lllllcllllllll llllllllllllllllllllllllllllllll 3 1293 00109 9500 THESIS i‘m “- mear 33%qu ig‘fiate I n-.-._i‘. ._ - This is to certify that the thesis entitled CAPRINE PLASMA 0- AND B-MANNOSIDASE ACTIVITIES: ASSAY ANALYSIS AND EVALUATION OF THE EFFECTS OF AGE, SEX, AND REPRODUCTIVE STATUS AND POTENTIAL USE IN HETEROZYGOTE DETECTION OF B-MANNOSIDASE presented by Robert W. Dunstan has been accepted towards fulfillment ' of the requirements for Masgerg degree in m MWBGM N 0 Date bli‘gl” 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from —3——. your record. FINES will be charged if book is returned after the date stamped below. @1113 U j‘ ggflfi‘ Ch 2 p ., ' ’5’ . 0 5‘9 SEP 1 512007 l Maze WW 5" G vfll f) a K ",1“ V t 'J .. ’7‘ T ,i’ 1.1 “him {if} ‘33. w' £23m CAPRINE PLASMA a— AND B-MANNOSIDASE ACTIVITIES: ASSAY ANALYSIS AND EVALUATION OF THE EFFECTS OF AGE, SEX, AND REPRODUCTIVE STATUS AND POTENTIAL USE IN HETEROZYGOTE DETECTION OF B-MANNOSIDOSIS BY Robert W. Dunstan A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1982 ABSTRACT CAPRINE PLASMA 0- AND B-MANNOSIDASE ACTIVITIES: ASSAY ANALYSIS AND EVALUATION OF THE EFFECTS OF AGE, SEX, AND REPRODUCTIVE STATUS AND POTENTIAL USE IN HETEROZYGOTE DETECTION OF B-MANNOSIDOSIS BY Robert W. Dunstan The effects of age, sex, and reproductive status on caprine plasma a- and B-mannosidase activities as well as the potential use of plasma assays for heterozygote detection of caprine B-mannosidosis were investi- gated. Optimal conditions for the assay of caprine plasma a- and B-mannosidase activity were determined. The pH optima were 4.0 and 5.0 for caprine plasma a- and B-mannosidase activity, respectively, and substrate hydrolysis was proportional to time beyond the incubation periods used in these studies. Age and sex affected caprine plasma a— and B-mannosidase activities, while plasma 8-mannosidase activity was affected by reproductive status. Obligate heterozygotes for B-mannosidosis had plasma 8-mannosidase values which were intermediate between those found in animals affected with B-mannosidosis and Controls. Putative heterozygotes for B-mannosidosis could not be definitively identified, but likely candidates for future inbreeding experiments were discerned. ACKNOWLEDEGEMENTS This thesis is the result of a team effort requiring the help of many. I therefore would like to acknowledge the following individuals and groups for their assistance: Dr. Margaret Jones for always giving me time when she had none to give, for inspiring me to give this project the best that was in me, and for showing me the joys and trials of doing quality research. Dr. Kevin Cavanagh, my colleague and friend, for his patience in teaching me the techniques used in this study. Drs. Allan Trapp and James Cunningham, for serving on my guidance committee. James Malachowski for his instruction in statistics and photography. Ronald Vanderhayden for making the graphs used in this report and for helping with the assays. Dr. John Gill for statistical consultation. Drs. Richard Evans, Adalbert Koestner and Kathy Lovell for editorial comments. 0 Julie Haddad for serving as my goat holder.and assistant. Janice Fuller for typing this thesis. Lastly, I would like to thank the many goat breeders who so gener- ously allowed me to use their time and animals, especially Jan Kelly, who taught me much about "goat people" and life in general. ii TABLE OF CONTENTS IMRODUCT ION I O O O O O O O O O O O O I O O O O O O O I O O O O O REVI Ew OF m LI TEMTUM O O O O O O O O O O O O O O O O O O O O 0 Overview of Lysosomal Storage Diseases. . . . . . . . . . . Biochemistry of Glycoproteins . . . . . . . . . . . . . . . Basic.Structure. . . . . . . . . . . . . . . . . . . Metabolism of Glycoproteins. . . . . . . . . . . . . The Genetics of Lysosomal Storage Diseases. . . . . . . . . The Genetics of Heterozygote Detection. . . . . . . . . . . The Methods and Materials of Heterozygote Detection . . . . Heterozygote Detection Using Blood Plasma and Serum. Heterozygote Detection Using Blood Cellular Elements. . . . . . . . . . . . . . . . . . . . . Heterozygote Detection Using Fibrbblasts . . . . . . The Measurement of Serum.and Plasma Lysosomal Hydrolase Activity . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting th 4-Methylumbelliferyl- an p-Nitrophenol Assays. . . . . . . . . . . . . . . The Use of Reference Enzymes . . . . . . . . . . . . Current Knowledge of Acidic a-Mannosidases. . . . . . . . . Non-Caprine B-Mannosidase . . . . . . . . . . . . . . . . . Caprine B-Mannosidosis. . . . . . . . . . . . . . . . . . . Introduction . . . .‘. . . . . . . . . . . . . . . . Mode of Inheritance. . . . . . . . . . . . . . . . . Gross Changes and Clinical Features. . . . . . . . . Morphologic Alterations. . . . . . . . . . . . . . . Ultrastructural Alterations. . . . . . . . . . . . . Nature of the Storage Product. . . . . . . . . . . . Nature of the Enzyme Defect. . . . . . . . . . . . . Summary of Literature Review. . . . . . . . . . . . . . . . OBJECT IWS C . O O O O O O O O O O O O O O O I O O I O O O O O O O MERIALS AND WTHODS C O O O O O O O O O O I O O 0 O O O O O O O 0 Processing Plasma from Control and Breeding Herd Populations. . . . . . . . . . . . . . . . . . . . . . . Determination of Plasma a- and B-Mannosidase Activities . . Determination of the Effects of Assay Incubation Time on Enzyme Activity . . . . . . . . . . . . . . . . . . . Determination of the pH Optima. . . . . . . . . . . . . . . iii Page 0) P‘F‘k‘ F4P4C>¢>Ulbabtu 14 15 16 21 22 24 27 27 27 28 29 30 31 31 32 35 36 36 37 38 38 RESULTS 0 O O O O O O O O O O O O 0 O O O O O O O O O I 0 O O O O The Effect of Incubation Time on Plasma a- and B- Mannosidase Activity . . . . . . . . . . . . . . . . . pH Activity Curves. . . . . . . . . . . . . . . . . . . . Analysis of Control Animals . . . . . . . . . . . . . . . Effects of Age on Plasma a- and B-Mannosidase Activity. . . . . . . . . . . . . . . . . . . . Effects of Sex and Reproductive Status on Plasma a- and B-Mannosidase Activity . . . . . . . . . Analysis of Breeding Herd Goats . . . . . . . . . . . . . Use of Plasma B-Mannosidase Activity as a Means for Detecting Heterozygotes for B-Mannosidosis. The Relationship Between Plasma a-Mannosidase and. B-Mannosidase Activities. . . . . . . . . . . . DISCUSSION 0 O I O O O O O O O O O O O O O O O O O O O O O O O O The Effect of Incubation Time on Plasma o- and B- Mannosidase Activities . . . . . . . . . . . . . . . . pH Activity Curves. . . . . . . . . . . . . . . . . . . . Analysis of Control Animals . . . . . . . . . . . . . . . Use of Plasma B-Mannosidases as a Means for Detecting Heterozygotes for B-Mannosidosis . . . . . . . . . . . SUMRY C O O O O O O O O O O O O O O O O O O O O C O O O C O O O BIBLIWMPHY O - O O O O O O O O O O O O O O O I O O O O O O O O 0 iv Page 39 39 39 39 39 44 49 49 54 55 55 55 56 59 62 63 Table LIST OF TABLES Page Biochemical characteristics of non-caprine B-mannosidases . 25 Correlation coefficients (r) relating age in months to (1) plasma a- and (2) B-mannosidase activity in adult male and female goats . . . . . . . . . . . . . . . . . . . 47 Mean plasma a- and B-mannosidase activity in neonatal, young, and adult control goats according to age, sex, and reproductive status . . . . . . . . . . . . . . . . . . 48 LIST OF FIGURES Figure Page 1 The synthesis of glycoproteins (Sharon and Lis, 1981) . . . 6 2 Inborn errors of glycoprotein cataboliem (after Dawson, 1979) O O O O I O O O O O O O O O O O O O O O O O O O 0 O O 8 3A Effect of assay incubation time at 37 C on caprine plasma B-mannosidase. . . . . . . . . . . . . . . . . . . . 41 33 Effect of assay incubation time at 37 C on caprine plasma a-mannosidase. . . . . . . . . . . . . . . . . . . . 41 4A Effect of pH on caprine plasma B-mannosidase. . . . . . . . 42 4B Effect of pH on caprine plasma demannosidase. . . . . . . . 43 5 The effects of age and sex on plasma (A) a-mannosidase and (B) B-mannosidase activity in the control subpopula- tions 0 I O O O O O O O O O C I O I. O O O O O O O O O O O Q 46 6 Comparison of plasma B-mannosidase activities of obligate and putative heterozygotes with age- and sex- matChed contrOIS O O O O O O O O O O O O O O O O O O O O O O 51 vi INTRODUCTION Recently, a new inherited disorder of glycoprotein catabolism, B-mannosidosis, was defined in the caprine species. B-Mannosidosis is an autosomal recessive disorder characterized by profound neonatal neurological deficits and facial dysmorphia (Jones et al., 1979). Neurovisceral storage of the oligosaccharides Man(Bl-4)GlcNAc(Bl-4)GlcNAc and Man(Bl-4)GlcNAc was associated with tissue deficiency of B- mannosidase (Jones and Laine, 1981; Jones and Dawson, 1981; Matsuura et al., 1981). Recent studies suggest that affected animals also have a complete deficiency of plasma B-mannosidase activity (Cavanagh et al., 1981). However, the determination of optimal conditions for the assay of caprine plasma a- and 8-mannosidases, the influence of age, sex and reproductive status on the activities of plasma mannosidase in a clinically normal caprine population, and the use of plasma as a source of enzyme activity for heterozygote detection of B-mannosidosis have not been studied." To determine optimal conditions for evaluating the activities of caprine plasma a- and B-mannosidase, theeffects of reaction time and pH optimum on a colorimetric assay were investigated. Because age, sex, and reproductive status significantly affect the activity of specific lysosomal hydrolases in man and cattle (Erickson et al., 1972; Griffiths et al., 1978; Annunziata and Di Matteo, 1978; Lombardo et al., 1981; Lowden, 1979; Jolly et al., 1974a; Jolly et al., 1973), these factors were examined in relation 1 2 to plasma a- and 8-mannosidase activities in a population of clinically normal goats, and the results were compared with those from a herd of known and putative heterozygotes for B-mannosidosis. Plasma has been used successfully as a source of enzyme activity for heterozygote detec- tion because it is easy to collect and has been successful in detecting carriers of other inborn errors of glycoprotein catabolism. In bovine a-mannosidosis, for example, heterozygotes were found to have enzyme values between those of normal and affected subjects (Jolly et al., 1974a: Jolly et al., 1973; Jolly and Desnick, 1979).- Plasma a-mannosidase activity was assayed in order to determine both its relationship to plasma B-mannosidase activity and its useful- ness as a reference enzyme for plasma B-mannosidase activity in further characterizing heterozygotes (Jolly and Desnick, 1976; Winchester et al., 1976; Saiffer et al., 1975). a-Mannosidase seemed a logical choice for reference-enzyme studies, since its activity in tissue has been shown to be elevated in a goat with B-mannosidosis (Jones and Dawson, 1981). This thesis is the first to study assay conditions for the caprine plasma mannosidases. It is also the first study to investigate the effects of age, sex, and reproductive status on plasma B-mannosidase activity in any species, to examine the effects of these factors on plasma a-mannosidase activity in the goat, and to use a readily available body fluid for detecting B-mannosidosis in caprine heterozygotes. REVIEW OF THE LITERATURE Overview of Lysosomal Storage Diseases Inherited metabolic diseases have been studied extensively during the past two decades. As the pathogenesis of these diseases has been clarified, so has the understanding of normal biochemical pathways. This is particularly true of the lysosomal storage disorders. These diseases were first envisaged by Hers in 1963, when, from his study of Type II glycogen storage disease (an acid maltase deficiencY): he pro- posed a category of diseases characterized by the absence of specific lysosomal hydrolases and concomitant accumulation of incompletely degraded macromolecules within lysosomes (Hers, 1963). ' Hers also established four criteria for defining these diseases (Hers, 1. 1965): As seen by the electron microscope, the cellular inclusions are bound by single membranes and stain positively for acid phosphatase, i.e., they are in lysosomes. The disorders are progressive. Multiple organs are affected. The stored material is heterogeneous. Currently, there are more than 35 recognized recessive and X-linked inherited inborn errors of the lysosomal apparatus with a morphological hallmark of hypertrophy and hyperplasia of lysosomes_(Desnick et al., 1978a; Hers, 1973). Although there is considerable overlap, these 4 diSeases can be divided into five major categories based on the bio- chemical nature of the accumulated metabolites: l) the glycogen storage diseases, 2) the lipidoses, 3) the mucolipidoses, 4) the mucopolysaccharidoses (errors of glycosaminoglycan catabolism), and 5) the oligosaccharidoses (disorders of glyc0protein catabolism). This report will concern itself primarily with disorders of glycopro- tein catabolism. Biochemistry of Glycoproteins Basic Structure There are two basic classes of compounds which contain proteins covalently bound to sugars: the glycosaminoglycans and the glycopro- teins. Glycosaminoglycans are high molecular weight linear carbohydrate polymers that are generally composed of disaccharide repeating units of a uronic acid (D-glucuronic acid or L-iduronic acid) and a hexosamine (N-acetylglucosamine) and, with the exception of hyaluronic acid, con- tain sulfate esters or sulfate amines. In contrast, glycoproteins do not contain uronic acid, lack a serially repeating unit, contain a relatively low number of sugar residues in the heterosaccharide portion (which is often branched), and contain several sugars which are not characteristic components of glycosaminoglycans (e.g., fucose, sialic acid, galactose) (Harper‘et al., 1979; Margolis and Margolis, 1979). Oligosaccharide chains on glycoproteins are classified according to their amino acid linkages as well as their inner core structure. Although there are five major types of carbohydrate-peptide linkages, only the structure, synthesis, and catabolism of glycoproteins with N-glycosidic linkages are germane to this discussion. 5 The N-glycosidic linkage, which consists of N-acetylglucosamine B-linked to asparagine (GlcNAc-Asn), is the most ubiquitous carbohydrate- peptide linkage, being widely distributed in animals, plants, and microorganisms (Sharon and Lis, 1981). The oligosaccharides linked to asparagine typically have the following branched core structure: MAN (cl->3) MAN (Bl-*4) GLCNAC (81+4)GLCNAC—-ASN MAN(a1+6) Glycopeptides containing this structure fall into two broad categories: the neutral or oligomannosidic type, which contain only mannose and N-acetylglucosamine (as is found in chicken ovalbumin), and the acidic or complex type. The latter contain, in addition to mannose and N-acetylglucosamine, sialic acid, galactose, and fucose (Kornfeld and Kornfeld, 1976; Sharon and Lis, 1981). Examples of this type of com- pound are found in human or bovine immunoglobulin G. Metabolism of Glycoproteins The biosynthesis of the asparagine-linked oligosaccharides is initiated by the transfer of UDP-GlcNAc to the polyisoprenoid lipid, dolichol phosphate. Sequential addition of sugars from their nucleo- tide derivatives to the N-acetylglucosaminylpyrophosphoryldolichol occurs to form the unit Man(Bl-4)-G1cNAc(Bl-4)-GlcNAc-P-Pédolichol. Additional mannose reSidues and three glucose residues are added to this structure to form the completed dolichol intermediate. The entire oligosaccharide moiety is then transferred to an asparagine residue on the nascent polypeptide chain. This asparagine-linked oligosaccharide is the precursor for formation of both oligomannosidic and complex glycoproteins (Figure l). Man Man-Man/ \Man-GlcNac —GlcNac —Asn Glc -Glc - Glc -Man— Man— Man 1 a-Glucosidases Man - Man Man Man -Man/ Man - GlcNAc -G1cNAc - Asn Man - Man - Man a-Mannosidase Man Man 7 / Man Man - GlcNac - GlcNac Man/ Oligomannosidic UDP-GlcNAc,GlcNAc Transferase I, Chains a-Mannosidase Man Man -G1cNAc -GlcNAc GlcNac -Man i UDP-GlcNAc, GlcNAc Transferase II GlcNAc-Man Man - GlcNAc - GlcNAc GlcNAc -Man/ Further processing of complex-type chains Figure l. The synthesis of glycoproteins (Sharon and Lis, 1981). 7 The three glucose units are now sequentially removed. After removal of the glucose units, four mannose units are excised followed by the addition of GlcNAc to the a(l-3)-mannose. The transfer of this GlcNAc residue then signals an a-mannosidase to cleave two mannose residues linked a—l-6 to the B-linked core mannose (Tabas and Kornfeld, 1978). Subsequently, one or two additional GlcNAc units are added to the a-l-G-linked mannose. Further processing may then occur in the endoplasmic reticulum or the Golgi apparatus where the glycoproteins assume their final structure (Sharon and Lis, 1981; MaFVel, 1980). Glycoproteins are catabolized within lysosomes by the sequential action of exoglycosidases (neuraminidase I, neuraminidase II, B-galactosidase, N-aspartyl-B-glucosaminidase B, a-mannosidase, B-mannosidase, and a-fucosidase), and an endoglycosidase (B-N-acetyl- glucosaminidase) (Dawson, 1979). Due to the sequential and primarily exoglycosidic nature of this catabolic scheme, a deficiency of any one enzyme precludes further processing by the more terminal catabolic glycosidases, and the oligosaccharide remnants remain stored within lysosomes. With the discovery of B-mannosidosis in goats, enzymatic deficiencies of all the exoglycosidases in glycoprotein catabolism have been discovered (Figure 2). Of interest is the fact that only deficiencies of B-mannosidase, a-mannosidase, and N-aspartyl-B- glucosaminidase result in the storage of products limited to glycoprotein catabolism. The remaining exoglycosidases act on glycolipid and/or glycosaminoglycan substrates in addition to glycoprotein substrates (Harper et al., 1979). INBORN ERRORS OF GLYCOPROTEIN CATABOLISM Near-«Ac 2 Gal 2 61:51:: E Mm Flue g G B B B MESH!“ eGlchc — Asn -mtido Neuflfic 2'(ku-wuom oE>Nco :fl cos» Ioooou wva momsmo coHuomoH ou oooom Dawn 25 N «Moommw 0c mm: «Bow .m.z o.m ooo.ova mzm H.m mnma ocom meHOMwosw mwwosohs memmumoumaouzo Eoum oocamuno one mood» locum N «uowmwm 0: mm: meow .m.z m.m ooo.vo mzm o.H whoa cos ms0H3MHsm mshommwom umoH oumuownonumo >uw>fiuom o: ooHHM£OOMmfiQ v.m whoa sow mcamocoo mun mule mm m.m ooo.OMH mza m.o umsmaesom some: mseqwsumamm ooaumsoommdua o.a puma nu- ...m.z o.v-m.m ooo.oma .mza o.~ amends some: mzaawmummme Hozsm mucoesoo Aumoa auw>au0< we mm unmwoz REES EM monouowom condom sueaenaaosuone Hoseumo Masseuse: mommoflmoccmalm ocflummoicoc mo moaumeuouomumno HMOflEoAUOAm .H wanes 26 oouuomou no: u .m.z«« >mmmm Hocotmouuficnm n mzm« mwmo n how . . O v+ no 0 om: mmmmm um magnum “coda so uoommo o: o>ms Im>fiuomcw mooa Hmma «sum .~+:N .~+oo see metro om m~.e .m.z mza m.m noumoeo ssuom sass: m.>um.v mm coo3uon ooum>auomca magnum ismmma co smm m.o mm on mama uoommo o: no: m+cN cwa omllo mm o.v .m.z mzm 0.0 owommmq uo>fla umm >nmmumoumeouno Scum oocamuno mc0auomum N «mm now: as magnum «>Mmmm onu :0 uoommo o: o>mn ooum>wuomcw aooa whoa ones can mean sea oouno mo m.v ooo.ooa mzo m.~ ocoxsm uosoe>o so: m.m mm zoaon o cm: on manaum oabmumco .uoowmo ooum>fluomcfi mom hood 0: we: mean sea miIO ow o.¢ .m.z Aawcocmv H.h nauseous: mscsuuoo cause O om: um magnum .ooum>wuomca mom «in no on .ooum>wuomcw . wma him mm as tha II: sea mIIO mm m.v .m.z “Hacocmv m.o mumsmmom newmsm mswumcoe qmsze mucoeeoo “smog >uw>auo¢ we no unmaoz Azsv as oocowomom . oousom sueaenaaosuoae awesome Hassooaoz reanasoeooe H assay 27 The kM using pNP substrate is considerably higher for rat liver than other species evaluated, including man. In all cases where examined, B-mannosidase appears to be unstable at low pH, stable upon freezing and, in contrast to d-mannosidase, does not appear to be a metalloenzyme. Caprine B-Mannosidosis Introduction B-Mannosidosis is a rapidly fatal neurovisceral storage disease due to a deficiency of the terminal enzyme in glycoprotein catabolism, B-mannosidase. The disease was originally observed in New South Wales (Hartley and Blakemore, 1973) and arose independently in a herd of goats in Michigan (Jones et al., 1979). The disease was first defined by Jones et a1. when oligosaccharide (Jones et a1, 1980; Jones and Laine, 1981; Matsuura et al., 1981) and enzyme (Jones and Dawson, 1981; Cavanagh et al., 1981) studies were performed on tissue, urine and plasma obtained from neonatal Nubian goats exhibiting severe neurologic defects and facial dysmorphia (Jones et al.,l979). Mode of Inheritance. B-Mannosidosis appears to be a Mendelian disorder which exhibits autosomal recessive inheritance. This is suggested by results of both inbreeding and outbreeding experiments on a herd of goats in which the gene for B-mannosidosis is known to exist. The experiments exhibit the following features typical of an autosomal recessive disorder: 1. The B-mannosidosis phenotype has only been expressed experi- mentally by inbreeding animals which are either known heterozygotes or putative heterozygotes. 2. No first generation breedings of known heterozygotes to randomly selected controls has produced affected animals. 28 3. Siblings of an affected goat appear to have a one in four chance of being affected (of 24 offspring currently pro- duced by inbreeding, 6 have been affected). 4. Both males and females have been produced by inbreeding animals in which the phenotypic expression was not present. Gross Changes and Clinical Features The primary gross abnormalities in B-mannosidosis are present at birth and are characterized by facial dysmorphia and joint abnormalities (Jones et al., 1979; Jones et al., 1981). A depressed nasal bridge, frontal bossing, narrowed palpebral fissures, and an elongate, narrow muzzle comprise the facial dysmorphia. The joint abnormalities are progressive and are characterized by carpal contractures and hyperexten- sion defects of the pastern joints. On necropsy, in addition to the above changes, cardiac hypertrophy, pectus carinatum, enlargement of spleen and kidney, and cerebral ventricular dilatation were noted. Lack of myelin is evident in the cerebrum and cerebellum, and proximal musculature appears atrophic. With computerized axial tomography, affected animals exhibit obvious cerebral ventricular dilatation but no other consistent radiographic lesions. 0n neurologic examination, goats affected with B-mannosidosis exhibit a prominent intention tremor, pendular nystagmus, spasticity, and inability to walk. Patellar reflexes are typically not present at birth but were accentuated in an animal surviving into the second month. Electromyographic abnormalities include spontaneous potentials resembling positive sharp waves and fibrillation potentials suggestive of denervation. No significant alterations are present in electro- encephalographic or funduscopic examination. 29 Clinicopathologic data, including calcium, phosphorus, BUN, crea- tinine, SGOT, CPK, alkaline phosphatase, and complete blood count, are not consistently altered. In recent studies, using serum electro- phoresis, there was a prominent decrease in a and Y-globulins in 2 neonatal affected animals. This difference was explained by the fact that these animals did not obtain a significant amount of colostrum postpartum. Morphologic Alterations An evaluation of the morphologic alterations of B-mannosidosis is currently in press (Jones et al., in press). They will be only briefly discussed here. Light microscopy. The hallmark of B-mannosidosis under the light microscope is diffuse PAS-negative cytoplasmic vacuolation. This altera- tion is prominent on toluidine blue-stained semithin sections but may be difficult to discern on routine HaE sections. The vacuolar altera- tions are consistently present in fibroblasts, macrophages, endothelial cells, and perithelial cells. All parenchymal cells, with the exception of epidermis, are also affected to some degree. Prominent examples of the pathologic alterations of B-mannosidosis are observed in the brain and the proximal tubules of the kidney. The lesions in the brain are most extensive in, but not limited to, the cerebral cortex and the cerebellum. These lesions could be grouped into three major types of pathologic alterations (Hartley and Blakemore, 1973; Jones et al., 1979; Jones et al., 1981; Jones et al., in press): 1. Neuronal lesions. There is a fine to coarse, typically cell- specific vacuolation of neuronal cell bodies. The greatest percentage of neuronal cell bodies exhibiting vacuolation is 30 in the cerebral cortex, but the most severely affected cells are the cerebellar Golgi type 2 cells and neurons of the dentate nucleus. 2. Axonal and myelin lesions. There is a severe lack of myelin of the cerebrum and cerebellum and optic nerves. This myelin paucity is not present in peripheral nerves. The central nervous system axonal lesions include "spheroids" which consist of aggregates of dense bodies and mitochondria. 3. Glial alterations. In addition to gliosis, the changes in the glial cells are characterized by primarily cytoplasmic vacuolation. Oligodendroglial cells were sparse and appeared to be extensively vacuolated. In the kidney, the epithelium of the proximal convoluted tubules is characterized by numerous large vacuoles which appear to entirely replace the normal cytosolic parenchyma. In contrast, the distal tubules, the collecting ducts, and parietal and visceral glomerular epithelium exhibit only a fine vacuolation typical of that observed in most other organs (Jones et al., 1981; Hartley and Blakemore, 1973). Ultrastructural Alterations All tissues exhibited essentially similar ultrastructural alterations. These are characterized by cytoplasmic vacuoles .2-.8 um in diameter. The vacuoles are lined by a single membrane, often containing membranous and floccular material, and are related to the forming face of the Golgi complex (Jones et al., 1981). 31 Nature of the Storage Product Recent studies on the nature of the storage product in B-mannosidosis have shown a pathologic storage of the oligosaccharides Man(Bl-4)GlcNAc- (Bl-4)GlcNAc and Man(Bl-4)G1cNAc in the brain (Jones and Laine, 1981) and kidney (Matsuura et al., 1981), as well as elevated levels of mannose and GlcNAc residues in the urine (Jones and Dawson, 1981). The accumu- lation and excretion of these compounds presumably arises from the turnover of glycoproteins containing N-linked glycosyl chains. The storage of the trisaccharide Man(Bl-4)GlcNAc(Bl-4)GlcNAc is of interest because of the chitobiosyl linkage, which is apparently not broken down by endo-B-glucosaminidase. The reason for this lack of cleavage is not yet known; however, its presence brings to question the function and activation factors required for endo-B-glucosaminidase action (Jones and Laine, 1981; Matsuura et al., 1981). Phospholipid, neutral lipid, glycolipid and gangliosides all are apparently not specifically altered in animals affected with B-mannosidosis. In B-mannosidosis the storage product is limited to oligosaccharide products of perturbed glycoprotein catabolism. Nature of the Enzyme Defect A deficiency of both tissue (Jones and Dawson, 1981) and plasma (Cavanagh et al., 1981) B-mannosidase activity has been demonstrated in goats affected with B-mannosidosis using the synthetic substrate p-nitrOphenol-B-mannoside. With respect to tissue activity, no enzyme activity was reported in the brain or kidney. In the liver, 8- mannosidase activity was markedly deficient but not completely absent as in other organs. The presence of this significant activity was due to an overlap from a B-mannosidase with a more neutral pH (Dawson, 32 1982). On evaluation of a number of other glycohydrolases involved in glycoprotein catabolism in an affected goat, tissue a-mannosidase and a-fucosidase activity varied from 2 to 20 times normal depending on the tissues analyzed. Interestingly, an obligate heterozygote which was examined had tissue B-mannosidase, a-mannosidase, and a-fucosidase activity levels all intermediate between levels in the affected animal and control levels, suggesting the possibility of using these assays for heterozygote detection. 1 Similarly, plasma assays have shown affected animals to have essentially no B-mannosidase activity. However, unlike tissue studies, the plasma a-mannosidase activity in affected goats is similar to that of control goats (Cavanagh et al., in press). Complete characterization of caprine B-mannosidase is currently in progress. However, the hepatic enzyme reportedly has an optimum tissue pH of 5.0 with a second activity peak at a more neutral pH (Dawson, 1982). Summary of Literature Review Lysosomal storage diseases are a diverse class of enzymopathies characterized by the functional deficiency of a catabolic gene product leading to the intralysosomal accumulation of substrate or substrate precursors. The oligosaccharidoses are a category of lysosomal storage diseases which involve errors of glycoprotein catabolism. All glyco- protein storage diseases are autosomal recessive with heterozygotes exhibiting approximately one-half the enzyme activity of normal indi- viduals. Attempts to detect carriers of these disorders, therefore, typically involve measuring the activity of the partially deficient enzyme and comparing this to putative carrier and control values. The 33 analysis of enzyme activity most frequently used for heterozygote detection involves the use of either chromogenic or fluorimetric sub- strates on blood plasma, serum, or cellular elements. Although most carriers can be separated from genotypically normal subjects using these methodologies, definitive diagnosis of all putative carriers may not be possible due to an overlap in activity levels between normal subjects and heterozygotes. There are three major causes for this overlap: 1. Genetic factors. These refer to the fact that at any locus there may be variant alleles producing active gene products with slightly different activity, stability, or kinetics. These different gene products may lead to considerable activity variance in both controls and heterozygotes. 2. Variability inherent within the assay itself. These factors include the tissue used as an enzyme source, the choice of substrate and the substrate concentration, the pH at which the enzyme is assayed, the type of buffer used, the tempera- ture at which the enzyme is assayed, and the stability of the enzyme to freezing. 3. Extrinsic factors related to the metabolic and physiologic differences inherent within the subjects examined. These factors include age, sex, reproductive status, and environment. B-Mannosidosis, the most recently described disorder of glycopro- tein catabolism, is a rapidly fatal neurovisceral storage disease due to a deficiency of the terminal enzyme in glycoprotein catabolism, B-mannosidase. This disorder has currently only been defined in the caprine species, where it exhibits autosomal recessive inheritance. The major features of goats affected with this disease are: 34 Grossly, there are facial dysmorphia, joint abnormalities, and profound neurologic deficits. Histologically, there are diffuse cytoplasmic vacuolation and demyelination of the central nervous system. Biochemically, there are neurovisceral storage of the oligo- saccharides, Man(Bl-4)GlcNAc(Bl-4)GlcNAc and Man(Bl-4)GlcNAc, and negligible B-mannosidase activity in plasma or tissue. OBJECT IVES The objectives of this research were: 1. To establish the pH optimum for the assay of caprine plasma a- and 8-mannosidases. To determine the effects of assay incubation time on caprine plasma a- and B-mannosidase activities. To determine the range and mean plasma a- and B-mannosidase activities in a control population of Michigan goats with respect to age, sex, and reproductive status. To determine whether the plasma B-mannosidase assay and/or the use of the plasma a-mannosidase to 8-mannosidase ratio can serve as diagnostic tests for detection of heterozygotes for B-mannosidosis. 35 MATERIALS AND METHODS Processing Plasma from Control and Breeding Herd Populations Specimens were randomly selected from 12 goat herds in 3 Michigan counties during April and May, 1981. Blood was collected from 27 males and 25 females aged 1 to 7 days (mean age 4 days), defined as neonatal goats; from 27 males and 25 females, aged 22 to 28 days (mean age 24 days), defined as young goats; and from 24 males and 26 females, aged 6 to 96 months (mean age 36 months), defined as adult goats. All animals were clinically normal and their reproductive status was noted at the time of obtaining blood. Blood was also collected in a similar manner from a breeding herd of known and putative heterozygotes for B-mannosidosis at Michigan State University. This herd included 8 males and 7 females, all putative carriers, whose blood was drawn twice, first as neonatal goats and then again as young goats (except for 1 male) when they had reached the age of 22 to 28 days. The adult population from this herd consisted of 4 O males and 10 females aged 3 to 40 months, of which 1 male and 2 females are known through inbreeding to be heterozygotes, with the remainder being putative carriers. The blood was collected from the jugular vein in sterile heparinized vacutainers and stored at 4 C during transportation to the laboratory. The plasma was separated by centrifugation (500 g, 15 minutes, 4 C), 36 37 usually within 1 to 2 hours of collection. Specimens were then ali- quoted in 0.5 m1 volumes and stored at -20 C until enzyme analysis. All analyses were performed within 4 months of the collection date. Determination of Plasma o- and B-Mannosidase Activities The assays for plasma a- and B-mannosidases were performed using the appropriate pNP derivative (Dawson and Tsay, 1977). Each assay mixture contained the following: 20 ul of 10 mmol substrate, 100 ulof 0.1 14 sodium citrate-phosphate buffer (pH 4.4 for the a-mannosidase assay and pH 5.0 for the B-mannosidase assay, except where noted), and 50 ul of diluted plasma 1:2 (v/v) for both a- and B-mannosidase assays of goats 28 days or less and 1:1 (v/v) for both assays of adults. The reaction mixture in the a-mannosidase assay was incubated for 1 hour at 37 C. The reaction mixture in the B-mannosidase assay was incubated for 20 hours at 37 C. Following incubation, the reaction was stopped by the addition of 0.5 m1 of 3.5% trichloroacetic acid (w/v) and subsequent addition of 0.8 m1 of 0.25 M glycine NaHCO buffer, pH 3 10.0. The mixture was then centrifuged in a Clay-Adams Serofuge and the supernatant was used for obtaining spectrophotometric readings at 410 nm. Plasma enzyme activities represent the average of 3 replications and are expressed as nanomoles of p-nitrophenol formed per hour per milliliter of plasma (nmol/ml/hr). As a means of minimizing experimental error, no data were accepted unless the range of the 3 replications was less than 0.020 spectrophotometric absorbance units. All statistical comparisons were performed with the improved Bonforoni t-test (Games, 1977) and, unless otherwise stated, data were considered significant fdr p<0.05. 38 Determination of the Effects of Assay Incubation Time on Enzyme Activity The effects of assay incubation time on plasma a-mannosidase activity were determined by performing the assay at 15 minute intervals through the range of 15 to 120 minutes. The effects of incubation time on B-mannosidase activity were determined by performing the assay using 2, 4, 8, 10, 24, and 25 hour incubation times. Determination of theng Optima The pH optima for the a- and B-mannosidase assays were established using 0.5 M HCl-KCl (pH 2.0), 0.5 M glycine-HCl (pH 2.5), 0.1 M citrate- phosphate buffer (pH 3.0 to 7.0) at 0.5 intervals,and 0.1 M acetate at 0.2 pH intervals through the range of 3.8 to 5.6.. RESULTS The Effect of Incubation Time on Plasma a- and B-Mannosidase Activity Figure IBUU ,illustrates that the hydrolysis of pNP-B-mannopyranoside by plasma diluted 1:1 with saline was proportional to time up to 24 hours.. Short reaction times with undiluted plasma were not routinely used because of high sample blanks. Similarly, hydrolysis of pNP-a-mannopyranoside by plasma diluted 1:1 was proportional to time up to 2 hours (Figure 3[B])- pH Activity Curves The dependency of plasma a- and B-mannosidase activity on pH was evaluated through the range of 2.0 to 8.0 using HCl and KCl, glycine HCl and citrate-phosphate buffers (Figure 4[A,B]). From the pH activity curves, plasma S-mannosidase exhibits activity between pH 4.0 and 7.0 with optimal activity at pH 5.0. In contrast, plasma a-mannosidase exhibits activity between pH 3.0 and 7.0 with an optimal activity at pH 4.0. Analysis of Control Animals Effects of Age on Plasma a- and §7Mannosidase Activity The mean plasma a- and B-mannosidase activities in both sexes decreased with maturity. Male and female adult goats had significantly lower mean plasma a- and B-mannosidase activities than either neonatal 39 40 Figure 3A. Effect of assay incubation time at 37 C on caprine plasma B-mannosidase. Figure 3B- Effect of assay incubation time at 37 C on caprine plasma a-mannosidase. 41 Time (hrs) Figure 3A 1 60 90 /20 Time (m in.) .30 3000- 3.3.5 32223233.... .25. 0.3.35.2...Q 6000'- 4000- 2000- oEmEQ \E\3eocqotteiq BE: 3623.32.76 , Figure 3B 42 IZOP p-monnomou activity a o 1 Q 0 Figure 4A. Effect of pH on caprine plasma B-mannosidase. [500‘ 0 0 O I 500— “*Mannosidasc act/wry Figure 4B. 43 Effect of pH 1 5.0 pH on caprine plasma a-mannosidase. 44 or young goats (p<0.10 for the mean B-mannosidase values for young male and adult male goats) (Figure 5[A,B]). Except for significantly higher B-mannosidase values in neonatal male goats when compared to young male goats, mean plasma mannosidase levels did not differ in these age groups. The lack of any correlation between age and either plasma a- or B-mannosidase activity in adult goats (Table 2) suggests that there is no progressive increase or decrease in these two enzymes beyond sexual maturity. Effects of Sex and Reproductive Status on Plasma u- and B-Mannosidase Activity» The effect of sex on mean plasma a- and B-mannosidase activity seems related to sexual maturity (Table 3, Figure 5[A,B]). In adult goats, males had significantly higher mean plasma a- and B-mannosidase activities than females, but in young goats only the mean plasma a- mannosidase level was higher. Sex appears to have no significant effect on mean plasma a- and B-mannosidase activities in neonatal goats. Reproductive status affects mean plasma B-mannosidase levels more than mean plasma a-mannosidase levels (Table 3). Gravid goats had sig- nificantly higher mean plasma B-mannosidase activity than lactating goats, and intact young male goats had higher levels than their neutered counterparts. Of the 2 neutered adult male goats and the 2 adult female goats not pregnant, none had mean plasma values appreciably different from its respective sex-matched population as a whole. However, the small sample size of each subpopulation precluded meaningful statistical . evaluation. 45 Figure 5. The effects of age and sex on plasma (A) a-mannosidase and (B) B-mannosidase activity in the control subpopulations. Each vertical bar is the mean i std. error. Neonates (0-7 days); Young (22-28 days); Adult (6-96 months). 46 2600 - 7 % uMalu IFamalu D i??? iii??? adult 2000 '- 23:: 0333536-! young Ago class aaaaalu I400 '- 800 Figure 5A '/ % sMalu adult J1 young Age class 22/22, "0000108 120- _ O 8 IOO '" .23.: cautions—em. 60- 40 Figure 5B 47 Table 2. Correlation coefficients (r) relating age in months to (1) plasma a— and (2) B-mannosidase activity in adult male and female goats Age to Plasma Age to Plasma a-Mannosidase B-Mannosidase Class _ N Activity (1) Activity (2) Adult males 24 0.001 0.025 Adult females 26 -0.248 -0.561 The lack of any correlation between age and either plasma a- or B- mannosidase activity in adult goats suggests that there is no progres- sive increase or decrease beyond sexual maturity. 48 Table 3. Mean plasma a- and B-mannosidase activity inneonatal, young, and adult control goats according to age, sex, and repro- ductive status Mean Plasma Activity (nmol/ml/hr) r Std. Error Class N a-mannosidase B-mannosidase Neonatal Goats (177 days old) Male 27 2,206i117 113.1i6.4 Female 25 2,158f136 98.7i6.7 Young Goats (22-28 days old) Male 27 2,3871108 88.8:2.2 Intact 16 2,3472133 93.412.8 Neutered ll 2,4451188 82.2i2.7 Female 25 2,019i104 91.7i4.3 Adult Goats (6-96 months old) Male 24 1,814i116 77.2:3.7 Female 26 1,262i 63 64.1:3.l Gravid 14 1,3271 95 70.5:3.2 Lactating 10 1,186: 97 ‘ 55.7i5.6 49 Analysis of Breeding Herd Goats Use of Plasma B-Mannosidase Activity as a Means for Detecting Heterozygotes for B-Mannosidosis The 3 adult obligate heterozygotes in the B-mannosidosis breeding herd had values approximately one-half of those of the sex-matched adult control goats. The lone male carrier had a plasma B-mannosidase . value of 39.7 nmol/ml/hr, compared to the mean value of 77.2 nmol/ml/hr for the adult male controls (Figure 6[A]). The 2 carrier females had plasma B-mannosidase values of 31.7 nmol/ml/hr and 35.0 nmol/ml/hr, compared to 64.1 nmol/ml/hr for the adult female controls (Figure 6[B]). Although the large overlap of plasma B-mannosidase values in relation to age, sex, and reproductive status (data not shown) between the breed- ing herd and the control populations prevented a distinct separation of heterozygotes from genotypically normal goats,:2adult male and 1 adult female putative heterozygotes had values lower than the sex- matched obligate heterozygotes and distinct from their respective adult control populations (Figure 6[A,B]). Attempts to detect heterozygotes in neonatal and young goats were hindered because of the absence of known heterozygotes in these age groups and because of the large differences in the individual plasma B-mannosidaSe values obtained from the breeding-herd animals at 1 week and at 4 weeks of age. Only 2 males and 1 female from these populations had low activity levels as both neonates and young goats (Figure 6[C,D,E,F]). 50 Figure 6. Comparison of plasma B-mannosidase activities of obligate and putative heterozygotes with age- and sex-matched controls. A - adult males (6-96 months); B - adult females (6-96 months); C - young males (22-28 days); D - young females (22-28 days); E - neonatal males (1-7 days); F - neonatal females (1-7 days). Numbar of goal: Number of goats 51 DaContrals Adult male: .8Putaliva Heterozygotes l2 - .uouigou Heterozygotes Fr . // / e-zs 53-33 38-63 53-68 cm: 03-90 9w: Haaufldaau aatlvlly Figure 6A l6 - 77 Adult females l2 - [flu-Controls 8 ' I'Pulativa Heterozygotes I-Obligala Heterozygotes __1 4 - 8-23 23-38 38-53 53-68 68-83 83-98 98-ll3 A—mannasidau activity Figure 6B Number at goal: Numbor of goals '2)- 30 52 Young moloa D-Conlrolo I'Putotivo Hotorozygoloo I20 p—momooldaoo «mm Figure 6C Young fomaloo D-Controlo .‘Putativo Notorozygoloo 11" / 'A 1‘// 38-53 53-68 68-83 83-98 98-ll3 Il3-l28 lZB-l43 p-m onnooidaoo activity Figure 6D Number of goole Number of goals 53 I6— Neonalal males l2— D-Controle 8 7% =Pulotive Heterozygotes —" 4_ -—l H e Z A? Z e pm r1 38-53 53-68 68-83 83-98 98-ll3 ll3-l28 l28-l43 2l8-233 233-248 p-mann oeidaee activity I Figure 6E I6— Neoaotal females l2+- D-Controle T ’sPutative Heterozygolee 8— 4.. / f g A 5%; l l lyfil—l 38-53 53-68 68-83 83-98 98-ll3 ll3-l28 l28-I43 2|3-233 p—mannosidoee activity Figure 6F 54 The Relationship Between Plasma a-Mannosidase and B-Mannosidase Activities The correlation coefficient relating plasma a-mannosidase to plasma B-mannosidase for all the control goats is 0.284. Correlation coefficients were also low when the relationship between the 2 plasma mannosidases was evaluated according to age, sex, and reproductive status, and this suggests that there is no relationship between the activities of the 2 plasma mannosidases. Despite these low correla- tions, the ratio of a- to B-mannosidase activities was determined for controls and compared to the ratios obtained from obligate and putative heterozygotes. Although these values varied considerably and offered no better separation on an individual baSis than that obtained from plasma B-mannosidase alone, the mean a- and B-mannosidase ratio for the control population (22.4) varied considerably from the mean ratio for the known heterozygotes (50.4). DISCUSSION The Effect of Incubation Time on Plasma a- and B-Mannosidase Activities Substrate hydrolysis by the plasma mannosidases was prOportional to time well beyond the respective incubation periods used in this study to evaluate control and B-mannosidosis breeding herd goats. This implies that the long incubation times used, primarily with respect to B-mannosidase assay, should not detract from the validity of the assay. Long incubation times are common when colorimetric assays are used to measure lysosomal hydrolase activity. A more sensitive plasma 8- mannosidase assay with a shorter incubation time may be possible now that the 4-MU derivative of B-mannopyranoside is commercially available. pH Activity Curves The pH optimum of 5.0 for caprine plasma B-mannosidase was the same as has been_reported in caprine tissue using both pNP and 4-MU substrates (Jones and Dawson, 1981). This value is somewhat higher than values reported in human serum (pH 4.25) and in rat liver (pH 4.6) (Chester and Ockerman, 1981; LaBadie and Aronson, 1973). Other workers have shown the pH optimum of B-mannosidase from diverse, non-mammalian sources to be in the range of 2.8 to 5.0 (Sone and Misake,l978; Bouquelet et al., 1978; Elbein et al., 1977; Wen et a1, 1976;Li and Lee, 1972; Sugahura et a1. , 1972; Sukeno et al., 1972; Muramatsu and Egani, 1967) . An additional pH optimum for caprine B-mannosidase reported from crude 55 56 liver preparations (Jones and Dawson, 1981) was not observed. The instability of caprine B-mannosidase at low pH corresponds to reports in rat liver, Tremella fuciformis, and Turbo cortunus, where partially - purified B-mannosidase activitywwas shown to fall rapidly below pH 3.5 (LaBadie and Aronson, 1973; Sone and Misake, 1978; Muramatsu and Egani, 1967), as well as for human serum B-mannosidase, which was stable between pH 4 and 6 for at least 24 hours at 37 C (Chester and Ockerman, 1981). These results differ from pineapple bromelain B-mannosidase, which appears to be relatively stable at low pH (Li and Lee, 1972). The pH optimum.of caprine plasma a-mannosidase was 4.0. This corresponds with the pH optimum of plasma a-mannosidase of 4.0 to 4.5 in other mammalian species (Phillips et al., 1974; Opheim and Touster, 1978; Burditt et al., 1980b). The lack of an additional pH optimum in the plasma a-mannosidase activity is not surprising. Separation of acidic and neutral forms of d-mannosidase generally requires the use of DEAR-cellulose chromatography (Winchester et al., 1976). The a-mannosidase assays performed on controls and B-mannosidosis breeding herd goats were carried out at pH 4.4 because this value cor- responded to the pH optimum for plasma a-mannosidase in the bovine (Jolly et al., 1973). The difference between caprine plasma a-mannosidase activity at 4.4 and the pH optimum of 4.0 is less than 5%; therefore, the results of this study are valid. Analysis of Control Animals The effects of age, sex, and reproductive status on plasma a-mannosidase in cattle have been reported previously (Jolly et al., 1974a; Jolly et al., 1973), as have the effects of age and sex on this 57 same glycohydrolase in man (Erickson et al., 1972; Griffiths et al., 1978; Annunziata and Di Matteo, 1978; Lombardo et al., 1981). Earlier values of caprine serum a-mannosidase activity with p-nitrophenyl-a-D— mann0pyranoside as substrate were higher than the levels reported here (Garcia et al., 1979). This may be due to the lower substrate concentration in our study. Our data suggest that the mean plasma a— and B-mannosidase levels in the control population tend to remain constant in goats through 4 weeks of age and then decrease sometime during sexual maturity (Figure 6[A,B]). The mean male plasma B-mannosidase values are the only excep- tion in that they begin to decrease at an earlier age. The absence of any relationship between the age of the adult goats and plasma a- and B-mannosidase values suggests that the mean plasma levels of the mannosidases do not change significantly after sexual maturity. The age dependence of a-mannosidase levels in goats through maturity is exactly opposite from that observed in cattle, which exhibit more than twofold increase in acidic a-mannosidase activity from birth to maturity (Jolly et al., 1973; Jolly and Desnick, 1979). For man, most reports state that there is no change in plasma a-mannosidase activity with age (Erickson et al., 1972; Griffiths et al., 1978; Annunziata and Di Matteo, 1978). However, the findings in a recent study suggest that age does affect most plasma lysosomal hydrolases, including a-mannosidase, as follows: there is an absolute maximum activity at birth, an absolute minimum activity at puberty, a gradual rising and then falling through early and middle adulthood, and a final increase that remains constant through old age (Lombardo et al., 1981). 58 This report is of interest because our values for goats appear to follow this course, at least initially. This study indicates that the effects of sex on mean plasma a- and B-mannosidase activities in goats are most pronounced in mature animals. Adult males have significantly higher mean plasma mannosidase levels than females (Table 2, Figure 6[A,B]). This distinction is less clear in the non-adult control populations. The results for plasma @- mannosidase values in goats also differ from those in cattle, i.e., mature bovine females have higher d-mannosidase values than mature males (Jolly et al., 1974). Reports on man indicate that sex has no effect on plasma a-mannosidase or plasma glycohydrolase values in general (Erickson et al., 1972; Griffiths et al. , 1978; Annunziata and Di Matteo, 1978; Lombardo et al., 1981). The reproductive status of the goats affected plasma B-mannosidase activity more than plasma a-mannosidase activity. Gravid females had significantly higher mean plasma B-mannosidase values than did lactating females, and intact young males had significantly higher values than young neutered goats. In contrast, the mean caprine .a-mannosidase values are not apparently affected by reproductive status. The only study on reproductive status in cattle reported no discernible differences between mean plasma a-mannosidase levels of gravid and non-gravid cows (Jolly et al., 1974). There are no reports on plasma d-mannosidase values in steers or on the effects of reproductive status on plasma a-mannosidase activity in man. 59 Use of Plasma B-Mannosidases as a Means for Detecting Heterozygotes for B-Mannosidosis Heterozygotes for B-mannosidosis have plasma B-mannosidase activi- ties between the mean values for sex- and age-matched control populations and those for animals affected with B-mannosidosis (Cavanagh et al., 1981). These results are consistent with an earlier report that an obligate heterozygote for B-mannosidosis showed a partial deficiency of tissue B-mannosidase activity (Jones and Dawson, 1981). In this study, a definitive diagnosis of putative carriers could not be made because of the small number of obligate heterozygotes in the B-mannosidosis breeding herd and the wide range of values obtained in the control population. However, 2 adult male and 1 adult female putative carriers that have values lower than their sex-matched heterozygotes appear promising for future inbreeding studies, as do 2 male and 1 female non-adult putative carriers that had low plasma B-mannosidase levels as both neonatal and young goats. The use of plasma lysosomal hydrolase activity in detecting hetero- zygotes for glycoprotein storage diseases has previously been reported, and the results compare quite favorably with our own observations (Jolly and Desnick, 1979). These studies typically show that plasma as a source of enzyme activity is highly successful in diagnosing individuals affected with glycoprotein storage disease (Jolly and Desnick, 1979; Winchester et al., 1976; Stanbury et al., 1978). However, detection of the carrier state may be rendered difficult by the range of values from normal subjects, which overlap and obscure the values of putative hetero- zygotes (Jolly and Desnick, 1979; Stanbury et al., 1978; Desnick et al., 19768; Johnson and Chutorian, 1978). 60 Because of the low correlation coefficient between plasma a- and B-mannosidase activities, there is an indistinct separation between putative heterozygotes and controls with the a- to B-mannosidase ratio as the principal criterion, and thus a-mannosidase should not be used as a reference enzyme in detecting B-mannosidosis heterozygotes. This is unfortunate, since relating the activity of B-mannosidase to an enzyme with which it is highly correlated would negate the variation of this enzyme's activity introduced by age, sex, and reproductive status (Winchester et al., 1976). In spite of the lack of correlation, the mean a- to B-mannosidase ratio for the obligate heterozygotes does differ considerably from that of the control population as a whole. This suggests that even though individual obligate heterozygotes cannot be identified from the a- to B-mannosidase ratio, population differences may still exist. It is now clear that future evaluation of plasma a- and B-mannosidase levels in goats should consider age, sex, and reproductive status. The marked contrast between age and sex effects on plasma a-mannosidase values in goats and cattle suggests that factors affecting plasma a- mannosidase activity are species-specific and that results obtained from cattle cannot be assumed valid for other species as well. _Future studies on the regulation of mannosidase activity indifferent species are important to the understanding of cell regulation of mannosidase activity in glycoprotein catabolism. Finally, although neither plasma B-mannosidase activity alone nor plasma a- to B-mannosidase ratios distinguish B-mannosidosis heterozygotes from the control populations, the 3 obligate heterozygotes did have plasma B-mannosidase values intermediate between those of affected and control animals and had plasma d- to B-mannosidase ratios higher than controls. Thus, plasma 61 mannosidase assays may be used to select the most likely heterozygous candidates for the B-mannosidosis breeding program. Future results from the breeding program that establish whether goats with low 8- mannosidase values are heterozygotes will be important in determining the validity of this approach. SUMMARY The effects of age, sex, and reproductive status on caprine plasma a- and B-mannosidase activities as well as the potential use of plasma assays for heterozygote detection of caprine B-mannosidosis were inves- tigated. Optimal conditions for the assay of caprine plasma a- and B-mannosidase activity were determined. The pH optima were 4.0 and 5.0 for.caprine plasma a- and B-mannosidase activities, respectively, and substrate hydrolysis was proportional to time beyond the incubation periods used in these studies. Age and sex affected caprine plasma a- and B-mannosidase activities, while plasma B-mannosidase activity was affected by reproductive status. Obligate heterozygotes for B-mannosidosis had plasma B-mannosidase values which were intermediate betWeen those found in animals affected with B-mannosidosis and controls. Putative heterozygotes for B-mannosidosis could not be definitively identified, but likely candidates for future inbreeding experiments were discerned. 62 BIBLIOGRAPHY BIBLIOGRAPHY Annunziata, F., and Di Matteo, G. (1978) Study of influence of sex and age on human serum lysosomal enzymes by using 4-methyl- umbelliferyl substrates. Clin. Chim. Acta 90, 101-106. Bouquelet, 8., Spik, G., and Montreuil, J. (1978) Properties of a B—D-mannosidase from Aspergillus niger. Biochtm. et Biophys. Acta 522, 521-530. Burditt, L. J., Chotai, K., Halley, D., and Winchester, B. (1980a) Comparison of the residual acidic a-D—mannosidase in three cases of mannosidosis. Clin. Chim. Acta 104, 201-209. Burditt, L. J., Chotai, K., Hirani, S., Nugent, P. G., and Winchester, B. G. 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