myo - §NOSITOL METABOUSM DURING DEVELOPMENT AND LACTAHON 1N 'E’HE RAT Dissertation for the Degree of Ph. D. MECHSGAN STATE UNWERSWY Lfib‘is E’JGENE BURTSN 1976 »_ LIBRARY This is to certify that the thesis entitled m—Inoaitol Metabolic. During Development and lactation 1n the Rat presented by Innis E. Burton has been accepted towards fulfillment of the requirements for Ph.D. degree in Blochuintry I W Major professor Date /’- 04639 WC. LIBRARY END! RS WWI-9W“: 6‘ M/ 'f 34 ABSTRACT m-INOSITOL METABOLISM DURING DEVEIDBENT AND IACTA‘I‘ION IN THE RAT 3! Louis Eugene Burton The disposition of m-inositol in rat tissues use examined dur- ing the last four days of gestation. during neonatal development and during lsctstion. Develop-ental profiles were obtsined for the enzynes Q-glucose-éophosphete: kminositol-l-phosplnte synthsse (EC 5.5.1.“ and km-inositol-l-phospmtea rpm-moan“ pimpmme (EC 3.1.3.25) in fetel end neonetsl liver and brain. nsternsl liver and nemry tis- sue, end plecents. Developmental free m-inositol levels were sees- ured for piss-s, liver and brain of the fetsl end neonatal rat sui for plune. liver, placenta. and usury tissue of the Internal nt. anyntic studies. notshly in fetsl liver. suggested tint eleve- ted fetal please. levels (7—8-fold thet of the den) of m—inositol nsy originete from liver ensynetic synthesis and subsequently be replaced by nutritioml (silk m-inositol end senery m-inositol synthesis) sources during neonetsl developent. Fetsl please m-inositol levels were not significantly effected by dietary gig-imitol deprivation. however. 2-deoxy-2—glucose adsinistretion use shown to depress m- inositol levels in fetsl pleas and lost fetel tissues (in the presence or absence of dietary m—inositol) suggesting that hiosynthesis ac- counts for at least 605 of the high fetel content of m-inositol. Louis Eugene Burton Coaparisons of fetal. weanling and adult rat tissue/places wmsitol ratios suggested developental increases in the abil- ity of tissues to retain intracellular m-inositol. Dietary studies were undertaken to ennine the effects of diet- ary minositol deprivation on the growth and developent of fetal. neonatal and young adult rats and on lactation in dens. These studies suggested that intestinal flora contribute significant anounts of m—inositol' to the animal during deprivation. Dietary m-inositol strongly influenced the levels of m-inositol in the silk, nannary tissue and liver of the lactating den. Levels of silk 6-p-galactinol were also shown to be directly related to levels of silk m-inositol in rats and in a single hunan subject. The feed- ing of a gig-inositol deficient diet and the physiological stress of lactation produced a fatty liver after four days of lactation which was alleviated by minositol supplenentation or by cessation of lac- tation. Ninety-seven percent of the lipid deposited in the m-inositol deficient lactation-induced fatty liver was shown to be triglyceride. Plasna of lactating rats fed a minositol deficient diet had reduced levels of lipoprotein-associated lipids and elevated free fatty acid levels during the course of lactation and. subsequently. elevated lipo- protein-associated lipids levels and noraal free fatty acid levels dur- ing involution. These observations have suggested that moinositol deficient lactation-induced fatty liver results from a block in holo- lipoprotein synthesis or secretion caused by a reduction in liver phos- pholipids and phosphatidylinositol during m—inositol deprivation. Additional work was carried out exanining the effects of 2-deoxy- Q-glucose and 5-thio-2-glucose adninistration on m-inositol levels Louis Shagene Burton in testes amt liver of nice. The 6-phosphate of 5-thio-2-glucose was ensynatically synthesized and shown to be a conpetitive inhibitor (‘1'0'33 :14) of Q-glucose-6-phosphatea p—m-imeitoi-l-pmapmte 3mm. (ac 5.5.1.4. B—glucose-é—phosptnte, xii-o. 51 as) g m. g 1i_v_g adninistration of 2-deoxy-2-glucoss resulted in reduction of minositol levels in the testes and liver of nice while 5-thio-2- glucose adninistration resulted in significant elevations in testi- cular m-inositol. Prelininary evidence suggests that in 5-thio-2- glucose treated animls. m-inositol elevations are a result of the indirect effects of 5-thio-Q—glucose ministration on glucose netab- olisn and that inhibition of m-inositol bioeynthesis by 5-thio-2- glucose-é-phosphate _i_n vivo does not occur. mlNOSITOL METABOLISM DURING DEVELOPMENT AND LACTATION IN THE RAT By Louis Eugene Burton A DISSERTATION Subnitted to Michigan State University in partial fulfillment of the requirenents for the degree of DOCTOR OF PHILOSOPHY Department of Biochenistry 1976 DEDICATION I wish to personally dedicate this work to Iris Ann, my wife. May our lives benefit from this accomplishnent as much as we hoped. ‘ It will always be as much here as it is mine. 11 AQCNOUIEDGW‘I‘S The author would like to especially thank Dr. Iillian I. Hells for his thought-provoking discussions. unfailing financial support and for suggesting this particular area of study. I would also like to thank the nenbers of any guidance comnittee, Doctors Clarence Suelter. Robert Cook, and especially Richard Luecke and Loren Bieber. for their support. suggestions and useful criticisns. Special thanks must go to Chao—Hen Kuo. Dr. Richard H. Wagner and Rita a. Ray. as well as the other nenbers of the laboratory. for their stinulating discussions and moral support. In addition. I would like to extend ny appreciation to Mrs. Betty Smith for her cooperation and assistance in the care of the experinental aninals. Finally. I cannot forget the unfailing support of ny wife. Iris Ann. my children. Eric and Heather, and our fanilies; for it was their love and tolerance which added purpose to this goal. iii TABLE OF CONTENTS HST OF mm 0 O I 0 O O O O O O O O O O O O O O O O 0 LIST OF FIGIIRE O O O C O O I O O O O O O O 0 O O C O O O HST 0F mnous O O 0 O C O O O O O O O O O O 0 O C INTRODUCTION “an“ Smy O C O O I I O O O O O O O O 0 O O m—i-Inositol and its Nutrient Role . Biosynthesis ofn m—Inositol . . . . m—Inositol and Lipids. . . . . . m-Inositol Metabolism and Other Proposed Functions Objectives and Rationale . Organisation......... Edema. O O O O O O O O 0 Chapter I . STUDIES ON THE DEVEIDHENTAL PATTERN OF THE mm mmxnnc ammmsmm TO wmosrmr. IN mmTOOOOOOOOI0.0.0.0000... Aumt O O O O O C O . Introduction......::::::::::: Naterialsandhethods............. among 0 O O O O O O O O O O O O O O O 0 m1. 0 O O O C O O O O O O O O O O O 0 Enzyme Preparation: Syntlnse and Phosptntase SynthaseAssay................ m-Inositol-l-Phosphate Phosphatase Assay. tenination of Phosplnte. . . . . . ProteinDeternination. . . . . . . . Gas Chromatograplv of m-Inositol. . WTissueeeeeeeeeeeee 00110611101! Of Milk. e e e e e e e e e Preparation of muosiml-l-anplnu iv \OVO\ MUNH H Clupter Rama I I I I I I I I I I I I I I I I I I I I I I I m-Inositol Content of Selected Tissues in the Heanling,FetalarflPregnantRat. . . . . . . . m—Inositol Content of Rat Plasna. . . . . . . m—Inositol Content of Rat Milk and Henry cm I I I I I I I I I I I I I I Hunan Milk molnositol . . . . . Develop-ental Pattern of Synthase Developmental Pattern of the Phosphatase Effects of Diet and 2-Deoxy-D-Glucose Administra- tion on Fetal Rat Tissue m-Inositol Content . . Discussion . . . . . . Acknowledgements . . . References.. . . .. II'. EFFECTS OF DIETARY m—INOSITOL 0N TISSUE LEVELS OF mwrnosrmmem'r. . . . . . . . . . . . Aumt I I I I I I I Introduction . . . . . Materials and Methods. Rum“. I I I I I I I I I I I I I I I AninalsandDiets.... ...... Tissue and Fluid m-Inositol Contents. Roam“ I I I I I I I I I I I I I I I I I I I Effects of Dietary m-Inositol on Free Pool m‘hoaitol in 8.1”” T188138. e e e e e e Discussion I I I I I I I I I I I I I I I I I I I I References I I I I I I I I I I I I I I I I I I I I I I III . mINOSITOL manousa DURING neutron AND namep- m IN m RAT. m PREVENTION or IACTATION mau- mm mm BY DIETARY m-Iwosnoz. . . . . . Abstract . .... . . . Introduction e e e e “8.1311313 ”fl RIMS. mamnigtseeeeeeeeeee Hill: Collection and Amniotic Fluid. . . Tissue and Fluid Content of m-Inositol. Lipid Content of Tissue . . . . . . . . Phospholipid Phosphorus levels. . . . . Fatty Acid Quantitation . . . . . . . . 3‘2 $8 $§B§8W£¥§ 818:5? ‘S Chapter Smcountseeeeeeeeeeeeeeeeeee sutnticseeeeeeeeeeeeeeeeeeee new“ I I I I I I I I I I I I I I I I I I I I I I I I Body'lelghtPrOfile. e e e e e e e e e Amniotic Fluid Free m-Inositol Levels Plasma Free gin-Inositol Lavels . . . . Brain and Kidney Free m—Inositol. . . Intestinal Free m—Inositol Levels . . FertilitysttdiosI I e e e e e e e e e Effects on Hilkm m-Inositol. . . . . . Effects on 6-9 -Calactinol Content of Milk. Effects of Lactation on Dams. . . . . . . . Development of Fatty Liver in lactating Dams Developing Rat Liver Free m—Inositol. . . Developing Rat Liver Lipid, Lipid-Bound 319- Inositol and Phospholipid Phosphorus Content. . . Fatty Acid Distribution in Total Liver Lipids . . D “was ion I I I I I I I I I I I I I I I I I I I I I I Refamnua I I I I I I I I I I I I I I I I I I I I I I IV. CHARACTERIZATION OF THE LIPID IN m—INOSI'NL DEFI- CIENT IACTATION-INDU- FATTY LIVER AND AmmCt . I I I I I I Introduction . . . . . Materials and Methods. “and“! I I I I I I I I I I I I Animal Treatments anl Diets . . . Total Lipid Extraction and Quantitation Free and Esterified Cholesterol . Triglycerides and Glucose Levels. Phospholipid Quantitation . . . Phosphatidylinositol levels F1... pgtty AC1d8e e e e e e Summation of Total Lipids . Elecmn Kimmy e e e e Raul“ I I I I I I I I I I I I I PLASMA IN RATS. Effects of Dietary m-Inositol on Liver Height as a Percent of the Total Body Height . . . Lipid Constituents of the htty Liver . . . El.ctmmmcopyI I I I I I I I I I I I I Characterization of Plasma. Lipids . . . . . m-Inositol Deprivation and Liver Lipid in lactating Dams Fed a m-Inositol Deficient DietflithoutSulfa................ vi $ .92 ‘O‘O‘O UWN 8 8 Xfifigfigflfigfi §§s Te? V. VI. EE-Inositol Deprivation and Kidney and IntestineLipidLevels............. Discussion. . . . . . . . . . . . . . . . . . . . . . Refanncoa I I I I I I I I I I I I I I I I I I I I I I mmosrror. IN THE nevemmc an mm. BIOSYN- mrc mm AND DIETARY wears on an AND LIPID- BOUND m—INOSITOL AND F'UMARASE AND 2'. y-crcuc NUCW-B'JHOSPHOHYDROIASE ACTIVITIES. . . . . . . AthtI I I I I I I I I I Introduction........... murals m Hetms e e e AnimalsandDiets................ TiasuePreparation............... 30m“ I I I I I I I I I I I I I I I I I I I I I I I Free m1nositol in Developing Rat Brain. . . . Developental Profile of Rat Brain mlnositol Synthase and Loam-hositol-l-Phosphate Phos- muses I I I I I I I I I I I I I I I I I I I I Effects of Dietary mlnositol on m-Inositol LIVIIBinNImlTIIIuII. e e e e e e e e e e e Central Nervous Systen Myelination and Mito- chondriogenesis. . . . . . . . . . . . . . . . . D18“81°neeeeeeeeeeeeeeeeeeeeee Rd.r.n°.'eeeeeeeeeeeeeeeeeeeeee STUDIES ON THE mas 0F S-THIO- moose AND 2-03)“- woosa ON m-Iuosr'ror. mm sn IN arcs AND m MIBITION BY 5-THIO-g-CIBCOSE-6-HIOSPIMTE OF Pam- HOSINIPI-MMR “WIS, E m e e e e e e e Abstract. . . . . . . Introduction. . . . . Materials arr! Methods mumhI I I I I I I I I I I I I I I I I I I I Synthesis of 5-Thio-D-Glucose-6-Phosphate. . . . Isolation and Charactarization of 5-Thio-2- Glucmw‘mapmueeeeeeeeeeeeeee Measurenent of V for Hexokinase and D—Glucose- 6-Phosphate DehyHIgenase Using 5-Thio-fi-Glucose or its 6-PhosphateasSubstrate. . . . . . . . . Preparation and Assay of g-Glucose-6-Phosphate c m-IMlel—l-Hbspflu sumo. e e e e e e ‘Liqudcmumphyeeeeeeeeeeee SpenCounts.................. V11 Page .108 .108 .112 .1110 .115 .115 .115 .117 .118 . 118 . 118 .121 .121 .121 128 . 130 . 130 . 130 . 132 . 132 . 132 . 133 . 133 . 13b . 134 . 135 Chapter Anmh I I I I I I I I I I I I I I I I I I I Quantification of Clucose-6-Phosphate and Adenosine—5'-Triphosptnts . . . . . . . . . . R’sulu I I I I I I I I I I I I I I I I I I I I I I Synthesis of 5-Thio-D-Glucose-6-Phosphate . . Isolation, Identification and Quantification of 5-Thio-D-Glucose-6-Pl'iosphate . . . . . . . Inhibition of D-Glucose-é-Phoszmate: Inositol-l-Phosphate Synthase ynthase) by 5-Thio-D-Glucose—6-Phosptate ( 5T6-6-P). . . . V for 5-Thio-Dbclucose with Hexakinase and sphate with.Glucose-6-Phosphate Dehydro- gemsaI I I I I I I I I I I I I I I I I I I I Effects of the Administration of S-Thio-Q- Glucose. ZnDeoxbe-Glucose. and D-Glucose on HunchwuLwds............ ‘Ig Vivo Experimentation . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . Ref.rence a I I I I I I I I I I I I I I I I I I I I DMSSION I I I I I I I I I I I I I I I I I I I I I I I I I Reannccs I I I I I I I I I I I I I I I I I I I I Amn I I I I I I I I I I I I I I I I I I I I I I I I I I viii Page . 135 . 135 . 135 . 135 . 137 . 137 . 139 . 139 . litZ . 151 . 157 . 160 . 166 Table Chapter 1. Ctapter 1. Chapter 1 . Ctnpter 1. LIST OF TABLES I Lam-Inositol Content of Selected Tissues in the Weanling,FetalandPregnant........... Effects of 2-Deoxy-D-Clucose and Diet on Fetal Ratm gig-Inositol Content of Plasma and Selected TissuesI I I I I I I I I I I I I I I I I I I I I I III Effects of Dietary m-Inositol on the Free 33- Inositol and 6- -Galactinol Content of Rat Milk at 1“ my: of “cation. I I I I I I I I I I I I I Fatty Acid Distribution of Dietary Lipid and Total Lipids in Liver of gig-Inositol Deficient andSupplemented lactating Rats. . . . . . . . . . IV Relationship of Liver and Body Height During Lactation and Involution for Dana Fed a 512‘ Inositol Supplenented or Deficient Diet. . . . . . VI A Comparison of V for -Glucose and 5-Thio-2c Glucose and Their-85M te Derivatives in Two Emylesyauueeeeeeeeeeeeeeeese Quantification of 5-Thio-2—Glucose-6—Phosplnte . . Effects of 5-Thio-D-Clucose and 2-Deoxy-D-Glucose Administration on m-Inositol Levels of Mouse PlasmaandTestes-Experinentl . . . . . . . . . Effects of S-Thio-Q-Clucose aui 2-Deoxy-D-Clucose Administration on m—Inositol Levels of Mouse PlasmaandTissues- ExperimentII . . . . . . . . Page .136 .136 . 143 . 1‘6 Table 5. 7. Effects of the Administration of 5-Thio-2- Glucose . 2-Deoxy-2-Glucose . and Q-Glucose in Mice on Plasma Glucose and gin-Inositol Levels . . Effects of 5-Thio-2-Glucose . 2-Deoq-D-Glucose and D—Glucose Administration on Free and Lipid- Bound LIE-Inositol Levels of Mouse Testis azrl Liwr-1veseeeeeeeeeesesseeoe Effects of S-Thio-D-Glucose , 2-Deoxy-2-Glucose and ID-Glucose Administration on Q-Glucose-é-Phos- plate and Adenosine-5'-Triphosphate Levels of Mouse Testes arr]. Liver - Experiment IV . . . . . . Effects of 5-Thio-D-Glucose ani 2-Deoxy-2—Glucose Administration on Testes m-Inositol Content with The. I I I I I I I I I I I I I I I I I I I I Page . 147 .149 . 149 .150 Figure LIST 01“ FIGURE Ctmpter I 1. 2. Chapter 1. Clupter 1. 3. m-Inositol Content of Rat Pup Plasma arri Maternal Plasma During Developent . . . . . . . . lam-Inositol Content of Rat Milk and Mammary GlandDuringlactation.............. Developmental Changes in Human Milk m—Inositol “"18 I I I I I I I I I I I I I I I I I I I I I I Developmental Changes in Activity of D-Glucose -6-Phosphatex L-m-Inositol-l-Phosphate Synthase of Fetal and Neonatal Rat Liver. Maternal Liver. PlacentaandMammaryGlarai............ Developmental Cinnges in Activity of rpm-Inos- itol-l-Phosphate Phosplntase of Fetal and Neonatal Liver. Maternal Liver. Placenta and Mammary Gland. II Free m-Inositol Content of Plasma. Liver. Lung. Heart. Kidney and Intestine in Female Rats Fed a m—Inoeitol Supplemented or Deficient Diet. . . . Free mtg-Inositol Content of Spleen. Pancreas. BrainandMemnaryGland inFenale Rats Fedagfl- Inositol Supplemented or Deficient Diet. . . . . . III Body Heights of Developing Rats Fed a m-Inositol Supplemented or Deficient Diet . . . . . . . . . . Free momenta Levels in the Plasma of Devel- opingRatsandlactatingDams. . . . . . . . . . . Free LIB-Inositol Levels in Selected Tissues of Developing Rats Fed a m-Inositol Supplemented or Def1C1°nt Diet I I I I I I I I I I I I I I I I I I xi Page 25 30 32 35 47 61 67 Figure Clupter 1. Effects of Dietary m-Inositol on Milk and Mammary Gland Free mlnositol Levels. and Milk6-D-GalactinolLevels. . . . . . . . . . . . Effects of Dietary aye-Inositol on Free and Lipid-Bound m-Inositol. and Total Lipid, and Phospholipid Phosphoms Content in the Liver of “cut“ M I I I I I I I I I I I I I I I I I I Effects of Dietary gin-Inositol on the Free and Lipid-Bound mlnositol. Total Lipid. and Phos- pholipid Phosphorus Levels in the Liver of the DIVIlopm ht I I I I I I I I I I I I I I I I I I IV Triglyceride and Phospholipid Content of Liver Lipids in Lactating Dams Fed a m—Inositol Supplemented or Deficient Diet . . . . . . . . . . Free and lsterified Cholesterol Content of Liver Lipids in Lactating Dams Fed a m—Inositol Sup- plemented or Deficient Diet. . . . . . . . . . . . Electronmicroscopy of the Livers of lactating Dams Fed a gig-Inositol Supplemented or Deficient Diet at 1“ my. Of moutionI I I I I I I I I I I I I I Triglycerides and Free Fatty Acids in the Plasma of lactating Dams Fed a m—Inositol Supplemented orDeficientDiet................. Free and Esterified Cholesterol Content of Plasma of Lactating Dams Fed a _m_yg-Inositol Supplemented or Def1C1Int DictI I I I I I I I I I I I I I I I I Phospholipid and Phosphatidylinositol Content of the Plasma of lactating Dans Fed a m—Inositol Supplemented or Deficient Diet . . . . . . . . . . Glucose and Lipoprotein-Associated Lipid Content of the Plasma of lactating Dams Fed a aye-Inositol Supplemented or Deficient Diet . . . . . . . . . . Chapter V 1. 2. Free Len-Inositol Content of Developing Rat Brain. km-Imsitol-l-Phosphate Synthase and m-Inos- itol-l-Phosphate Phosphatase Activities Rat BrainDuringDevelopment. . . . . . . . . . . . . xii Page . 101 .10“ .107 .107 .120 .120 Figure P580 3. Free gig-Inositol and Lipid-Bound gig-Inositol Content of Cerebrum and Cerebellum From Rats Fed a m-Inositol Supplemented or Deficient Diet. . 123 1+. Comparison of Cerebellar and Cerebral 2'. 3'- c-AMP-3'-Phosphohydrolase and Fumarase from Rats Fed a gig-Inositol Supplemented or De- fiCient Diet I I I I I I I I I I I I I I I I I I I I125 Chapter VI 1. Gas Chromatographic Analysis of Q-Glucose and S-Thio-D-Glucose and 2-Clucose-6-Phosphate and 5-Thio-g-Glucose-6-Phosphate . . . . . . . . . . . .138 2. Lineweaver-Burk and Dixon Graphic Analysis of the Inhibition by 5-Thio-2—Glucose-6-Phosphate of the Synthesis of L—m—Inoeitol—l-Phosptnte from 2- G1uwse.6.Pm8mu I I I I I I I I I I I I I I I I I lac 3. Effects of the Administration of a Sizzle Dose of 5-Thio-2-Glucose, 2-Deoxy-2-Glucose . Q—Glu- aces and Saline on Plasma Glucose Levels in Mice . .141 4. Effects of D-Glucose. 2-Deoxy-2-Glucose, and 5-Thio-2-Glucose Administration on Sperm Count 1n Nine I I I I I I I I I I I I I I I I I I I I I I 12‘s Discussion 1. m—Inositol, 6-P-Galactinol and lactose Levels in Hm ”inI I I I I I I I I I I I I I I I I I I .163 Appendix 1. The Physical Appearance of the Livers of Dane Fed a m—Inositol Supplemented or Deficient Diet after 21 “ya at mention I I I I I I I I I I I I I I I I 168 xiii LIST OF ABBREVIATIONS Adenosine-S'-triphosphate Cholesterol. cholesterol esters 2'. 3'-Cyclic nucleotide 3'-phosphohydrolase 2-Deoxyhp-Glucose and its 6-phosphate Dithiothreitol Ethylened iaminetetracetic acid Free fatty acids Q-Glucose-é-phosphate International unit Inhibition constant Michaelis constant Oxidized and reduced nicotinaside adenine dinucleotide Oxidized and reduced nicotinaside adenine dinucleotide phosphate Phosphatidylinositol Phospholipdd, phospholiptd phosphorus Standard deviation Trichloreacetic acid Triglyceride SeThio-Q-glucose and its 6-phosphate Trishwdrexymethylaminomethane Maximu- velocity xiv INTRODUCTION A brief survey of the literature concerning mygyinositol is presented in order to supplement a discussion of the objectives and approach of this project. IJTERATURE SURVEY Although the existence of cyclitols and especially mygrinositol have been known for more than 100 years, the study of their biochemis- try has developed only in the last 40 years. Interest in.mygrinositol increased following the discoveries implicating its importance to living systems; i.e.. growth factor or vitamin qualities, and its role as a component of phospholipids. Several reviews of interest have since been written including those by Heidlein (l) on the biochemistry of cyclitols and a more recent survey by Posternak (2). Another publication (3) worthy of attention is a survey of recent advancements in the area of cyclitols and phosphoino- sitides assembled by the New York Academy of Sciences. MXOf;QQSit01 and its Nutrient Role. After the discovery of £192 inositol by Scherer (4) in 1850, most of the initial work was directed at the isolation of mygrinositol from a large variety of tissues (5). The more recent studies of this compound have centered around its es- sential nutrient role in microorganisms, mammals, and man. Although it has been proven to be an essential nutrient of yeast and fungi (6-9), its vitamin-like function in mammals has not yet been established. Eagle 91. $.00, 11) have demonstrated that minositol is essential to the growth and survival of 18 normal and cancerous human cell lines and two normal mouse cell lines. More recently (12). they have also presented evidence that of 22 mammalian cell strains, only one can be '1 2 grown indefinitely on minositol free mediums. The role of m—inositol as a vitamin in animals was first ex- amined by woolley (13) in 1940. In this study, he demonstrated that mice on a m-inositol deficient diet displayed symptoms of inadequate growth and alopecia. and death followed in two to three weeks. In later studies (11+), he found that supplementation of the diet with 100 mg of minositol or phytic acid/100 g of diet relieved the deficiency symp- toms. Hartin (15). however, found less chronic alopecia in mice fed the deficient diet and found the deficiency symptoms somewhat remedied by pantothenic acid. Hoolley (16) confirmed this observation in later experi ments and explained the apparent discrepancy by synergism between pantothenic acid and minositol (17). As a result of this work. re- quirements for m—inositol by other species luvs been investigated. Studies on the requirements of the rat for m-inositol gave con- tradictory results (19-26). In earlier studies. diets were found to contain contaminating m-inositol in starch and casein (27. 28) and sucrose (26). gig-Inositol supplied by intestinal flora (29) has also been shown to be an important exogeneous source in dietary studies. These sources undoubtedly contributed to the earlier contradictions and the difficulty of interpretation of sttflies. in this area. In addition. en- dogenous contributions. which include .131 _x_r_i_v_q synthesis (28) and possible tissue mobilization of m-inositol. present problems in the study of its nutrient or vitamin-like role in growth and specific tissue requirements. Biosmthesis of mm—Inosito . Suspicion tl'nt animals may have the capability to synthesize m-inositol in m began with Needham's ob- servations of continued inositoluria in rats maintained on low m- inositol diets (30). m-Inositol biosynthesis in 1112, however. re- mained uncertain due to inconsistent results (26) and suggestions that 3 intestinal flora may have been a potential source (1?). later work in rats and mice utilizing radioactive glucose as a precursor and intes- tinal antibacterial agents (28. 31) or germ-free animals (32) definiti- vely demonstrated the biosynthesis of m-inositol from glucose in mammalian tissues. Although biosynthesis in animals had been shown. neither the sites nor the pathway of m—inositol synthesis had been elucidated. In ex- periments utilizing tissue slices and radioactive glucose. Hauser and 1“C from glucose into Finelli (33) demonstrated the incorporation of gig-inesitol in liver. kidney and min of the rat. At the same time. an _i_n _\_r_i_t_r_9_ enzymatic biosynthesizing system was being uncovered by Eisenberg and Bolden in rat testis (34 - 36). Chen atxi Charalampous in yeast (37. 38) and Pins and Tatum in Neurospora crassa (39). Discovery of biosynthetic activity for m-inositol resulted in attempts to elucidate the mechanism of the enzymatic formation of m- inositol from glucose. The biosynthesis of m-inositol from Q-glu- cose-6-phosphate is catalyzed by g-glucose—é-phosptmte a L—m-inos- itol-l-phosphate syntlmse (synttmse; EC 5.5.1.“) and lam-incend- 1-phosprste phosphatase (phosplntase; ac 3.1.3.25) (no). The syntisse has been shown to be rate limiting with a requirement for run" (41) and has been the most extensively studied of the two enzymes regarding properties (#2 - Mt) and mechanism (ll-0. 1+5 - 47). The synthase from higher fans of life has been classified as a type I cycloaldolase requiring an intramolecular oxidation-reduction sequence at C- 5 using run" and a Schiff's base intermediate (ti. at. 1+5). Synthase from N. crease and yeast is a type II cycloaldolsse requiring Zn2+ (1+2. 1&8). mlnofitol and Lipids. The major role m-inositol is known i. to play is as a component of tissue phospholipids. The majority of studies involving phosphoinositides have been centered in min tissue. The three classes of known phosphoinositides include monophosphoinosi- tide. diphosphoinositide and triphosphoinositide. Excellent reviews concerning bioeynthesis. chemistry and metabolism of this group of phospholipids have recently been presented (49. 50). Suggested physi- ological roles of polyphosphoinositides in neural membranes have in- cluded affinity for divalent cations (51) and possible involvement in postsynsptic events during transmission in sympathetic ganglia (52). Since 19%. conflicting reports lave been published on the role of m—inositol as a lipotropic agent. Gavin and McHenry (53) first re- ported the lipotropic action of m-inositol prevented fanation of a special biotin fatty liver. Other workers supported these observations and reported interactions and ' comparisons between gig-inositol and cho- line (20. 51+. 55) in alleviating fatty livers as well as showing m- inositol's ineffectiveness against thiamine type fatty livers (53). Best and his oo-workers (56), however. differed with these views and could produce no lipotropic action that could be attributed to mimos- itol. Hegsted. gt 9;. (57. 58) produced a dietary oil dependent intes- tinal lipodystrophy in female gerbils by withholding m—imsitol. here recently. flayashi. g_t_ 2.2.. (59) demonstrated fatty liver formation in male weanling rats in the presence of sulfa drug when fed a mines- itol deficient diet containing a saturated cottonseed oil. Utilizing a diet similar to Hayashi. Burton aai Hells (60) have produced a fatty liver in female rats which is dependent upon m-inositol deficient diet and lactation and in the presence of an unsaturated fat some. work to elucidate the characteristics of the m-inositol deficient fatty liver 5 by Burton and wells (this thesis) and others (59a, 61) suggests it to be very similar to the fatty liver produced by choline deficiency. gmlnositol Metabolism and Other Prom functions. This brief many would not be complete without a discussion of m-inositol cata- bolism and wasted functions beyond those previously mentioned. The catabolism of m-inositol is believed to progress through the pentose cycle via the glucuronic acid pathway (62, 63). These reactions ulti- mately lead to Q-xylulose-S-phosphate, glycoly‘tic intermediates and po— tential gluconeogenesis (62). Proposed functions of m-inositol secondary to its major role as a component of membnne prospl'loinositides are many and varied. Some of these functions include: cofactor of galactosyl transferase in the synthesis of verbascose (64) and stachyose (65); a contraction factor in mitochordria as a phosphoinositide (66); a role in cellular polarity (67-70). as a substrate in the production of 6-(5-gslactinol in milk and usury gland (71). uronic and pentose write and phytic acid in plants (72 ). indolesoetic acid esters in plants (73) and the synthesis of blue- nomycin (7n) and streptomycin (75); and a potential role as a stabilizer of microtubules (76). Finally. Iajor observations have been made re— garding minositol. diabetes and insulin release. Diabetes he been shown to be accompanied by marked increases in plasma and urinary m- inositol levels (77) and these elevations as well as the resulting de- crease in nerve tissue m-inositol levels may be responsible for the impaired motor nerve conduction velocity observed in the peripheral nerves of diabetics (78). with regard to insulin release. minositol has been proposed to be involved in the process as the phosphoinosidide (79) and some preliminary work in that area has begun (80). 6 OBJECTIVES AND RATIONAIE Aside from the apparent role which glorinositol phosphatides play as important membrane constituents (h9-52) other potential functions which have been examined include: a lipotropic function (53—59), a growth factor for microorganisms (6-9) and mammalian cell lines (lo-12), and an animal vitamin (81). It was the goal of this research to examine the role of exogenous and endogenous sources of myg-inositol and their effect on growth, development and mygyinositol metabolism in the rat. Initial work (Chapter I, 82) in these studies involved an investigation of the origin of the concentration difference between fetal and neonatal plasma and maternal plasma. As a first step in the understanding of the relatively higher levels ofqugrinositol in fetal and neonatal rat plasma as com- pared to the levels in maternal plasma, the examination of the develop- mental changes in the level of this compound was essential. A deter- mination of the effects of the two major sources, bioeynthesis and diet. would aid in evaluating the significance of these differences. The approach used here involved examining changes in the levels of milk aggrinositol during lactation and gigginositol and its biosynthetic en- zymes in plasma and tissues during the course of fetal and neonatal development. In addition, dietary supplementation versus denial of glorinositol was used as an approach to ascertain what proportion of gygrinositol in the adult and developing rat might be of dietary origin and what proportion might be due to ggugggg_synthesis. During the course of the dietary studies, a‘mygrinositol deficient lactation-induced fatty liver was discovered that was prevented by dietary supplements of gygrinositol or by termination of lactation (Chapters III, IV). Recent work, therefore, has emphasized the 7 character of the lipid deposits as an approach to understanding the mechanism of formation of the fatty liver. Investigation of this problem may lave relevance to humans and especially maternal nutrition during lactation. Studies regarding the relationship between dietary and milk levels of m-inositol may prove to be of interest. Infants who cannot be nursed normally (for example. premature and galactosemic infants) may require more than the low levels of minositol observed in bovine milk and commercial formulas (82). The latter portion of this thesis set out to examine potential inhibitors of biosynthesis of m-inositol in order to supplement the dietary work and aid in the interpretation of the role of gzgfinositol in developent. Establishment of 5-thio-2-glucose-6-phosphate as a new _i_n_ _vi_t._r3 inhibitor of the biosynthesis of arm-inositol-l-phos- phate and the study of the effects of S-thio-g-glucose and 2-deoxy-2- glucose (the 6-phosphate is a known potent inhibitor of m-inositol synthesis .12 m (83) ) administration on m—inositol in the mouse culminate the studies presented here. With the establishment of E 1112 inhibitors of m-inositol bioeynthesis. a better understanding of the role bioeynthesis plays in m-inositol metabolism should develop. ORGANIZATION The text of this thesis has been presented as chapters. each following a format used in most biochemical journals. Several of the crepters or portions thereof represent material already published or suhnitted for publication at the present time and reflect the literary style of the journal for which they were written. Chapter I. with the exception of the 2-deoxy-2-glucose work and the tissue m-inositol survey, is presented as it appears in Develomntal 1310ng1 31, 3542 8 (1974). under the title of ”Studies on the Developmental Pattern of the Enzymes Converting Glucose-6-Phosphate to winesitol in the Hat" by Louis E. Burton and William H. Hells. Chapter III and Chap- ter V rave been submitted to the m1 of Nutri_t_i_._o_n_ for publication. Chapter III has been suhIitted under the title of "gig-Inositol Me- tabolism During Lactation and Development in the Rat. The Proven- tion of lactation-Induced Fatty Liver by Dietary LIE-Inositol" by Louis E. Burton and William V. Hells. Chapter V represents a selec- ted portion of a suuitted article entitled ”gig-Inositol Metabolism in the Neonatal and Developing Rat Fed a gin-Inositol Deficient Diet” by Louis E. Burton. Rita E. Bay. James R. Bradford, Joanne P. Orr. Jeffery A. Nickerson and Iilliam I. Hells. Chapter VI is presently being submitted to Alc_h_ives of Biochefimirstrj and BioMics for pub- lication under the title of ”Studies on the Effect of S-Thio-Q-Glu- case and 2-Deoxy-2-Glucose on pig-Inositol Metabolism in Mice and Inhibition by 5-Thio-2-Glucose-6-Phosptnte of Iran-Inositol-l-Phos phate Synthesis. _i_r_i_ vitro" by Louis E. Burton and Hilliam H. Hells. 2. 3. 7. 9. 10. 11. 13. 11+. 15. 16. 17. REFERENCES Heidlein, E.R., Biblflc. Ser, Mellon Inst, ind. Res, no. .6. (1951). Pasternak, T.. ”The Cyclitols", Hermann, Holden-Day, Inc” San Francisco, California (1965). Eisenberg, F., Jr., Consul. Ed. ”Cyclitols and Phosphoinositides: Chemistry Metabolism, and Function”, wN,YL Acad. sci. 1_6§. Art. 2 (1969). Scherer, J.. Liebigg Annln. 21. 322 (1950). Henry, P.. and Balatre. P.. "Les Inositols", hasson et Cie Paris (1947). Hilliams, R.J.. Eakin, 3.3. and Snell, 5.22., J. AllL Chem. Soc. §_2_. 12011 (1940). Kogl, 1",. and van Hasselt, N.. z. _1_’_hy_s.iol. Chem, gig. 71+ (1936). Buston, 11.11.. and Premanik, 3.. Biochem. J. g5. 1656 (1931). Hackinnon, J .E. , and Artagaveytia-Allende, R.C. . J Bacteriol 19. 91 (1948). Eagle, 1%., Oyama, V.I.. and Le11y, 11., Science l2}, 8116 (1956). Eagle, 11., Oyama, V.I.. levy, M” and Freeman, A.E.. J: Biol, Chem, _2_2§_. 191 (1957). Eagle, H., Agranoff, B.V., and Snell, 3.3., J, Biol, Chem, £15, 1891 (1960). woolley. 11.11.. J Biol Chem 116. 113 (1940). woolley. 11.11.. J Biol Chem 129. 29 (19111). Martin, G.J., Science 22. 422 (19111). woolley. D.w.. Pros. Soc, Exp. Biol. Med, 96. 565 (1941). woolley. n.w.. J. Exptl, Med, 25. 277 (19%). 10 18. Pavcek, P.L., and Baum, H.L., Science a. 502 (1941). 19. Nielsen, E., and Elvehjem, C.A., Proc, Soc, Exp, Biol, Med, 99. 349 (19%). 20. Forbes, J.C., Proc. Soc. Expt, Biol. Med, 2. 89 (1943). 21. Fournier, P.. ch. hebd. SeancLAcadL Sci.._ Paris 231. 182 (1956). 22. Jukes, T.H., goo. Soc. ExptLBiol Med 5+5 625 (19%). 23. Richardson, L.R., Hogan, A.C., Long, B., and Itschner, K.I., Proc, Soc, mt. Biol, Med, _ug. 530 (19111). 21». Alstrom. 1.. Legged. Seani _lfl. 433 (199+). 25. Pfaltz, H., zII Vitaminforsch, _1_2. 193 (19u2). 26. McCormick, 14.11., Harris, P.H., and Anderson, 0.8., J, Nutr, 2, 337 (195“). 27. Pasternak, T., Schopfer, H.H., and Boetsch. B., Helv Chim Acta go. 2676 (1963). 28. Halliday, ML. and Anderson. L.. J, Biol, Chem. 2.1.2.797 (1955). 29. Nielsen. 2.. and Black, 1.. Proc. Soc. Expt, Biol, Med, 55. 1h (19%). 30. Needham, J.. Biochg. J. _1_a_, 891 (1921»). 31. Daughaday, 3.11., learner, J. and Phrtnett, 0.. J. Biol, Chem. 21.2,. 869 (1955). 32. F'reinkel, N. ami Dawson, R.H.C., Biocmm, J, 2;, 250 (1961). 33. Hauser, G., and Finelli, N.N.. J. Biol. Chem. 238, 3221+ (1963). 3k. Eisenberg, Jr., P. and Bolden, A.H., Biochem. Biophys. Res. Commun. _1_2_. 72 (1963). 35. Eisenberg. Jr., F., Bolden, A.N. and Loewus, F.A., Biochem, Biopm, Res, Commun. lit, #19 (196“). 11 36. Eisenberg, Jr., P. and Bolden, A.H., Biochem. Biophys. Res. Commun, 21. 100 (1965). 37. Chen, 1.3. and Charalampous, F.C., J Biol Chem. £22, 1905 (1961»). 38. Chen, LU. and Charalampous, F.C., Biochem. Biophys. Res. Commun. 12. 521 (1964). 39. Pina, E. and Tatum, E.L.. Biochin. Biophys. Acta 116. 265 (1967). no. Eisenberg, Jr., F., J. Biol. Chem. 242. 1375 (1967). 1+1. Sherman, U.R., Stewart, ILA. and Zinbo, 14., J. 13319;. Chem. 224. 5703 (1969). 1+2. Pina, B., Saldana, Y., Brunner, A. and Chagoya, V., Ann, N,Y, Acad. Sci. 16;. 5a], (1969). 43. Pine, 11.2., Brunner, A., Chagoya de Sanchez, V., and Pins, 13., Biochem. Biophys. Acta 122. 79 (1973). 141+. Naccarato, H.F.. Ray, R.E., and Wells, HAL, Arch. Biochem. BioLhE, 133. 19» (1971+). #5. Loewus, F.A. and Kelly, 8.. 133,9th Bighfi. ResL Commun. 2. 2m (1962). no. Barnett, J.E.G. and Corina, D.L.. Biochem, J, _1_0_8. 125 (1968). 27. Chen, C-H. J. and Eisenberg, Jr., F., J. 3:131. ChemL 290, 2963 (1975). 48. Charalampous, F.C. and Chen, 1.8., Heth. Enzymol. 2, 698 (1966). 49. Hawtl'nrne, J.N. and Kemp, P., Advan, L12, Res, g, 127 (1964). 50. Hawthorne, J.N. and Kai, $1., Chap. 17 in Handbook of Neuro- Chemistgy. Vol. 3. p. 491, Plenum Press, New York, New York (1970). 51. Kai, n. and Hawthorne. J.N., m. N.YLAcad. Sci. 165. 761 (1969). 52. 53. 58. 59. 59a. 60. 61. 62. 63. 64. 65. 66. 12 Iarrabee, 11.6. and Leicht, 8.3.. J. Neurochen. _1_2. 1 (1965). Gavin, c. and McHenry, B.w.. J, Biol. Chem. _1_32. (+85 (1961). Gavin. 0., Patterson, J.H. and NcHenry, E.H., J. Biol, Chem, 6 138. 275 (1993). Engel, R.H.. J. Nutr. 29,. 175 (1992). Best, C.H., Lucas, C.C., Patterson, J.R., and Ridout, J.R., Biochem. J. 5+8. £51 (1951). Hegsted, D.M., Hayes, K.C., Gallagher. A. and Hanford, B., J. Nutr, _1_03. 302 (1973). Knees. J.F., Hegsted, D.H. and Hayes. K.C., W L02, 1% (1973). Hayashi, E., Maeda, T. and Tomita, T.. Biochim. Biophys. Acta 369. 131+ (1974). Hayashi, 3., Needs. T. and Tomita, T.. Biochim, B10252, Acta M! 146 (1974). Burton, L.E. and Hells, B.R., J. Nutr. (1976) in press. Burton, L.E., Ray, B.R., Bradford, J.R., Orr, J.P, Nicherson. J.A.. am Hells, HJL. m (1976) in press. Hankes, L.V., Politzer, 8.14.. Touster, O. and Anderson. L., Anni NJ. Acad. 89,1; 165. 561+ (1969). Freinkel. N.. Cabor, A., Williams, R.R. and Landau, B.R., Biochim, Biomg, Acta 231. 425 (1970). Tanner, V., Lehle. L. and Kandler. 0.. Biochem, Biom. Res, Commun, 32. 166 (1967). Tanner, w. and Kandler, 0.. Eur- J. Biochem. g. 233 (1968). Vig'nais, P.V.. Vignais. MN. and Lehninger, A.L., J, Biol, Chem, 222- 2011 (196“). 67. 74. 75. 76. 7?. 78. 80. 81. 82. 83. 13 Hokin. L.E. , in Structure and Function of Nervous T1 ssue (Bourne, 0... ed.). Vol. 3.. p. 296. Academic Press, New York (1970). Kai. n. and Hawthorne, J.N., Ag. n.1, Acad. Sc_1_,_ _1_65. 761 (1969). Schacht, J. and Agranoff, 3.8.. J, Biol, Chem, 237. 771 (1972). Freinkel, N. and Dawson, R.M.C.. aggro; _253. 535 (1973). Naccarato. v.72. Ray, 3.5:. and wells. 11.11.. J, Biol, Chem, _259. 1872 (1975). Loewus, B., Ann N Y Acad Sci 1_65. 577 (1969). Labarca, 0.. Nicholle, P.B. and Bandurski, R.S., Biochem, Biophys. Res. Commun. 29, 6H1 (1965). walker. J.B.. J Biol Chem 222. 2397 (1979). walker. J.B.. m 2. 363 (1971). Wagner, R.H., Nickerson, J.A. and Bells, 3.3., Fed. Proc. 35, 1766 (1976). Daughaday, 11.11. and Lerner. J.. J Clin Invest 22. 326 (199+). Greene, D.A., DeJesus, Jr., P.V., and Hinegrad, A.I., J, Clin, Invest, 55. 1326 (1975). Hokin, L.E., Intern, Rev, Cxt_ql, 23. 187 (1968). Freinkel. N., Elyounsi. 0.. Bonnar. J. and Dawson, R.H.C.. 9y}. 333., 22. 568 A (1974). Posternak, T., Chap. XVI.“ in The C litols. p. 303. Holden-Day, Inc. , San Francisco, California (1965). Burton, 1.1:. and wells. w.w.. Devel, Biol, 31. 35 (197B). Barnett, J.B.G., Brice. R.E.. and Corina. D.L.. Biochem, J, £92. 133 (1970). CHAPTER I STUDIES ON THE DEVEIOPMENTAL PATTERN OF THE ENZYMES CONVERTING cmmsM-mosmm 'ro m—INOSITOL IN THE RAT ABSTRACT The migrinositol level of plasma was determined during pre- and post-natal development of the rat. Fetal concentrations exceeded those of maternal rats by nearly lO-fold. Immediately after birth, the 5197 inositol level decreased but was maintained at values 3-4 times that of the lactating dams. The cyclitol content of rat milk was high and rose during lactation to a maximum of 1.6 mm. The biosynthesis of mygrinositol from glucose-6-phosphate is cata- lyzed by 2—glucose-6-phosphate: Lgmygyinositol-l-phosphate synthase (EC 5.5.1.4) and ngygrinositol-l-phosphate phosphatase (EC 3.1.3.2h). The activity of both enzymes was monitored in fetal and neonatal liver, maternal liver, placenta, and mammary gland. Results indicated that fetal liver accounted for over 48% of the total fetal carcass synthase and 26% of the total fetal carcass phosphatase activity. Developmental changes correlated well with the pattern of gigginositol in fetal rat plasma. Similarly, the enzymes of the gygyinositol biosynthetic path- way increased in rat mammary gland in close agreement with the aggrinosi- tol content of milk and diminished to prelactation activities within 2“ hours after the onset of involution. The mygyinositol levels in selected tissues including brain, heart, lung, liver. kidney and intestine were compared for two day prepartum, weanling and pregnant female rats. Data were consistent with increased retention of aggrinositol during development in the rat. Effects of 14 15 Zpdeoxybg-glucose administration and dietarngygrinositol on fetal tissue gigginositol were also examined. Potential contributions of diet and biosynthesis of gxgyinositol to fetal tissue gigginositol levels are discussed. The gigginositol level of colostrum and milk of five human sub- jects was highest (2.8 mM) before birth and decreased to 140% of that level five days postpartum, where it remained for at least three weeks. Even after seven months of lactation. the milk of one subject contained 3-4-fold more mygyinositol than all commercial infant fore mulas analyzed. INTRODUCTION Previous studies of Q-glucose-6-phosphate: gfigygrinositol-l- phosphate synthase (synthase, formerly cyclase, EC 5.5.1.4) and L- m—inositol—l-phosphate phosphatase (phosphatase. EC 3.1.3.25) in mammalian tissue have been largely confined to rat testis (l-u). Although the pathway leading to mygginositol is assumed to occur in virtually all mammalian cells. though at variable rates, little is known about the existence and regulation of the enzyme system in other tissues, e.g.. brain (5). In slice experiments with radio- active precursors. Hauser and Finelli (6) demonstrated incorporation of 1"c from glucose into m—inositol in liver, kidney and brain. In studies of a number of animal species, Nixon (7) confirmed an older observation by Offergeld (8) that the mygrinositol content of fetal blood consistently exceeded that of the maternal blood by nearly tenpfold, suggesting a critical function forggygrinositol in development. Studies by Nixon (9) on exteriorized sheep fetuses 16 provided indirect evidence to support the hypothesis that fetal blood m-inositol was derived from the fetus, not the placenta or mateml circulation. Furthermore, Andrews 91 3;. (10) found that perfused fetal sheep livers were capable of synthesizing m- inositol. Since the origin of the fetal and neonatal blood m- inositol has not been fully established, an investigation of the developmental pattern of the synthase and phosptntase in fetal. neonatal, and maternal rat liver as well as in the mammary gland at various stages in lactogenesis was conducted to provide direct evi- dence for the ability of these tissues to synthesize m—inositol and to correlate the enzyme activities with the levels of m-inositol in plasma and milk. In addition. effects of 2-deoxy-2—glucose adminis- tration. the 6-phosphate of which is a strong competitive inhibitor (K ,-20 uh) of the synthase reaction (12 ), on fetal m-inositol levels were studied in order to seek further evidence for synthesis as a significant source of fetal m-inositol. MATERIAIS AND METHODS Ragents. The following materials were obtained from the indicated sources. Glucose-6-phosphate dipotassium salt. nicotinamide adenine dinucleotide (Ref). and ascorbic acid from Sigma Chemical 00.; y- 9hi_re_;_-inositcl-3—phosphate and dithiothreitol (Dl'l‘) from Calbdoohem: ethylenediaminetetraacetic acid (mm) from Matheson, Coleman 1: Bell. winesitol and 2-deoxy-2-glucose from Nutritional Biochemicals; et- . methyl mannoside from General Biochemicals, Inc.z 3% OV-l (w/w) on Chromosorb 8 (100-200 mesh) from Applied Science Laboratories, Inc.; and trimethylchlorosilane and hexamethyldisilazane from Pierce Chemical Co. 17 Animals. Pregnant rats of the Holtzman strain (Madison, wis- consin) weighing 2&0 g were used in the study. Litters were ad- justed to 7 or 8 pups, and all dams were fed a commercial pellet diet and water ad libitum. For experiments with 2-deoxy-2-glucose (ZDG), fetuses from dams fed a m-inositol supplemented or deficient diet as descriMd else- where (see Chapter III) were sampled at three days pre-partum. Each dam was injected intraperitoneally with 100 mg/kg body weight 2- deoxy-Q-glucose or saline daily for two days prior to sampling. Sampling occurred 21+ hours after the last injection. Fetal age was estimated from the sperm positive date supplied by the Holtzman Com- pany. Fetuses were removed from the dam under other anesthesia and liver. brain and kidney samples were subsequently removed and stored at -80° 0. Blood samples were collected by cardiac puncture with heparin- ized syringe or hematocrit tubes, cells removed by centrifugation and the resultant plasma pooled from each litter and stored at -80° C. E__n_zme Preparation: Synfimése and Phosfiiatase. As noted by earlier workers (1. ll). extracts from mammalian tissues normally contain very active phosphatases and phosphoglucose isomerase which compete for the substrate. 2—glucose-6-phosphate. causing serious difficulties in the measurement of total synthase activity. These problems can be partially obviated by controlled heat treatment. and synthase can be conveniently assayed by the periodate oxidation procedure of Barnett _ep 9;. (12). Synthase was prepared according to the method of Barnett 23 9,1,. (12) with the following modifications. The heat treatment to inacti- vate nonspecific phosphatases and phosphoglucoseisomerase was carried out immediately after extraction in a Potter-Elvehjem homogenizer and 18 prior to the centrifugation at 105,000 xg. The inactivation was extended to 10 minutes at 60° C. since this was shown to provide a higher yield of total enzyme activity. Ammonium sulfate fractions from 0 to W of saturation were used in order to recover as much of the enzyme as possible. Dialyzates were centrifuged at 11+, 500 xg for 15 minutes to remove any insoluble protein appearing during dialysis. The samples were either frozen at -80° C for up to one week or as- sayed at once. both procedures giving identical results. The phosphatase was isolated from the supernatant fraction of the synthase preparations by adjusting from 40% to 60% of saturation with ammonium sulfate. The precipitated proteins were dialyzed overnight at 6° C against four liters of 50 mM Tris-acetate, pH 7.5. and 1 an P-mer- captoethanol. Synthase Assay. Synthase was assayed according to Barnett gt 9_1. (12) with minor alterations. These variations included a orange to 10 In ETA to minimize unremoved phosphatase activity, 14 mn ammonium ace- tate, and the addition of l mu dithiothreitol. Samples were assayed for free phosphate as described below. One unit of synthase is defined as 1 mole of L—m-inositol—l-phosphate produced per hour at 37° C. L-mInositol-l-Phosfl'gte Phosgtase Assay. The phosphatase procedure followed was essentially that of Eisenbers (3) . The reaction mixture contained 1&0 ml! Tris-acetate. pH 7.1” 100 ml! KCl, 3 mM Mg012'6 H20, 0.2 ml! L-m—inositol—l-phosphate or L-ghi_r_o-inositol-3-PhOBPMte and 0.1 ml enzyme extract in an assay volume of 0.5 ml and was incubated at 37° C for 30 minutes. The reaction was terminated by adding 0.25 ml of 20% TCA, and the protein was removed by centrifugation. A 0.5 m1 aliquot was analyzed directly for free phosphate as indicated below. A 19 blank in which TCA was added prior to the addition of the enzyme ex- tract was used to account for free phosphate and that released by action of the TCA. One unit of enzyme activity is defined a l umole of phosphate released per hour at 37° C. Determination of thsphate. Phosphate was determined using the method of Ames (13) with minor variations. Phosphate color reagent (2.1 ml) containing six parts 0.42% ammonium molybdate-4 H20 in 1.0 N sto, and one part 10% (w/v) ascorbic acid were added to 0.9 ml of the sample to be assayed. The color reaction was incubated at 45° C for 20 minutes and absorbance was measured at 700 nm in a Gilford Model 300 spectrophotometer. Protein.Deternination. Protein was determined by the method of Iowry gt 9,1. (14) using bovine serum albumin as a standard. Gas Chromatgfim of m-Inosito . Plasma and tissue samples were deproteinized according to Somogyi (15). The suspension was centri- fuged to remove the precipitated protein and «methyl mannoside was added to an aliquot of the supernatant as an internal standard. The sample was mixed thoroughly and taken to dryness on a rotary evaporator. Samples were then analyzed for m—inositol according to wells gt 2.3,. (16). . (Mm T1 ssue. For analysis of mammary gland free m-inositol levels. tissue was removed, minced, and soaked for 60 minutes at 0° C in an oxytocinasaline solution (10 IU of oxytocin per liter of 0.% NaCl) to release contaminating milk. Collection of Milk. In the morning. lactating rats were lightly anesthetized with sodium pentabarbital (60 mg/kg) and injected with 0.15 ml of an oxytocin solution (10 IU/nl). ‘ Milk was collected by a 20 gentle suction apparatus employing a water aspirator. At various periods pre- and postpartum, milk was also collected from five human volunteers ranging in age from 22 to 33 years. Preparation of gemyo-Inosito15;;Phosphate. We are indebted to Dr. Laurens Anderson, University of Wisconsin, for a generous gift of a mixture of approximately 67.&% gggygrinositol-l-phosphate and 32.2% ‘mygrinositol-2-phosphate as determined by gas-liquid chromatography of the fully trimethylsilylated compounds. The retention times were fur- ther verified by authentic standards of the monophosphate esters gen- erously provided by Dr. C.E. Ballou, University of California. Berkeley. For further supplies of the substrate which is unavailable commercially, we carried out large-scale conversion of g-glucose-é-phosphate to ;- mygrinositol-l-phosphate with partially purified synthase from rat testis (30-fl0% ammonium sulfate fraction). A typical reaction contained 50 mH Tris-acetate, pH 8.0, 10 mM sodium.EDTA, in mM ammonium acetate. 5 mM potassium g1ucose-6-phosphate. 0.1 mM NAD+, 1 mM DTT, 20 mg of a commercial antibiotic preparation, and 10.3 units of synthase, in a final volume of 500 ml. After 169 hours the reaction was stopped by boiling. and the protein was removed by centrifugation. A yield of ap- proximately 135 of gggygrinositol-l-phosphate was achieved under these conditions. The product was recovered and separated from Q-glucose-6- phosphate by passage over a BioRad AB 1x8, 200~400 mesh column (1.5 x 60 cm) in the formats form and equilibrated with 0.1 M ammonium formate, 0.02 M sodium borate. pH 9.5. at a flow rate of 0.1; ml/minute (17). The sugar phosphates were eluted with a gradient consisting of two chambers of 0.1 M ammonium formats, 0.02 M sodium borate, pH 8.5 (150 ml each) and a third chamber of 0.625 M ammonium formats in 0.02 M 21 sodium borate, pH 8.5. Fractions of 10 ml were collected and the £12, inositol-l-phosphate peak which preceded the glucose-é-phosphate from the column was found routinely in fractions 35-#9 and glucose-6- phosphate at tubes 50-55. Glucose-é-phosphate was detected spectro- photometrically (18). Tubes containing the m—inositol—l-phosphate were pooled and treated with 0.1 volume of saturated Ba(0H)2: the barium salt of the phosphate ester was precipitated by the addition of fbur volumes of 95% ethanol and isolated by standard techniques (19). To prepare stock solutions of the inositol phosphate, barium was ex- changed with hydrogen by Dowex 50 (11+) and contaminating boric acid evaporated as methyl borate after two to three successive additions of methanol. The product displayed an identical retention time with that of authentic gemygrinositol-l-phosphate as the trimethylsilylated deri— vative on a 1.8 m by 6 mm column of Chromosorb H coated with 3%10V-l at 230° 0 (20) and was quantified by the periodate phosphate elimination reaction (12). RESUIES mygzInositol Content of Selected Tissuestip the Ieanligg. Fetal and Wt Rat. Table 1 displays free gum-inositol contents of selected tissues in the weanling, fetal and pregnant rat. Generally, the fetal-maternal tissue glorinositol levels reflected the large (7-8- fold) fetal-maternal plasma Elm-inositol gradient. Fetal tissue pyg- inositol contents were 2- to h—fold higher than that of the corresponding maternal tissue (significance as seen in.Tab1e 1): however, several tissues, notably brain and lung, showed no significant differences in mygrinositol content. TABLE 1 m-Inositol Content of Selected Tissues in the Heanling. Fetal and Prognant Rat Animalsa'b Tissue Pregnant Female Fetus Ueanling Liver 0.159 i 0.01?; 0M9 r 0.0913 0.235 r 0.064 Brain 5.0a e 0.5? £1.51 a 0.66 3.52 r 0.106 1 Lung 0.816 r 0.166 0.799 r 0.0% 0.872 a 0.155 Kidney 5.83 : we 5 1.58 a 0.06 5 6.9!» r 0.35 1 Intestine (small) 0.902 a 0.2581 1.50 r 0.16 1.26 1 0.10 heart 0.306 e 0.034s3 0.5» r 0.0533 0.284 r 0.013 Mammary 0.937 t 0.016 ....- ..... Ovaries 0.75“ t 0.184 ---- «_- Testes ----- ----- 5. 03 i 0.22 Placenta 0. 531 t 0.189 ..... ..-.. Carcass (-tissues) --- 0.857 t 0.029 n... Plasma 82.3 r 2.8 614.5 t 04.7 5 86.2 i zu.0 °Values are thememntSJ). terudase. awesnlingsorupoolsor fetuses ( one pool from each dsm ) expressed as umeles of m-inos- itol/ g fresh weight tissue or u! m—inositol for plasma. Dams and wesnlings (21 days of age) were maintained on a commericisl pellet diet and later 93 libitum. Fetuses were 2 days prepartum and the dams their corresponding mothers. Deteninstion of m-inositol as as described in the Methods section I’Stetistice - Superscripts in each column refer to the following com- parisons: l) premt fenle column - female vs. fetus; 2) fetal column - weanling vs. fetus; 3) weanling column - female vs. weanling. The following numbers refer to the level of significance observed using the Student's t test: 1. p<0.053 2. p<0.023 3. p<0.01: it. p<0.0053 5. p<0.001 . 23 Uhen weanling rats were compared with pregnant dams, significant differences were noted for brain (p<0.025) and kidney (p<0.05) in the tissue levels of Lalo-inositol. As the rat developed from fetus to weanling, significant changes were observed for liver (p<0.01). kidney (p (0.001) and heart (p<0.01) but not lung, intestine or brain. Other tissues, including mammary, the gonads and placenta, as well as fetal carcass, are listed for comparative purposes. Tissue-plasma mtg-inositol concentration gradients were observed in the fetus (T/P range: 7-fold to 0.7-fold) and the weanling (T/P range: 80-fold to 3-fold) rat. weanling tissue-plasma differences reflected those observed in the pregnant dam and were generally 2 3. Fetal gradients, however, revealed smaller tissue-plasma ratios generally 5 3 and were always lower than in the corresponding weanling tissue ratios, respectively. lilo-Inositol Content of Rat Plasma. Figure 1 shows the develop- . mental pattern of the Elm-inositol content of rat plasma for pups and dams. In the maternal blood, a small decrease in the level of plasma gum-inositol occurred shortly after parturition which returned to pre- partum levels by eight days postpartum. The lilo-inositol in the plasma of the fetuses dropped abruptly prior to parturition, but plateaued after birth at concentrations approximately four—fold higher than maternal blood which persisted for nearly 16 days postpartum. mic-Inositol Content of Rat Milk: and Mammary gig. A comparison of the pig-inositol content of rat milk and the free Lam-inositol con- tent of rat mammary gland is shown in Figure 2. A three-fold increase in the milk minositol content was observed during the developmental periods examined, and a similar increase was noted in the mammary tissue. .meofiez neg genomes me one geeoosn 483.334 .33 a can 095 o no eaoom : no .96 H seem 33 3:39.88 endow seen .Qezvoeneen .ghenveoa made no sameness shes 3 women swam s 035.? no 5.? can? s were: an 3833 shun .anelmoaesen means AOV seeds dances. ace 2! seeded ass as. we essence 3385.94 .H 53a 25 71 20 l 1 I "2 *- '0. O O O 0.2 - (Wm) 1N31N00 lollsom-m VWSV'ld iva AGE (Days) .133 has .A lllll V Edam . v .3303 messes: e5 5 seasoned es ewe 33.33.30 asses use model—en 33.3. .335 a .«o .:.a.m « sees on» message.» can? seem 53.3303 dosage seam-.3 spun one is assessed e585 ends awe-es: is can see me essence 83393 .N enema 2? 0035;; lo 6 led segoum ("$119 AHVWWVW l EV T fY'l 1 C! N (Wm)lN3.LNOO lOllSONI-m >I'IIIN 1V8 l2 l4 l6 l8 20 IO (Days) AGE 28 A similar observation of increased gygrinositol in mammary glands during lactation was reported by Dawson and Freinkel (21), but sepa- rate analysis of milk was not reported for their study. Human Milk syn-Inositol. The level of free glorinositol was found to be relatively high in the colostrum and milk of five human subjects (Figure 3), and this was distinctly different than the pattern for rat milk gigginositol. within five days postpartum, the mean value was approximately 40% of the highest level and this was followed by a period of relative constancy. One subject was observed at three and seven months postpartum. at which times glarinositol levels had de- creased even further. It is interesting to note, however. that even the lowest levels recorded for human milk in this study are several- fold higher than the values we have determined for commercially available for-ulas which ranged between 0.1“ and 0.21 ml. Developgental Pattern of Synthase. The highest liver synthase activity was observed in fetal rats. Thereafter, a decline occurred reaching adult levels by day 12 postpartum (Figure 4). As a comparison. the livers of four pools of three fetuses, two-day prepartum, contained over “8% of the total body synthase although the liver represents only 7.8% of the body weight. Prior to parturition, rat mammary gland syn- thase activity was low and similar to that of adult liver and placenia, but rose during lactation in an inverse relationship with that of news born rat liver. In either tissue, the activity of the synthase core related well with the level of free gygyinositol in fetal plasma or rat milk. Similarly. it appears that a slight decrease in maternal liver cyclase activity followed parturition and was accompanied by a parallel decrease in maternal blood glorinositcl level. The synthase activity of .3333 25m e5. no one knee sown seam uses ewe no.2: 358 om» 75m e5 mom vaeowe 30033 n no .x.l.m « seem e5. sense .eesen panes seen 525 H3281.“ fie: sees: as senses geese-moaned .n 8.3m 30 LI Estuazoa ease ms: Esteaoaa ml 0.! Si To w m o_~ o _ _ _ _ _ _ _ u _ _ _ _ _ (w w) 1N31N00 loilsom-m mm vanH 31 0.3.30 hence use 333.39% 3639 .5303 scenes: .3 5 ass—«moses es see 33» .emseswo .eseseosnmmmoeaoeme .ensnmuo Soon a we» .n.m « sees e5 senescence 3? seem .23 go hum-Is: use A. 5:00 IHQHNMMnHmwumga figs: A.v .Hg «es 388: use gem no eessssm 8. has: . 3838.883 «o 53632 5 scenes gee-senses .s ensur— 32 Am><9 mod _llcotsoss. +1 82333.. 4—“ 82.829— VN on N. o o «I VI A. a a _ q _ _ _ 1 1 v e — T (Glll n‘ — e 'el _ _ _ . r _ _ -36 . _ . _ _ _ _ _ T _ 1 0.6 _ _ _ i _ 1 _ 1 e3 _ _ _ unemd bui/ suun 33 the mammary tissue diminished to prelactation levels within 24 hours after the onset of involution. Developggntal Pattern of the Phosphatase. Analysis of 513197 inositol-l-phosphate phosphatase revealed a profile similar to that of the cyclase in both fetal and neonatal liver and in mammary gland (Figure 5). In the newborn liver, the phosphatase diminished more rapidly than did the cyclase activity. Another minor variation between the two enzymes can be seen in maternal liver phosphatase shortly after birth of the pups; i.e.. an increase was found in maternal liver phos- phatase, while a small decrease was found in maternal liver synthase activity (Figure 4). Fetal liver contained a significant amount (26.6%) of the total body phosphatase. Under all conditions studied. however, the activity of the phosphatase was 5- to 10~fold greater than that of the synthase. The patterns for the two enzymes were sufficiently simi- lar to suggest a coordinated response for both enzymes to those factors which regulate their activities. Effects of Diet and 2-DeoxyeD-Clucose Administration on Fetal get Tissue nyo-Inositol Content. Administration of 2-deoxy-2-glucose (2m) intraperitoneally to the pregnant dam reduced the levels of mygginositol in various tissues in the rat fetus regardless of the availability of dietarngygfinositol to the dam. Table 2 shows the effects of 2DG ad- ministration on free gygyinositol levels in plasma, brain. liver and kidney of fetal rats whose corresponding dams were fed a mygrinositol supplemented or deficient diet. A significant (p< 0.01) dietary effect of aye-inositol on tissue free gygeinositol content was observed only for the kidney. Administration of 2DG to both supplemented and defi- cient dams resulted in the elimination of the fetal kidney dietary .5303 nee-fie: In. 5 coon-coed as one aeuvauseo home.- 3... coupes-noun 333. .3 a no 535039 m «c Aces e .aa 0 «e one...H e no dd « 5.... .5 seconds, 3a! eel .AOV 85 nice: one .AOV 8:85 . v ~33 fiancee: .AOV 253 H8382 3e 3... mo Sadness seedesmAAofleoa A no 334.2 5 83.5 3:328 .m enema 35 Am ><9mo< Ill. cot-:35 L1 Eateaaeea 010mg 6m/ suun 36 TABIE 2 EFFECTS OF Z-mXY-g-GLUGGSE AND DIET ON FETAL RAT gig-BIOSI'I‘OL CONTENT OF PLASMA AND SELECTED TISSUE Treatments 1' 2 Tissue Diet Fed3 Control 2-Deoxy-g-Glucose maternal + 115 t 20 139 '1' 34A 1’1”” - 1+5.2 3 22.0‘1 37A i 8.69' A fetal + 6&9 ’5 9a _ 430 1' 61B Pm - 482 ‘5 102‘ 167 ‘3 36‘" 0 fetal + 0.592 1'3 0.051 0.450 '5 0.034C 11"” - 0.506 3 0.051a 0.221 1' 0. 0561' E fetal + 1.87 1' 0.0144 1.03 1' 0.09F “an” - 1.51 ‘3 0.11(1 0.838 1' 0.1143" E fetal + n.02 i 0.16 3.75 ‘3 0.27A min " 3089 t 00528, 303“ 1. 0018A’ 8 1 Dams were injected with saline (control) or Z-deoxy-D-glucose as given in the Methods section. Statistics: a. not significant (p >005). b. p<0. 05. c. p<0. 02. d. p<0. 01. e. p<0. 005,f . p<0. 001; Upper case letters refer to comparisons between control and 2—doexy-D- glucose groups; lower case letters refer to comparisons within control or 2-deoxy-D-g1ucose groups. Statistical evaluations were made using the Student‘s two-tailed t test. 2 Tissue levels ofm gig-inositol are given as ill! for plasma or uncles/g wet weight tissue for tissues. Each value is the mean- *8. D. for four dams or four pools of fetuses (1 pool from 1 dam). 3 For diet constituents. refer to the Methods section Chapter III. Supplemented diet (4-), Deficient diet (- ). 37 difference. whereas significant nductions appeared in fetal plasma (p1 0 E 6.0h— _- 4.0— b d x a J. 2.0- "' -‘ fl- 1; 0 I l I l O m- 4 8 I2 I TIME (weeks) Figure 1. Free LIE-Inositol Content of Plasma. Liver. Lung. Heart. Kidney and Intestine in Female Rats Fed a lilo-Inositol Supplemented or Deficient Diet. Supplemented animals are represented by the tri- angular symbols. deprived animals by the circular symbols. Each value is the mean 1- S.D. for ’4 animals. In A) plasma is represent- ed by the solid symbols. hepatic tissue by the open symbols. In 13) lung is represented by the solid symbols, cardiac tissue by the open symbols. In C) kidney is represented by the solid symbols, intes- tinal tissue by the open synbols. Square symbols represent animals fed the stock diet. 1:8 Similar'differences were not observed. however. for*deprived animals. Renal tissue (Figure 10) demonstrated significant (p< 0.05) differences in aggrinositol content between the two groups throughout the regimen: with the deprived animals having levels significantly below (p<0.05) that of the stock diet controls. Levels for supplemented animals were not different (p<0.05) from the stock diet fed animals. Mean differen- ces were observed for the gyprinositol content of the small intestine (Figure 10) throughout the time course. becoming significant (p<0.02) by four weeks on the diet. Significant differences in intestinal 519: inositol were observed for supplemented animals when compared to stock fed controls after four weeks of diet feeding. however. at no time was this observed for'deprived animals. Splenic tissue (Figure 2A) re- vealed a dietary response pattern of gygrinositol similar to the kidney showing a decrease in the levels in the deprived animals below levels observed for the stock diet fed animals shortly after initiation of the diet regimen. However. significant differences (p<0. 05) in gig-inosi- tol levels of the spleen between supplemented and deprived animals were observed only between four and eight weeks on the diets. Pancreas free gygyinositol levels (Figure 2A) showed no significant differences be- tween the supplamented and deprived animals. with the exception of levels observed at eight weeks on the diet (p<0.05). The level of m- inositol in the pancreas of stock diet fed animals was not.determined. The increase in the gygyinositol content of pancreas from eight to 16 weeks might be due to plasma elevations during that period for both groups, however, the observation was not universal for all tissues ex- amined. Drain free gzgrinositol was not significantly'affbcted br'dietary {glarinositol. After two weeks of feeding. however. mean differences .0 co jg l :3: on _ \ 0.4 -1 (D 2 E o . . i n . ‘5 B .8 1 - E 6 11 3 E m 40— —os-g E 3 § ao— 414% o 1 1 1 1 1 o 0 4 e :2 l6 TIME (weeks) Figure 2. Free avg-Inositol Content of Spleen. Pancreas, Brain and Mammary Gland in Female Rats Fed a gin-Inositol Supplemented or Deficient Diet. Supplemented animals are represented by the tri- angular symbols. deprived animals by the circular symbols. Each value represents the mean 1- S.D. for 1+ animals. In A) splenic tissue is represented the solid symbols. pancreatic tissue by the open symbols. In B whole brain tissue is represented by the solid symbols. mammary tissue by the open symbols. Square symbols represent animals fed the stock diet. Stock fed pancreatic and 16 week supplemented and deficient diet fed values for m-inositol were not determined. 50 between supplemented and deprived animals, though not significant. were observed through 16 weeks on the diet (Figure 23). Mammary gland (Figure 23) showed wide variation in Lug-inositol content in response to the diets throughout the time course, the significance of which has not been determined. Levels of mammary gland Egg-inositol were not determined at 16 weeks for animals fed either of the puri- f led dicta. DISCUSSION Studies regarding the role of dietary lam-inositol in controlling tissue and body fluid gig-inositol levels are lacking. A significant amount of the dietary work concerning gig-inositol has been centered on its role as a lipotropic agent (1 - 5) without the benefit of diet or tissue analysis for measurements of intake or affects on tissue levels of lug-inositol. This fact has resulted in some difficulty in the interpretation of data and the subsequent evaluation of the role pyp-inositol plays in diet (6 - 11). The purpose of this work was to examine the effects of dietary m—inositol on tissue and plasma free gum-inositol levels and the use of diet as an aid in elucidating the metabolic functions of Egg-inositol. None of the reported symptomatic responses (8. 13. 15) to 212- inositol deprivation were observed during this study. This might be explained by the lack of a sufficient depletion of tissue gig-inositol for most tissues during exposure to the deficient diet. Two effects are reflected by the majority of the tissues examined with regard to the effects of dietary m-inositol on tissue levels of Lam-inositol. While the deficient diet affected kidney. heart an! to a 51 small extent, brain and spleen, by lowering tissue gig-inositol below stock diet controls. it did not affect the other tissues examined. At the sane time, the supplemented diet stimulated gig-inositol levels in intestine, lung, heart and plasma above the stock diet controls while showing no significant effects on kidney, spleen, ani brain. Risenberg's work (16) on synthase activity in several of the tissues exalined here would suggest a strong effect of diet on cardiac tissue while almost no dietary response would be expected in testicular tis- sue, a fact which has been verified (Chapter III, 17). The data pre- sented here might be explained in part by tissue/plasma‘gygrinositol ratios on the basis of tissue differences in the ability to retain m- inositol (Chapter I, 18). Earlier studies reported conflicting data (6 - 11) resulting in difficulty in evaluating gig-inositol dietary effects. Although pre- cautions have been taken with dietary constituents to avoid contami- nation, accompanied by diet analysis to show the availability of m- inositol in the diets, the inability of the deficiency diet to reduce levels of gig-inositol in certain tissues might be explained by the presence of microorganisms in the gastrointestinal tract which are known to contribute an exogenous source of m-inositol to the rat (13). Evidence presented here suggests that for the rat. gig-inositol de- privation symptonology is not observed in the presence of intestinal flora (i.e. the absence of antibacterial drugs). The inportance of the intestinal flora as a contributor of exogenous gig-inositol via in- testinal absorption or coprophagy is also supported by the absence of a m-inositol deprived, lactation-induced fatty liver in dams fed a deficient diet containing no antibacterial drugs (Chapter IV). However. .52 in the presence of gastrointestinal bacteria, effects of dietary'gyge inositol were observed in some tissues on tissue levels of gygrinositol and supports the importance of diet as a significant contributor of gigginositol in tissues. 2. 3. 10. 11. 13. 1". 15. 16. 17. 18. 53 mass McFarland. M.L. and Mcflenry. s.w.. J, Biol, Chem, _1_L6, 429 (1948). Beveridge. J.n.n. and Lucas, c.c.. J, Biol, Chem, m. 311 (1945). Best, c.n., Ridout, 3.11.. Patterson. J.H. and Lucas. C.C., Biochem. .1, fig. 1448 (1952). Hayashi. 3.. Needs. T. and Tomita. T., Biochim. 310m. Acta }_§_Q, 134 (1971»). Hegsted, D.h.. Hayes. K.C.. Gallagher, A. and Hanfond. B., JI Nutr, £92. 302 (1973). Martin. G.J.. Science 22. 422 (1941). woolley. 133.. J. Exp. Med. 2.5- 277 (191.2). Pavcek. P.L. and Baum. H.L.. Science 22. 502 (1941). Nielsen. E. and Elvejhem. C.A.. Proc Soc Ex Biol Med _ug. 3H9 (1941). Forbes. J 0.. Proc, SocI E_)_cp, Biol. Med, if. 89 (1943). McCormick. M.H.. Harris. P.H.. and Anderson. C.A.. J‘ Nutr, 2'. 337 (195a). Halliday. J.w. and Anderson. L., J, Biol, Chem, _2_11. 799 (1955). Nielsen. s. and Black. A., Pros, Soc, Exp, Biol, Med, 55. in (19%). wells. w.w.. Pittman. T.A., and wells. H.J.. Anal Biochem lg. ‘#50 (1965). woolley. n.w.. J, Biol, Chem. _112. 29 (19M). Eisenberg. Jr., F., J Biol Chem gig. 1375 (196?). Burton, L.E. and Bells. ".3” JI Nutr, (1976) in press. Dawson. 8.11.0. and Freinkel, N., Biochem, JI 8, 606 (1961). CHAPTER III m-INOSI’IOL METABOLISM DURING IACTATION AND DEVELOPMENT IN THE RAT. THE PREVENTION or IACTATION-INDJ- FATTY LIVER BY DIE-Tm mLo-INOSITOL ABSTRACT Effects of dietary gig-inositol deprivation were examined during prenatal and postnatal development and during lactation in the rat. The deficient diet contained no detectable m-inositol while the sup- plemented diet contained 0.5% (w/w) m—inositol at the expense of sucrose. Both diets contained 25% casein. adequate amounts of all known vitamins, choline. and essential fatty acids as well as O. 5% (w/w) phtulylsulfathiazole to depress gig-inositol contribution to the diet by microorganisms. Pregnant rats of the Holtzman strain were fed the respective diets during gestation and lactation and pups were fed the corresponding diet after weaning until three months of age. There were no significant differences in body weight between experimental groups. Supplementation of the diet with gym-inositol increased significantly the levels of m-inositol in plasma. liver. kidney. and intestine of pups at virtually all ages examined. and increased significantly the levels of m-inositol in the milk and mammary tissue during lactation. During lactation. the LIE-inositol deprived dams developed severe fatty livers (31% w/w) ornraoterized by diminished phosphatidylinositol (50%) and total phospholipid phosphorus (57%) levels as oonpared with controls. After weaning. the liver lipid content of the gig-inositol deprived dams returned to normal (4.36). The data suggest that a pos- sible threshold level of free minositol (approximately 0.15 umoles/g 54 55 lipid—free tissue) was required to prevent fatty liver in lactating dams under these dietary conditions. Effects of the deficient diet on fertility were also examined. Based on sperm count and production of offspring, there were no dif- ferences between the experimental and control males. Females of both groups showed equal ability to produce offspring. INTRODUCTION Metabolic functions for gig-inositol. other than a role in. mem- brane components. are obscure. m-Inositol is an essential nutrient for microorganisms (l), mammalian cells (2). and for animals under specific dietary conditions (3 - 9). Since 19%, conflicting reports have been published on the role of gig-inositol as a lipotropic agent. Handler (u) and. more recently. Kotaki. gt _a_1_. (5) have demonstrated that lipid deposition can be prevented in rats by dietary m-inositol. However. pre-treatment of the animals (5) with a low protein. B vi- tamin-deficient diet followed by supplementation with B vitamins without choline and lug-inositol was necessary. Other reports showed curative effects of gig-inositol on fatty liver in rats fed a fat-free. high carbohydrate. gum-inositol deficient diet (6). Hegsted. g_t_ 2.1. (7, 8) produced an intestinal lipodystrophy in female gerbils by with- holding lug-inositol. Recently, Hayashi. g_t_ _a_l_.. (9) demonstrated fatty liver formation in rats in the presence of a sulfa drug when fed a gig-inositol deficient diet containing hydrogenated cottonseed oil but not for one containing natural cottonseed oil. The purpose of this study was to examine the effect of a m- inositol deprivation in pregnant and lactating rats on the gig-inositol 56 metabolism in fetal and postnatal offspring. During the course of the study. a lactation-induced fatty liver was discovered that was pre- vented by dietary supplements of m-inositol or by termination of lactation. MATERIAIS AND METIDE Animals and Diets. Female rats of the Holtsman strain1 weighing 250—300 g were used in all experiments. On the seventh day of ges- tation. the pregnant rats were divided randomly into two equal groups and fed purified diets with or without egg-inositol for the specified periods. The diets2 used in this study were patterned after those of Hayashi 91., 9_1_. (9) and were fed 55; libitum. Analysis of the mixed diet showed no detectable (<0. 001 mg/100 g diet) free m-inositol in the deficient diet and 504.2 1' 28.4 mg (mean t S.D. . n-4) per 100 g of diet free m—inositol in the control diet (10). A supplemental salt mixture3 was added to the standard Phillips-Hart salt mixture“ to bring 1‘I-loltzman Company, Madison. Wisconsin 2The following dietary materials were obtained from the indicated sources: vitamin-free casein. It -cellulose and vitamin mix without 33- inositol from ICN Nutritional Biochemicals Corporation. Cleveland. Ohio; Phillips-Hart staniard salt mix from Teklad Test Diets, Madison. Miscon- sin; soybean oil from Swift Oil Company, St. louis, Missouri; choline chloride and phthalylsulfathiazole from Sign. Chemical Company. St. louis. Missouri 3The supplemental salt mixture consisted of (% by weight): 6.213% Ctfiou'S H20. 0. 500% COC12'6 H20. 14.004“ ZnO. 38.2“” m4.u20' am 41. 03 % non-nutritive fiber (cellulose) “The Phillips-Hart salt mixture consisted of (76 by weight): 30.00% 57 the salt content up to accepted levels (11). Phthalylsulfathiazole was employed at the 0.3% (w/w) level to prevent potential contri- bution of gygrinositol to the diet by intestinal flora. The vitamin mixtureS incorporated into the diet was of standard content from a commercial source. The major difference between the Mayashi diet (9) and the one used in this study was the incorporation of natural soyb bean oil rather than hydrogenated cottonseed oil. Diets were freshly made every one to two weeks as needed and stored at 40 C. Neonates were nursed by their respective mothers until 21 days of'age at which time they were fully weaned to solid diets and water'gg libitum. During the lactational period. however. pups had free access to the solid diet as they desired. Animals were housed at 200 C in poly- carbonate cages with wood shavings with a light cycle of 12 hours (6:00 A.M. - 6:00 P.N.). Fetal ages were estimated from the sperm-positive date supplied by the Holtzman Company and correlation with a mean birth time for each group studied. Gestation for the rat fetus was observed to be 01:003. 7. 50% season-2 H20. 0.005% 0o012°6 320. 0.003% ousou' 5 1120. 32.2% xznmu. 2.75% ferric citrate. 10.2% "5501.” 1120. 0.08% KI. 16.7% NaCI, am 0.025% chl2 5The vitamin mix composition was as follows (g/kg). vitamin A ester (palmitate and acetate) concentrate (200.000 units/g). 4.5; Vitamin 93 (400.000 unite/g), 0.25: d-tocopherol. 5.0: ascorbic acid. 45.03 choline chloride. 75.0. and menaquinone. 2.25. The following vitamins were at levels in mg/kgi p-aminobenzoic acid. 5.0; niacin. 4.5: riboflavin. 1.0: pyridoxine-HCl. 1.03 thiamine-H01. 1.0: calcium pantothenate. 3.03 biotin. 20.0; folic acid. 90.0. and vitamin B 1.35 12’ 58 21.5 to 22 days. After birth. the number of pups was adjusted to eight per litter. The sampling of tissue was accomplished as much as possible at the same time of the day ani in a random order: i.e. . no preference was given to sex or diet in the order of killing. Tissue samples were removed from animals under ether anesthesia. weighed at u° c. and stored at -80° c. Blood samples were collected in hepa- rinized syringes or hematocrit tubes (by puncture). centrifuged. and the plasma was stored at -80° 0. Age 0 was considered the 2a hour period beginning with birth and all other ages were calculated relative to that point. Mothers and/or pups were sampled at -3. -2. 0. +4, +8, +14. +21. +32. +49. an} 120 (adult) days of age with respect to the pups' birth. Four maternal samples were taken at each time point. Eight pups of unknown sex were sampled at each point from birth until 21 days. On day 21. four male and four female pups were sampled. After 21 days, three male and three female pups were sampled at each time point. Fetuses from four dams in each group were sampled at pre-birth time points. Milk Collection and Amniotic Fluid. Milk collection was accom- plished as previously cited (12). Amniotic fluid was withdrawn with a syringe prior to removal of the fetus from the uterus. Tissue and Fluid Content of aye-Inositol. Free gig-inositol content of tissues. amniotic fluid. milk, and plasma was measured by gas-liquid chromatography6 (l3). Lipid-bound gig-inositol in the form 6Materials for gas-liquid chromatography of gum-inositol were as follows: K -methyl-D-mannoside from General Biochemicals. Inc. . Cha- grin Falls. Ohio: 3% OV-l (w/w) on Chromosorla H (loo-200) mesh from Applied Sciences laboratories. State College. Pennsylvania: and tri- methylcholorosilane and hexamethyldisilasane from Pierce Chemical Com- pany. Rockford. Illinois 59 of phosphatidylinositol was quantified from a neutral Folch lipid extract of tissue by the procedure of Hells. gt 11. (10). When de- termining tissue Lin-inositol. identical areas were taken from each of the animals. After day +4, small intestine samples were cleaned of their contents prior to analysis. Lipid Content of Tissue. Liver lipid content was determined gravimetrically by the method of Folch.gtflg1. (14). All organic sol- vents used in the extractions were freshly redistilled. Phosfllipid Phosphorus Levels. Phospholipid phosphorus in the extracted lipids was measured by the method of Ames (15). Fatty Acid juantitation. The fatty acid composition of liver and dietary lipids was determined by gas-liquid chromatography of their corresponding methyl esters7. Peaks were identified by comparison of retention time with standards, and composition was expressed as area percent of the total observed peaks on the chromatogram. Sperm Counts. Sperm counts were made following the technique of Kirton g; 2;. (16). Statistics. The results were analyzed using the Student's two- tailed t test (17). Significance was acceptable when p<0. 05. REULTS Body Hgight Profile. No significant differences were observed between the body weights of the m-inositol supplemented animals ver- sus those fed the deficient diet (Figure l). (The number of animals (n) 7Gas chromatography of the fatty acid methyl esters utilized a 1.8 M by 3mm glass column containing 10% SP 2340 (100-120 on Supelco- port, Bellefonte, Pennsylvania, at 170° 0 in a Hewlett-Packard Model 402 Gas Chromatograph, Hewlett-Packard Company, Avondale. Pennsylvania 6O .hnosaaoeauou .33 05 as 30.3 2.3 83 so.“ one: mal—ass 5»: .8 as...» season» some you .30.? 3.300903 23 new name 3 venues rue: newsman Ho when HN mag .33 min .333 5.... .3 s «n :1: .de. .o~:s .r 59.: .£ a. .e .o anus .ease - Rnus .naee n- 3 .30 s new .n.m u. sees 83 secession #58 goes Joan Assamese.“ “as meal. we Hoe." s . G .moaeseu . O .31! . R V vseaoauen no Assad-em one means no Hoon e . 4 .eoassem . 4 and! . 4 V seasons” as Heeaeosanflm e sea 3cm ensconced no serene: seen A g 61 (5)1H9l3M mv sass mod mm 00. T OONI 00m ooen com com “Fl l r 1W 1 _1— (5) lH9l3M 62 ranged from 8 to 35 for animals up to 120 days of age: for’adults (120 days of age). n93.). However. the mean weight in each group for the supplemented animals was always greater than that of the deficient group. No unusual physical characteristics such as cataracts. alopecia. or deformities were noted in animals of either group. Amniotic Fluid Free myo-Inositol Levels. Although the mean for the supplemented fetuses was higher than that for the deprived ones in every case. the amniotic fluid mygrinositol levels showed no statis- tically significant differences. values observed for amniotic fluid gig-inositol (mean ‘3 S.D. . n-4) in dams fed the deficient diet were 0.215 t 0.043 mM at -3 days, 0.295 t 0.024 mM at -2 days, and one value of 0.252 mM at approximately eight hours prior to birth. Supplemented animals showed levels of 0. 307 1’- 0.043 mM at -3 days. 0.1143 '5 0.131 mM at -2 days. and one value of 0.425 mM at approximately eight hours prior to birth. Plasma Free myg:Inositol Levels. Figure 2A indicates the observed plasma gum-inositol levels for fetuses. neonates, and young adult rats. Hith the exception of -2 days of age. significant differences (p <0.025) in plasma gem-inositol levels occurred between the two diets after birth (four days) through +14 days of age. At 21 days of age no significant differences were detected when comparing sex differneces between dietary groups or within dietary groups. After 21 days of age (weaning). significant differences in the plasma levels ofqugrinositol were observed between males fed the deficient diet and males fed the supplemented diet (p<0.025): this was also the case for females fed the two diets (p<0. 025). These differences. however. were not observed when comparing males and females fed the same diet. As previously 63 Figure 2. Free m—Inositol Levels in the Plasma of A) Developing Rats and B) Iactating Dams. (:1) Refer to figure 1 for the symbol legeni. Each point represents the mean 1: S.D. for four pools of fetuses (each pool from one dam). four pools of eight pups each for animals up to 21 days of age. four animals for 21 days of age. or three animals for ages greater than 21 days. (B) Each point rep- resents the mean : S.D. for four dsu. A . supplemented diet: . . deficient dict. {IL PLASMA A O — E 0.4 .. 3? S .s é. E 0.2 \ - b." A . ‘. ‘b— ---.-——— o l J . l :2 24 36 48 I ADULT AGE(days) PLASMA B 0.3 - E 50.2 -— .72 "0': J. O a J I O at... . J l l O 7 l4 2: LACTATION (days) 65 reported (12). eight- to ten-fold differences in plasma Lug-inositol levels were observed when comparing fetal to maternal values regard- less of the gum-inositol content of the diet. An initial response to the milk of the supplemented dams (Figure 4A) was observed in the pups at eight days of age. but the decrease in plasma levels of m- inositol postnatally continued at 14 days of age. Figure ZB indicates the levels observed of free Egg-inositol in the plasma of pregnant and lactating dams fed the gyg-inositol deficient and supplemented diets. Supplemented dams showed an elevation in the mean plasma qu-inositol content from three days prepartum (0.10 mM) to four days of lactation (0.26 mM). After seven days of lactation. plasma m-inositol returned to prepartum levels though the difference in the means were not statis- tically significant between groups or with time. Deficient dams showed a similar pattern for plasma gulp-inositol but without the subsequent rise frem 0 to 4 days of lactation. Brain and Kidney Free myg-Inositol. Differences in the developing rat brain free Lug-inositol levels (Figure 3A) were noted throughout the period studied. Statistically significant differences between diets were observed at four (p<0.05). l4 (p<0. 02). and 32 days of age (p <0. 05). The physiological significance of the decrease in free _myg—inositol levels after birth and the subsequent rise to levels greater than or equal to fetal brain levels remains obscure. Simi- larities between these studies and earlier work by Hello. 3‘3; 51. (18) and Allison and Stewart (19). were observed. In addition. the evidence indicates that the postnatal m—inositol increase occurred more ra- pidly for the animals fed the supplemented diet than for the animals fed the deficient diet. .13“— "w .273 am can» Hosea some new has-ans some» can own no Enos 3 Mom eds—ass use.“ .0: mo mass .8 3 as wages Now some mama .530 no naoon use.“ A83 23 no.5 doom nonov usage.“ mo eaooa use new .n.m a some one. senescence peace seem .modumem use nodes mo H39 4. . e anodes...“ . 0 “means . . was.“ 625.33 .93 modems.“ use nodes mo Hood s . ‘ arcades.“ . 4 .moass . ‘ 3cm ceases—saga .333 25 359035 See... A3 genes. :3 Susan A5 .23 preseason no geeseaaasm Ganesha a one sees reassessed .ao nesnnae nonsense 5 doses Hoeaeosuuflm sens .m shaman 6? ADULT 1:15 _Jl I ADULT ' AGE (days) >- 1’ p. g *3 a ‘8 a? E I- E3 —&2 -& .o —O O (D l 1 l 1 l 1 o o Q 0. 8 2 o. 3 .. N (anssu b / sapm fl) [mm-m (my b/sqom n) WWW 36 43 I r—ADULT AGE (days) 24 I ' ' .5 I I I § 19- T If T - 3 -3 Z w 2 L8, 5 ._ u '— E 2 33 .. g E — u - o <[ 1 I a o 0 l 1 I 8 3 ~ g 3 8 ("”3" 6/”Im“ II) .0550"! "m (anssu 6/ salon] fl) |0&!$OU!;m 68 The free gigginositol content of kidney tissue is shown in Figure BB. The postnatal elevation of free gig-inositol in kidney and a pla- teauing of the levels after 21 days of age were observed for both diets. Differences in kidney free gygrinositol levels at birth were not significant. Significant differences were observed for free gig-inosi- tol levels at all ages examined prior to 21 days of age (p<0.05). There were significant differences (p<0.02) in free Egg-inositol levels between males fed the supplemented diet and males fed the de— ficient diet; this was also true when comparing the females fed the different diets (p< 0.02) with the exception of the 21 day old females. Intestinal Free myo-Inositol lavels. The developing rat showed an approximate two-fold elevation in the intestinal free pool of m- inositol regardless of the diet fed (Figure 30). No significant dif- ferences in gym-inositol levels were noted prior to eight days of age; however, these intestinal samples were contaminated with intralumenal contents. Differences in filo-inositol levels were not observed between males and females fed the same diet with the exception of 21 day old animals when sex differences were significant (p<0.02) in both sup- plemented and deficient diets. At all ages after eight days. there were significant differences (p<0.02) in grinositol content between females fed the supplemented diet and females fed the deprived diet; this was also true for the males fed the different diets with the ex- ception of the 21 day old males. The overall trend involving the in- crease in levels of Eye-inositol in intestine reflected that observed in kidney; however, the plateau began somewhat later. at 32 days of age. Fertility Studies. Because the free m—inositol level of rat gonads is normally high with respect to other tissues, we examined 69 the effects of the deficient diet on those levels and on the sperm count. Figure 3D shows the free glorinositol levels for the de- veloping rat testis. The diet significantly (p< 0.05) affected levels of aggrinositol in this tissue prior to 32 days of age. Prior to 49 days of age sperm counts were untimeasureable by the visual counting technique used. Supplemented 49 day old males gave counts of 1.30 t 0.11 x 108 sperm per testis or 0.94 t 0.0“ x 108 sperm per gram tis- sue. whereas corresponding‘gygfinositol deprived males were 1.21 t 0.17 x 108 sperm per testis or 0.93 t 0.11 x 108 sperm/g tissue. Supplemented adult males revealed higher values of 2.21 I 0.08 x 108 sperm per testis or 1.2h8 : 0.130 x 108 sperm/g tissue. Sperm counts for’deprived adults were 2.13 t 0.12 x 108 sperm per testis or 1.201 t 0.031 x 108 sperm/g tissue (n93 for'all of the above obserb vations). These observations neither support nor’discount potential effects of gig-inositol on the fertility of rate since the presence of sperm was observed only after early differences in'mygfinositol levels disappeared. Fertility tests were conducted to determine whether possible in- direct effects of early testicularggygyinositol differences affected fertility. 0f the 12 males and 12 females from each of the two diets examined. all were shown to be fertile with no significant differences in litter sizes. Effects on Milk myg:1noeitol. Figure 4A shows the content of free aggrinositol in rat milk as affected by the diets. For the animals receiving the deficient diet. low levels (0.30 to 0.75 an) were obser- ved throughout the lactational period. A slow rise in the mean free gygyinositol content of the milk from 8 to 21 days of lactation was 70 Figure 1+. Effects of Dietary m-Inositol on A) Milk and 3) m- mary Gland Free m—Inositol levels, eni A) Milk 6—9-Ga1actinol levels. m—Inositol levels in the milk or mammary gland are rep- resented as follows 8 A , supplemented dame; . . deprived dams. 6—9-Gm1actinol levels in the milk are represented by: A , sup- plemented dame; O . deprived dams. Each point represents the mean t S.D. for four dams. INV refers to involution of 28 days. 71 22,5 _eezegeede A O 2 O MILK — 0.6 _ _ 2| l4 LACTATION (days) 6.0- _ D 4 in: _eeeeewelfi _ 0. 2 J I MAMMARY _ INV n... o. 3 2 “came: o \ no.2: : v .0335 P o. la LACTATION ( days) 72 observed, however; this may be correlated with an increased rate of bioeynthesis as reported in previous work (12). The milk from those animals receiving the supplemented diet showed a 5. 5-fold increase in gig-inositol levels at day eight (p<0.001) when compared with day four of lactation. Hhen compared with deficient diet fed animals, the milk gig-inositol level from gig-inositol supplemented dams remained relatively constant (“.5 mM) to day 21 (p<0.001. supplemented versus deprived dams). Although both supplemented and deprived mothers re- ceived their respective diets continuously. no significant difference was noted in the gig-inositol content of their milk between birth and the fourth day of lactation. The mammary tissue content of free m-inositol (Figure hB) fol- lowed a pattern closely related to that of the milk and similar to previous observations (12). However. the plateau observed in the sup- plemented animals' milk (8 - 21 days of lactation) was not observed in the gland until somewhat later (11+ - 21 days of lactation). In the supplemented animals ' mammary tissue. however. no delay in the increase of free Ely-inositol levels from o to a days of lactation occurred. Involuted mammary tissue showed a small but significantly (p <0. 05) higher content of gig-inositol for the supplemented animals (0.63 umoles/g tissue) when compared with the deficient animals (0.43 umoles/g tissue). Effects on 6- e -Galactinol Content of Milk. Previous work (20) has shown a relationship between the milk free Lam-inositol content and the levels of 6- P-galactinol in the milk. Figure 4A shows that the pattern of 6- 9 -ga1actinol content follows the pattern for gum-inositol content very closely in both the milk from control or m-inositol 73 ThBLE 1 mar 01" DIETARY m—INOSITOL ON THE FREE moINOSITOL AND spannermm. 00m OF“ RAT MIIK AT 11+ DAYS OF IACTATION Dietary Level of m-Inositol 6-P-Galactinol lilo-Inositol (%) (IN) (UK) 0 0.192 1 0.253 (to) 36.4 1; 19.5 (’4) 0.01 0.617 t 0.012 40.9 t 2.6 0.05 0.961» t 0.0% 63.1 r 9.3 0.25 “.01“ t 0.240 292.0 1 16.8 0.50 u.u22 :- 0.366 (4) 365.6 1 76.5 (4) Values are the mean 1: S.D. for 3 dams unless otherwise indicated (u). Females were fed a synthetic diet with the appropriate level of free m—inositol and mfg-inositol and 6-0-9 -D-Ga1actopyranosyl- inositol were determined by gas-liquid chromatography as described in the Materials and Methods section. 74 deprived dams. 6-{5-Galactinol levels in the milk of the Elle-inositol supplemented dams was 3. 5 - 9 times greater than that in the milk of deprived dams (statistically significant at all points, p<0.05). Effects on 6-9-galactin01 were also shown by varying the myo- inositol content of the diet through a range of values (see Table 1). 6-P—Galactin01 was shown to increase as the gig-inositol in the milk increased and maximized when the higher dietary levels of Eta-inositol were fed. Effects of lactation on Dams. The free ale-inositol content of maternal liver from the third day prior to birth through 21 days of lactation and four weeks after weaning is shown in Figure 5’". Livers of supplemented dams showed no significant change in the level of free m—inositol during gestation and at birth (approximately 0.33 umoles/g fresh weight tissue). At four days of lactation, levels increased to over 0.50 umoles/g tissue concomittant with plasma Egg-inositol increases during the same period. After four days of lactation. mean free 912- inositol levels steadily decreased to below prelactational levels by 21 days of lactation (0.25 umoles/g tissue). However, when compared with animals at 0 days of lactation. these changes during the course of lactation were not significant. Mean gig-inositol levels in the liver of the dams four weeks after weaning were significantly (p<0.05) higher than at any other time examined for both the m-inositel sup- plemented and deprived animals with the single exception of supple- mented animals at four days of lactation. The livers of deprived dams showed a rising trend (not statistically significant) in mean free gig-inositol content during gestation and at birth. After birth and the initiation of lactation. however. liver mean free m-inositol 75 Figure 5. Effects of Dietary neg-Inositol on A) Free and C) Lipid- Bound m-Inositol. and B) Total Lipid. and G) Phospholipid Phos- phorus Content in the Liver of lactating Dams. Symbols represent as follows: lipid-bound gig-inositol levels; A . supplemented diet fed animls and . , deficient diet fed animals; phoafl'blipid phosphorus levels: A . supplemented diet fed animals and . deficient diet fed animals. Each point represents the mean t S.D. for four dams. In (0). values are expreseed in umoles/ g lipid-free tissue. INV refers to involution of 28 days. 76 o .33: a \ no.2: 5 Eastman. — _ — TIT Tlell. 1M 1 ”r L. l1 1| I] ,, , fl I I I I I I I I” R m -u .L I7 10 A p e 4. 0 o u LACTATION (days) — — q . -m h P .51.: I IN' a 4.- a \s . . as . \ . \\ . 1m 1H m -.. U 40 _ _ _ 3. 2 I. o Q o o is... 33 as. sea. .83: ex See 3 3282a carriage o o. - 20 0 d 50.0 0 m w q — m L- 1Q HH I_I Ifi N . -m m m N .m w ( m 1mm m m L m 17 lo .. . _ m m m o .38.. 88.8.5 stifle Boosts. 77 levels significantly (p<0.005, compared with day 0 of lactation) decreased to a level of 0.07 umoles/g tissue by eight days of lac- tation where it remained until weaning (21 days of lactation). Re- covery of free Eng-inositol levels to pre-lactational levels (0.38 umole/g tissue) was also observed for the Elm-inositol deprived dams. Develggnent of Fatty Liver in Lactating Dams. The change in the percent lipid content of the livers of lactating dams was the most dramatic orange observed during this study (Figure SB). exp-Inositol supplemented dams showed no change in liver lipid content (0. 06 g lipid/g liver) during gestation. lactation, or after involution. De- prived dams exhibited normal levels of liver lipid during gestation through the fourth day of lactation. after which a nearly linear rise in the lipid content of the liver was observed until 21 days of lacta- tion (0.31 g lipid/g fresh weight of liver). After involution, the lipid content of the livers from m-inositol deprived animals re- turned to normal levels. Analyses were undertaken to examine the effects of the changes in the free gig-inositol and lipid content on the phospholipid (Figure 5c) and the phosphatidylinositol levels of the livers of lactating dams. The livers of supplemented dams showed only insignificant variations from a mean value of 35 umoles of phospho- lipid phosphorus per gram of lipid-free liver; livers from deprived dams were equivalent to those from supplemented animals for the first four days of lactation. A steady decline in the content of phospholipid phosphorus after four days of lactation to a minimum (20 umoles per gram lipid-free liver, p <0. 005, when compring livers from supplemented and deprived rats) at 1% days of lactation in livers of deprived dams was followed by a partial recovery to 27 umoles per gram of lipid-free '3“ F-a: J“: 78 tissue at 21 days of lactation (p<0.02, day 11+ versus day 21). After weaning. livers from dams showed no significant change from the mean value of 35 umoles/g lipid-free liver for the supplemented dams and full recovery of the glarinositol deprived dams occurred by 28 days after weaning (38 umoles per gram of lipid-free liver). The mean level of lipid-bound inositol in the livers of £19: inositol supplemented dams tended to decline (not statistically significant) from two days prior to birth to a minimum of 1.8 umoles per gram of lipid-free liver at 1b days of lactation which persisted until after involution. A parallel decrease of phosphatidylinositol in livers of glorinositol deprived rats occurred during lactation a (1.0 umoles per gram of lipid-free liver at 11+ days of lactation (p< 0.001) when comparing supplemented and deprived animals). A partial recovery to 1.25 umoles per gram of lipid-free tissue occurred by 21 days of lactation, (p<0.01, supplemented versus deprived animals) and after weaning, the tissue showed levels of 1.85 umoles of phosphatidyl- inositol per gram of lipid-free tissue (P<£0.05. supplemented versus deprived animals). These observations suggest that lilo-inositol de- privation may delay the return of lipid-bound Elle-inositol to normal levels. Developing fiat Liver Free myo-Inositgl. Fetal liver levels of free m-inositol (Figure 6A) tended to parallel the levels observed in the fetal rat plasma (Figure 2A). Supplemented animals showed a precipitous drop in liver mygrinositol levels from fetal ages (-2 and -3 days) to a minimum of 0.3 umole per*gram of fresh tissue at birth. Following birth, a sharp rise (to a maximum of 0.8 umole/g tissue) in the liver free pool was observed closely following the dietary intake 79 Figure 6. Effects of Dietary avg-Inositol on the A) Free and 0) Lipid-Bound m—Inositol, B) Total Lipid. and c) Phospholipid Phosphorus levels in the Liver of the Developing Rat. Supplemented animals A , males; A . fenles; A , a pool of males and females and deprived animals . . males; 0 . females: 0 , a pool of males and femles. Each point represents the mean 1: S.D. for four pools of fetuses (each pool from one dam). four pools of eight pups each for animls'up to 21 days of age, four animals for 21 days of age and three aniuls for ages greater than 21 days. In 0), solid lines represent lipid-bound mtg-inositol levels all dashed lines represent phospholipid phosphorus levels expressed as umoles/ g lipid-free tissue. 80 a... .. F H . . _ _ a T T 1 l H S M H m a LI 1r it Ifiu I J] I:l. J1 . in 1a R 1“” . In“ W . a R . . d U M E ( E V we. 1%” U laA ill lo M .10 A B a _ a _ FJF e _ a a a a m o m m a 0 as»: 38.2.. a. 3.32% o .33: ex 3 re. s: 732.818. 3.2.85-4...” 2.2-3. O 0 a T m lrl Lr o)+ I I. a .u R E l 4 V 2 U l e. i 0 C . _ _ n D n o w m m . .28.: o x 3.0:. 3 score-oi seize-oi AGE (days) 81 until 14 days of age. From 14 to 32 days of age. there was decrease in the liver free pool of Egg-inositol followed by a subsequent rise 'to adult levels approximately equal to levels observed at 14 days of age. Deprived animals showed a lowered but essentially parallel pat- tern. The neonatal rise in gum-inositol levels observed in supple- mented pups did not occur in deficient pups until after eight days of age. and the levels were significantly (p<0. 02) lower than corres- r- ponding controls for both sexes at all ages after 14 days with the single exception of 21 days supplemented arrl deprived females. Developim Rat Liver Lipid, Lipid-Bound myQ-Inositol and Phos- pholipid Phosphorus Content. Figure 6B illustrates liver lipid con- “' 5'. 1&an .I tents in developing rat pups fed the deficient and supplemented diets. Differences in liver lipid content between groups were not significant. Males fed the m-inositol deficient diet showed a tendency for an in- crease in liver lipid from 5.0% to 7.0% on a fresh weight basis com- pared with supplemented males at 21 days of age. At 32 days of age. an increase from 5.0% at 21 days to 7.0% at 32 days for deprived males and from 5.0% at 21 days to 6.5! at 32 days for deprived females was observed. This was the only instance that significant differences (p<0.05) between dietary groups were noted for either males or fe- males. No significant differences were observed in the phospholipid phosphorus or lipid-bound gig-inositol content in liver due to diet. sex or age of the animal. with the exception of liver phospholipid phosphorus of supplemented and deprived females at 49 days of age (p (0.05) (Figure 6C). Fatty Acid Distrflution in Total Liver Lipids. Table 1 shows the 82 it“ move sopumo op wearuooos abandasoea ens unease Hashes eaos_hppsu one .meammsn seas :.wo even a song newness mo .n.m « same one ms eeeneunxe ens nesae> eke: madness uo>aq .mesop manned mo Hopes: .soSSoA me ease as so some. .Hepsss do «5 ed a; ma do we r.~ me are a do w HA « ad « «a. w are a rm « mg. a can a a; asanofiuflm H .o o .N H .o a .e N .m c A m .o .e. .s o .o roses-eases $6 a is u «.2 a me u as w QR « 03 a are a was a. we Hesseossnflm ad o.~ n4 is do :4 3o soeaaonv 26 u-.. 1... a me a stun « rim H or a. ed a mad .3. sea ass. 92 «.3 33 0.2 13 0.3 or: censor Jenni .eem worse A333 H88. 5 asses asses m9HA zH mQHmHH Q .586 .4u£..8.~ .0 .584 . I also .0 £- cmé . U .eohoanwe 3. 38:33st no 9835533 an» assurance 93.6.33 .5 m 5 .2: 84 u 0 £- on.o .O .5. mad . l .5 35 . 4 E- o . Sosa... 3 egauwnoeowsamunuogun mo msg#52350 05 passages wagons.“ 2.3 “a on .33.” afiafooooaeun sea sflooofiéésauofiuflmh mo 385:3 as so snaufiao-oooo3u .muoEaA B 83325 one so oagflg oBaoue gene 3 use Edfigofia 2 .N 8.3a -32. _.E-e.o._3o=_o-o.o_fi.9 73.5 La..e-:o8_e..9 o._ no 0 no- 9. n..- 8 9 Q. no 0 9- - owm o _ _ Kl _ _ a a _ ‘ uoE> m \ «HIRHII ” .ilulnlln. IIIIIIIIIIIIIIIIIII MN \\.v V I .I \e AT .1 V . \n; .1 a. I . m 1. n a s m I WI I. / l o a m s / m «_r (a I J N. m < e. l I u v. 141 A400 - - E O 9 \300 o — _ é Lé-I) \ I > 8 //‘ \ 3 200 —— . -1 (D 2 + 4 a: mo —' —-« 0 L J L J L 0 30 60 90 l20 MINUTES AFTER INJECTION Pigtm 3. Effects of the Administration of a Single Dose of 5-‘1‘hio- D-Glucose . 2-Deoxy-2-Glucose . LID-Glucose and Saline on Plasma Glucose levels in Kiss. The following represent the sugars or saline and their respective doses: . . saline: I . g-glucose; O . 2-deoxy- g-glucose; and A . 5-thio-D-glucose all at 50 eg/ kg body weight; and A . 5-thio-g-gluooee at 250 mg/ kg body weight. Dosage volumes were between 0.1 and 0.2 ml. Each value represents the mean 3; S.D. for 3 mice. 142 body weight. the volume of administration being kept less than 0.2 ml. Saline was administered as a 0.9% solution and an additional group of mice was given STG at 250 mg/kg body weight (319-5). Hy- perglycemia was observed to reach its peak value for glucose and ZDG groups from one to two hours after administration. S-Thio-Q- glucose showed a rapid decline after one hour in the high dosage group (SIG-5) while in the low dosage group (SIG-l. 50 mg/kg body weight) blood glucose was continuing to rise at two hours. In Vivo mmentation. 2-Deoxy-Q-glucose-6-phosphate (10) and. in this report, 5-thio-2-glucose—6-phosphate have been shown to be competitive inhibitors of ym-mositol-l-phosphate synthase. Therefore. experiments were designed to determine the effects of 5- thio-g-glucose (STG) and 2-deoxy-2-glucose (ZDG ) administration might have on is M minositol levels. Levels of glucose or glucose analog injected were all 50 mg/kg body weight/day for seven days. and tissue was taken 2“ hours after the final injection (Experiment I. Table 3). Plasma levels of ago-inositol appeared unchanged. although wider variations as compared with controls were noted in the 2m and 3m injected animals. Testicular tissue in response to ZDG. showed significantly (p<0.005) reduced levels of free m-inositol (amiss of control). S-Thio-Q-glucoee did not elicit a change in plan m-inmitol levels at the dose given. However. in this experiment. sampling of tissue 2“ hours after the last injection did not account for potential transient effects of 3m administration on tissue or plasma m-inositol levels. In Experiment 11 (Table 4). levels of glucose and ZDG injected were identical to those in Exper- iment I. and 5m administration was increased to five times that in 1'43 TABLE 3 Effects of 5-Thio-13-Glucose and Z-Deoxy-Q-Glucose Administration on m-Inositol Levels of House Plasma and Testes - Experiment I a TISSUEa ANIMALS n Plasma Testis Control 3 20.6 t 1.8 2.900 r 0.118 5 T0 1+ 23.7 i: 3.9° 2.757 t o.l7l° 2 Do 3 23.9 i: 5.7° 1.000 i: 0.02910 "Values represent the mean 1: S.D. for n aninls, plasma values are in uh and tissue levels are in umoles/ g of wet weight tissue. Animals were injected once daily intreperitoneally with 50 mg/ kg body we ht of glucose (control). 5-thio-2—glucose (5T6) or 2—deoxy-2-glucoee ZDG) for 7 days. Tissue sampling use performed 21$ hours after the last binjection. °p<0. 005 not significant TABLE lb Effects of 5-Thio-2-Glucose and 2-Deoxy-2-Glucose Administration on aye-Inositol Levels of House Plasma and Tissues - Experiment 11 TISSUES; Animals Days Inj. (n) Plasma Testis Brain Liver 3 (5) 31.6 i 5.0 2.90 i- 0.31 0.13 t 0.20 0.295 t 0.065 7 (5) 27.6 i 7.8 3.09 t 0.57 15.03 i 0.28 0.235 a 0.027 3 (5) 33.1 a 8.2° 1.3a {0:333:92 s 0.66° 0.227 1' 0.0340 2 no 7 (6) 1M3 t 3.6d 1.03 t 0.171’ 3.90 t 0.53° 0.185 t 0.022(1 a'Values represent the mean t S.D. of 11 animals. plasma values of m- inositol are in m an! tissue values are in umoles/ g of wet weight tissue. Animals were injected once daily intraperitoneally with ng/ kg body weight of glucose (control) or 2-deoxy-D-glucose (200 or with 250 mg/ kg body weight of 5-thio-D-glucose (310) for the in- dicated time period. Statistics: b. 1:45.001; c. p<0.0053 d. p< 0.02; and e. not significant at the 5% level. 1144 the first experiment. Tissues emined were the same with the addi- tion of brain and liver. Animals were sacrificed after three and seven days of treatment and tissue was sampled three to four hours after the last injection. Table to shows the results obtained for the determination of free pig-inositol in selected tissues. As in the first experiment. plasma variations were negligible except after seven days for ZDG treated animals. In 200 treated animals. the seven day plasma level of m-inositol was 51.8% of the control val- ue. Brain free m-inositol levels were unaffected by 2130 or 5TG ad- ministration. No significant differences were observed as measured by the Student's two-tailed t test. Liver showed no significant changes in free m—inositol after three days of treatment with 5T0 or with 200. After seven days. however. significant elevation (103% of control) of m—inositol in liver was evident for the 5m treated animals (p<0.02). Concomit- tantly. 2DG caused a significant decrease in liver free gum-inositol (78.6% of control. p<0.02). Although significant deviations from the control were observed for liver and plasma samples. the largest difference occurred in the testes of the experimental animals. In animals treated with ZDG. the decrease in testicular free m—inositol followed a time course that was nearly linear for seven days. Final values at seven days dupli- oated those in Experiment I at a level of 33.3% of the control value (p<0.001). Animals treated with 510 showed a significant elevation in testicular free m-inositol levels at both three (p<0.005) and seven (p<0.02) days of administration when compared with respective controls. . 86v.“ . + n~55!” .._. .mNodVe .3. .moan . * Exodus.“ as 3.82:: n." 0233.353 .333 m \luone Mo Hop-us we dosages. one .35 Am 5 3.3: «.33» Emma .«o Hopes: es sewage one need? A< 5 .335 63.3.5 enoosamumioflvun messengers a .33 3.3.23 333 oeoosamumuhwoevum 3:30.33 n .3823 33.3 onoosamlm 3:39.59.” 0 .wls 50.2: new nee—Hg eeoosdwoflv use eeoosfimbnoeu has a mo seavneoxe on... 5.3 00.? n Hon .n.m « sees 2.3 3:39.39» e39 seem .03: 5 9560 ahemm eo 538.223 eeeeeaoumuofisun 8e . oeoefleumubsenum . 88:85 so fleets .e 86E ZO_._.