5, or glycogen isolated from tissue was dissolved in 50 mM citrate, pH 5 (100 mg original tissue/ml). Amyloglucosidase dis- solved in buffer (20 mg/ml) was added, 20 ug/ml. If crude enzyme were used, the undissolved material was first removed by centrifugation. Samples were incubated with shaking at 37°C for 2 h or as Specified, placed in ice and assayed for glucose released by the enzymatic method previously described. Tissue samples were centrifuged to clarity before being assayed. Recovery experiments Standard glycogen (8 mg/50 ml) was added to the perchlorate pellet obtained upon deproteinization of 100 mg of tissue, and the glycogen was isolated and quanti- fied as previously described. Addition of glycogen before the perchlorate step resulted in incomplete precipitation of the standard presumably because of inefficient trapping of the glycogen by tissue proteins. If tissue samples were deproteinized both in the presence and absence of standard glycogen and the perchlorate extracts incubated 137 with amyloglucosidase, glucose release could be demon- strated only for those samples to which glycogen had been initially added. Enzymatic assay for debranching activity The method of Passonneau g£_al. (1967) was em- ployed except that BSA was omitted. The assay consisted of the following components expressed as final concentra- tion: 0.05 M recrystallized imidazole, pH 7; 0.5 mM MgAcetate; 1 mM NADP+; 0.1 mM AMP; 5 mM inorganic phos- phate; 0.25 mM glycogen (as glucosyl units); 6 ug/ml phosphoglucomutase; 4 ug/ml glucose-6-pho5phate dehydro- genase. Phosphorylase a and accompanying debranching activity (Sigma) were activated with BSA, dithiothreitol, AMP and heating at 38°C for 60 min as described (Passon- neau SE 31., 1967). The phosphorylase was added to the reaction to give a final concentration of 10 ug/ml. The reaction was initiated by addition of the enzymes and in- crease in absorbancy at 340 nm at 25°C was monitored. The initial rate of the reaction due to phosphorylase activity is very rapid (0.04 OD units/min) and gradually slows as the outer tiers of the glycogen are cleaved and becomes limited by slow debranching activity. 138 Purification of debranching activity Amylo-l,6-glucosidase was purified from frozen rabbit muscle according to the method of Cori (1955) through the first ammonium sulfate precipitation and dialysis. The procedure of Nelson, Kolb and Larner (1969) was also employed, isolating the activity from fresh rabbit muscle. The enzyme was used after the ammonium sulfate and dialysis steps. Upon assay of the activity, a significant reaction in the absence of added glycogen was observed.- This was attributed to entrapment in the ammonium sulfate crystals of glycogen that had been added during purifica- tion. This component was reduced considerably by repre- cipitation of the dialysate with ammonium sulfate (45% sat- uration). A portion of this material was then dissolved in 0.005 M Tris - 0.0005 M EDTA - 0.01 M 2-mercaptoethanol without further dialysis and was used in the assays. Purification of pullulanase activity Pullulanase was purified as an extracellular enzyme from the culture filtrate of batchwise-cultivated Aerobacter aerogenes according to the procedure of Bender and Wallenfels (1966). The enzyme was precipitated with acetone, dissolved in buffer (50 mg/ml) and dialysed against 0.02 M phosphate buffer, pH 6.8 for 24 h. This enzyme catalyzes the hydrolysis of a-l,6 linkages in the branched 139 a-glucan, pullulan. It also attacks glycogen and was of interest for this reason. Assay of pullulanase activity on glycogen To 12.7 mg of glycogen dissolved in 0.1 m1 of distilled water was added 0.1 m1 of the pullulanase solu- tion. The pH was corrected from 6.8 to 5 with l N HCl. The mix was incubated with shaking at 37°C for up to 3 h. Aliquots measuring 10 01 were Spotted at selected intervals on Whatman No. 1 paper and developed twice with ascending chromatography in butanol : pyridine : water (6 : 4 : 3, v/v/v). Oligosaccharides were detected with silver nitrate dip reagent (Mayer and Larner, 1959) as follows: the dry chromatogram was dipped into a solution containing 1 ml of a saturated silver nitrate solution, 6 ml of water and 200 m1 of acetone, air dried and then placed into a tray containing 40 ml of 10% NaOH and 200 ml of methanol. After black spots indicative of reduced Ag+ appeared, the chro- matogram was placed in 0.05 M NaZSZOB to wash out remain- ing silver ions. It was then air dried. Dilution of commercially available Karo syrup provided maltoligo- saccharide references. RESULTS Quantification of standard glycogen by acid hydrolysis and amyloglucosidase methods Comparison of standard glycogen determined as glucosyl units/ug either by acid hydrolysis followed by enzymatic quantification of glucose or with purified amyloglucosidase revealed the two methods to be identical (Figure 18). Assay of glycogen with crude amylogluco- sidase from either Rhizopus or Aspergillus reSulted in similar release of glucose. Time course study of the release of glucose from glycogen By amyloglucosidase Glycogen isolated from tissue was hydrolysed by amyloglucosidase with and without added glycogen (Figure 19). The reaction was completed within 1 h but samples were routinely incubated for 2 h. Recovery of glycggen added to tiSsue Table 14 illustrates that standard glycogen added to brain tissue after perchlorate deproteinization could be recovered and quantified by the amyloglucosidase method. 140 141 Figure l8.--Comparison of standard glycogen de- gradation by acid hydrolysis and amyloglucosidase. Hydrolysis in sulfuric acid or enzymatic cleavage with amyloglucosidase was conducted as described in METHODS. 142 400 ' O '8 0 Acid hydrolysis O m 8 300 - o Amyloglucosidase 713 L. a) m o o 2 O 200 - "5 «n 2 o E c IOO - 1 IO 20 30 4O 50 60 7O Glycogen (ug) 143 .omOOSHm MOM oommmmm one om>o€mu mmB posqfiao cm mHm>uoucH Samson pd .oofiuom n v w uo>o comoowam oumocmum m0 m1 m.mv mo oncomnm Ho oocmmmum map ca mmmowmoosamonem nuw3 poumnsocfl was mammfiu Sauna Eoum omuoHOmH comoowaw .mmmofimoosHmonEm an comoomam mo mflmxaouoxc on» mo mafia nuflz mwmwamcmll.ma ousmfim 144 .9505 9...... .v m N _ - u q u q \. .Iul it. . /oco_o 03mm...- 956 2853 c3330 /. . O/\ \ l . o o\0 \x venue concern £3 38.... o 9 O O o o to N pesoe|eJ esoon|6 10 se|0ulu O 0 ¢ 145 .mmmoflmoosHmoH>Em spas pomhaouomn was cmmoowam one .moomemz ca omnfiuommo mm poops was Unaccoum one manna» Sauna Eoum omDMHOmfl mm3 cmmoowao "ouoz S n E >6 boa HOH mNN mHH h mOH mOH NMN mHH m mm HHH HNN mHH m vm mm NvH 05 ¢ mHH mm boa mm m mHH moa CON an N vNH HOEG mOH HOEC vow HOEG an H what/000mm wwwwwawfiwmoosam. comoomHMDoflSmwwa .Mwwmw WMMMWMMW. ommnom .monEmm downwa Eoum comoomau pumpcmum mo >Hm>oommnl.va mange 146 Quantification of glycogen levels ififbrain by acid hydrolysis and amyloglucosidase methods Tissue samples from the "closed—system" studies described in the previous chapter were used. A discrepancy was noted between the levels of glycogen determined by the two methods; the values resulting from the amyloglucosi- dase method were consistently lower than those obtained by acid hydrolysis (Table 15). The differences measured ap- proximately 0.5 umol glucosyl units/g with the exception of the samples at 24 and 48 sec which differed by 0.2 umol/g. Assay of standard glyCOgen withgphosphorylase Phosphorolysis of standard glycogen with phosphory- lase a (Sigma) resulted in incomplete degradation as cal- culated from the weight of glycogen assayed (assuming a molecular weight of 162 for the glucosyl unit). This was attributed to inactive debranching enzyme. Attempts made to purify the debranching complex from rabbit muscle yielded active phosphorylase but the a-1,6-glucosidase- transglucosylase was inactive in the assay system we were using. Degradation of glycogen standards was consistently 42 r 8% (n=7), which corresponds to the figure of 40% reported for the percentage of glucosyl units composing the outer tier of the glycogen molecule. It would appear then that the phosphorylase was not proceeding beyond the first branch point. 147 Table 15.--Comparison of Glycogen Levels in Tissue Deter- mined by Acid Hydrolysis and Amyloglucosidase Methods. 5:321: Acid Hydrolysis Amyloglucosidase umol of glucose released/g of brain 3 sec 1.77 t 0.38 1.09 12 sec 1.59 i 0.08 1.08 24 sec 1.24 t 0.04 0.97 36 sec 1.12 t 0.19 0.71 48 sec 0.87 t 0.08 0.68 5 min 0.55 i 0.01 0.00 10 min 0.52 t 0.04 0.05 Note: Tissue samples were taken from pools of control brains subjected to the periods of ischemia indi- cated. The values for acid hydrolysis represent averages of 3 determinations x SD; those for the amyloglucosidase method are averages of 2 differing from each other by 10% or less. 148 The assay was also supplemented with pullulanase, an enzyme that hydrolyses the a—l,6-1inkages in pullulan, a highly branched a-glucan. The enzyme itself was judged active by the appearance of materials chromatographing with maltose, maltotriose, maltotetrose and maltopentose upon incubation with glycogen at 37°C for l to 3 h. How- ever, inclusion of it in the glycoqen assay resulted in no further release of glucose-l-phOSphate above that obtained with phosphorylase alone. Assay of tissue glycogen with pHOSpHOrylase and amyloglu- cosidase The partially purified glycogen from 100 mg of brain was dissolved in 1 ml of water and centrifuged to clarity. Aliquots of the supernatant were subsequently assayed for glycogen using the phosphorylase and amyloglu- cosidase methods. AS indicated in Table 16 glucose re— lease was observed for both methods for the 3 sec ischemic tissue but not for the 5 min samples. More glucose was released with amyloglucosidase as would be expected if degradation was more complete than with the phSOphorylase. 149 Table 16.--Degradation of Glycogen Isolated from Tissue by PhoSphorylase and Amyloglucosidase. Tissue PhoSphorylase Amyloglucosidase Sample Assay Assay nmol of glucose released 3 sec 1 45 194 2 50 161 5 min 1 7 none detectable 2 2 none detectable Note: The tissue for these analyses was taken from pools of control brains subjected to the periods of post- mortem ischemia indicated. The assays performed with phosphorylase and amyloglucosidase are described in METHODS. DISCUSSION Glycogen isolated from 100 mg of brain by a modi- fied Pflfiger technique contains impurities that are de- tectable from the milligram quantity of material recovered; the glycogen component itself would consist of 15 - 30 micrograms. Consequently a fairly specific method must be utilized for accurate quantification of the poly- saccharide. The enzyme amyloglucosidase was investigated for this purpose. It was demonstrated to hydrolyze standard glycogen as quantitatively as does 1 N H2804. The reaction was conducted at pH 5, the pH optimum of the enzyme. If the reaction were run at pH 7, enabling coupling with hexo- kinase and glucose-6-phosphate dehydrogenase, the amyloglu- cosidase activity became limiting, having been reduced by 80%. Lowering of the pH to 6, better favoring the gluco— sidase (decreased in activity by 25%) resulted in an in- complete conversion of the glucose. Thus the assay con- sisted of two steps. Determination of the glucose re- leased from tissue samples was accomplished with hexokinase and glucose-6-phosphate dehydrogenase rather than with 150 151 glucose oxidase, as a slight turbidity remained in the incubate after centrifugation. Also an important advantage is that due to the fluorescence of NADPH the method can be adapted for fluorometry for greater sensitivity (Lowry gg‘gl., 1964). Unfortunately because of the incomplete degradation of glycogen obtained with phosphorylase, any direct, quantitative comparison cannot be made between that pro— cedure and the one utilizing amyloglucosidase described here. The method is reproducible as illustrated by the good replication obtained both with standard glycogen (Figure 18) and tissue samples (Table 15). Comparison of the procedure with acid hydrolysis degradation of tissue glycogen (Table 15) reveals the greater specificity obtained with amyloglucosidase. The hydrolysis of the tissue glycogen is presumed to be com- plete Since glycogen added to tissue could be quantita- tively recovered. It is postulated that the additional glucosyl units released by H2804 at 100°C may originate from glycoproteins that survived the alkali-digestion. Brunngraber (1970) estimated that 2.7 nmol of hexose are derived from the glycoproteins in 1 g of rat brain. Quali- tatively, a non-glycogen component is indicated by the absence of any substantial release of glucose from the 5 min tissue samples reacted with phOSphorylase (Table 16). 152 The ratio glycogen level at 5 min based on acid hydrolysis N I U! 3 sec degradation approximates 1/3. If the 5 min material is indeed glycogen, this same relationship should be main- tained regardless of assay method employed, and from the data in Table 16 this did not occur. In conclusion, the data presented here suggests that values reported for the levels of glycogen in various tissues determined by acidic degradation of the polymer may be erroneously high, depending on the amount of non- glycogen, glucose-containing material present. ACKNOWLEDGMENTS Mr. Louis E. Burton is gratefully acknowledged for the purification of the pullulanase; Dr. Richard Anderson, for the Aerobacter aerogenes culture. 153 SUMMA RY Feeding a diet 40% in D-galactose (w/w) to newly hatched chicks results in a syndrome characterized by shivering, seizures, ataxia and eventual death. The rate of glycolysis was examined in the brains of these animals, employing the "closed-system" technique. Cerebral levels of phoSphocreatine, ATP, ADP, AMP, fructose-L6-diphosphate, glucose, glycogen and lactate were quantified for various periods of post-mortem ischemia ranging from "zero" time to 10 min. The changes in the metabolites at ”zero" time confirmed the previous observations: phosphocreatine, ATP, fructose-diphosphate, glucose, lactate and glycogen were reduced in the galactose group. Concentrations of phosphocreatine declined immediately in both galactose- fed and control animals upon production of ischemia. ATP expenditure occurred in the brains of animals fed galactose at 12 sec of ischemia whereas this occurred significantly later (24 sec) in control animals. In both situations AMP levels increased in prOportion to the de- crease in ATP and reflected the utilization of ATP more accurately than did ADP. The adenine nucleotide energy 154 155 charge declined more rapidly in the chicks fed galactose than in those fed control diets. Glycogen reserves were depleted sooner in the galactose-fed group. Cerebral glucose levels were initially low in the chicks fed galactose, 0.3 umol/g of brain; they decreased to 0.15 umol/g after 6 sec of ischemia and were maintained at this level for the remaining time. Metabolic control of cerebral glycolysis at the phoSphofructokinase point could be demonstrated for the control animals but not for the galactose-fed chicks. Lactate accumulation characteristic of the anoxic state was rapid and immediate in controls but delayed for 1 min in the galactose- intoxicated chicks. Inspection of the utilization of actual and potential high energy phosphates disclosed a depressed rate of energy expenditure in the brains from galactose-fed chicks. Galactose and galactitol were quantified and observed not to decrease during the 10 min ischemia, indicating they were not energy sources. The level of citrate in the brain, of interest as a modulator of phosphofructokinase, did not differ between the two groups. The striking paucity of utilizable carbohydrates in the brains of the galactose-fed animals appears to be major factor in the slow-down of glycolysis and in the inability of the chicks to replenish the high energy phosphates expended. Interference of glucose entry into 156 the brain by galactose is postulated as responsible for the severe lowering of the cerebral levels of glucose. 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