ANNALS CLINICAL AND LABORATORY SCIENCE, Vol. 11, No. 2 Copyright © 1981, Institute for Clinical Science, Inc.

Leukocyte in Birth Defects— A Review

MARGE A. BREWSTER, Ph .D.

Departments of Pathology, Biochemistry, and Pediatrics, University of Arkansas for Medical Sciences, and Arkansas Children’s Hospital, Little Rock, AK 72201

ABSTRACT The utilization of peripheral leukocytes, mixed preparations or specific populations for the detection of homozygous and heterozygous states of inborn errors of via assay or detection of accumulated metabolites are reviewed. Additionally, present knowledge of metabolic activities in leukocyte populations and areas of potentially fruitful investiga­ tion are discussed.

Introduction Peripheral circulating leukocytes often provide a convenient “tissue biopsy” for Glycolytic enzymes are detectable in detection of birth defects resulting in polymorphonuclear leukocytes (PMNs)

specific enzymatic deficiencies and/or and in lymphocytes, 1 5 ,2 1 their activities metabolite accumulation. Such ap­ being very dependent upon cell prepara­ proaches are reviewed in this paper. At­ tion technique and upon anticoagulant

tention is directed to the articles by Hsia, 24 choice . 1 5 The lymphocyte hexose Frie etal,18and Cone etal 11 promoting the monophosphate pathway is only slightly

utilization of leukocytes for the study of active22 while that of the PMN is quite

inborn errors of metabolism. Known active . 3 Deficiency of glucose-6 -phos- leukocyte physiology and biochemistry phate' dehydrogenase has been demon­

have been compiled by Cline , 10 and strated in granulocytes concomitant with

leukocyte separation techniques up to deficiency in the erythrocyte . 38 T ri­

1970 have been reviewed by Cutts. 12 In­ carboxylic acid cycle activity is present in

born errors detectable via leukocytes in granulocytes 10 and in lymphocytes . 22 the homozygous or heterozygous state are Lymphocyte citrate synthesis occurs at a summarized in table I, and discussed in rate comparable to that in skeletal muscle,

the text. lungs, and pancreas . 36 Blass et al 5 146 0091-7370/81/0300-0146 $00.90 © Institute for Clinical Science, Inc. LEUKOCYTE ENZYMES IN BIRTH DEFECTS 147

TABLE I

Inborn Error Detection via Leukocyte Assay

Leukocyte Population Leukocyte Population Lympho- Granulo­ Lympho- Granulo- Disease State (form) cytes cytes Mixed Ref, Disease State (form) cytes cytes Mixed Ref.

Carbohydrate Metabolism Lipid Metabolism Glucose-6-phosphate X 38 Hypercholesterolemia*t X 17 dehydrogenase deficiency* Juvenile ceroid X 37 Pyruvate dehydrogenase X 5 lipofuscinosis* deficiency* Batten's disease* X 1 Ketoglutarate X 5 Gaucher’s disease*+ X 28, 59 dehydrogenase deficiency* Niemann Pick X 28, 67 Glycogen synthetase X 19 disease*t deficiency* GM2~ganglios idos is * t X 66 Glycogen storage X 65, 51 Fabry's disease* X 29 disease II* Metachromat ic X 2 Glycogen storage X 62, 51 leukodystrophy* disease III* Tay-Sach's disease*t X 26 Glycogen storage X 6, 51 disease IV* Mucolipid, Mucopoly­ Glycogen storage X 61, 51 saccharide Metabolism disease VI* Fucosidosis*+ X X 4 Mannos idos is * t X 33, 54 Mucolipidosis II* X 30 *t X 32 Amino Acid and Urea Schie syndrome* X 32 Cycle Meatbolism * X 31 Maple-syrup-urine X 7 Maroteaux-Lamy X 52 disease* syndrome* Isovaleric X 7 Sanfilippo, X 40 aciduria* type A*t a-Ketoadipic X 63 B-glucuronidase X 46 aciduria* deficiency* Cystinosis*t X 34, 60 Hyperprolinemia, X 56 Purine, Pyrimidine and type II* Folate Metabolism Homocystinuria*+ X 20 Orotic aciduria X 49 Carbamyl phosphate X 64 Purine 5 ’-nucleotidase X 25 synthetase deficiency* deficiency Arginino-succinase X 64 Lesch-Nyhan syndrome X 9 deficiency* Folate dificiency X 14

♦Homozygous state. tHeterozygous state.

employed a platelet enriched blood fac­ and VI.6,51’ 61' 62 Wyss et al 65 discuss the tion containing 1/3 leukocytes for the choice of leukocyte preparation in diag­ diagnosis of pyruvate and ketoglutarate nosis of type II. Leukocyte glycogen dehydrogenase deficiencies. synthetase activity has been studied in the

Lymphocytes can synthesize gly­ hepatic deficiency state. 19 cogen 2 2 but do not store appreciable amounts. 42 The glycogen content of the Lipid Metabolism granulocyte is comparable to that of liver and muscle, 42 but Esman’s 16 demonstra­ Fogelman et al 17 utilized unfraction­ tion that neutrophil glycogen content is ated leukocytes from subjects with increased upon in vitro incubation heterozygous familial hypercholes­ suggests caution in using neutrophil terolemia to demonstrate an abnormal in­ glycogen to demonstrate tissue storage. duction of 3-hydroxy-3-methylglutaryl The neutrophil and the liver utilize simi­ coenzyme reductase. lar mechanisms of glycogen degradation, Altered concentration of leukocyte thus allowing the neutrophil to be utilized docosahexaenoic acid has been demon­ for demonstrating the enzyme defects of strated in the juvenile form of ceroid lipo­ glycogen storage diseases types II, III, IV, fuscinosis. 37 Leukocyte myeloperoxidase 148 BREWSTER activity is markedly deficient, 1 w hile Leukocyte incorporation of radio- p-phenylenediamine-peroxidase activity labeled substrate into mucopoly­ is normal41 in Batten’s disease. saccharides, following phytohemagglu­ Lipidoses owing to deficient lysosomal tinin stimulation, was utilized to detect enzymes are readily demonstrable in patients with Hurler, Hunter, Morquio, or leukocytes, several assays employing Maroteaux-Lamy forms of mucopoly­ convenient artificial substrates. Synthetic saccharidosis. 57 a-L-iduronidase of leuko­ substrate beta-glucosidase activity in cytes has been utilized to detect homozy­ leukocytes can detect Gaucher’s disease gotes with Hurler and Schie syndromes32 homozygotes 28 and heterozygotes . 59 and heterozygotes with Hurler syn­

Niemann-Pick disease homozygote and drom e . 58 Hunter syndrome has been heterozygote detection via leukocyte diagnosed by lymphocytic activity of

sphingomyelinase still requires radio- iduronate sulfatase.31 and labeled sphingomyelin substrate . 2 8 , 6 7 N-acetyl-galactosamine-4 sulfate sul­ Leukocyte beta-galactosidase activity is fatase, deficient in Maroteaux-Lamy, are deficient in GM 2 -gangliosidosis, 66 possi­ demonstrable in leukocytes. 52 Leukocyte bly secondary to mucolipidosis or myco- activity in sulfamidase can distinguish

polysaccharidosis. 53 Alpha-galactosidase, heterozygous and homozygous forms of present in neutrophil granules, is reduced Sanfilippo disease type A. 40 Mucopoly­ in patients with Fabry’s disease .29 Meta- saccharidosis owing to /3-glucuronidase chromatic leukodystrophy has been diag­ deficiency is expressed in leukocytes.46 nosed via leukocyte arylsulfatase A assay, 2 but low activity has been reported in a Amino Acids and Urea Cycle Metabolism family without the disease . 8 The carrier state of Tay-Sach’s disease, suggested by Enzymes for the oxidative decarboxyla­ serum assay, is reliably confirmed by tion of branched-chain keto acids of leukocyte A activity.26 leucine, isoleucine, and valine are active in the neutrophil. These cells have been Mucolipid and Mucopolysaccharide utilized to demonstrate enzyme defi­ Metabolism ciency in maple-syrup urine disease and

Peripheral blood leukocytes have been in isovaleric acidemia. 7 Wilson et al63 has utilized to detect the carrier state of optimized the assay for the pathway con­ fucosidosis , 4 with lymphocytes being verting a-(l- 1 4 C)-aminoadipic acid to

a more reliable indicator than serum 14 C 0 2 and recommends its use in further or unfractionated leukocytes. The elaboration on the nature of a-ketoadipic a-(s) of leukocytes appears to aciduria. Simell45 has utilized granulo­

differ from that of serum . 55 Mannosidosis cytes to study transport of di amino acids in and the heterozygote state are demonstra­ lysinuric protein intolerance, showing ble in mixed leukocytes. 33 Thompson et that one transport system is present for a l 54 have shown that mannosidosis lysine, ornithine and arginine. Cystinosis heterozygote detection is more reliable is manifested by increased cystine in neu­ when referenced to leukocyte protein trophils, with typical increases of 80-fold than to other lysosomal ac­ in homozygotes and 6 -fold in hetero­ tivities. I-cell disease, or mucolipidosis II, zygotes. 34 Unfractionated leukocyte up­

is detected by leukocyte decrease and take of 3 5 S-cystine has also been utilized to

serum increase of several lysosomal acid diagnose cystinosis.60 Cystinuric patients , including /3-galactosidase, have not demonstrated an accumulation of /3-glucosaminidase, /3-glucuronidase, cystine or a dibasic amino acid transport a-galactosidase, and arylsulfatase A. 30 defect in their neutrophils . 39 Type II LEUKOCYTE ENZYMES IN BIRTH DEFECTS 149 hyperprolinemia has been characterized tion. 5'-Nucleotidase does not appear to by deficient pyrroline-5-carboxylic acid be present in human polymorphonuclear dehydrogenase in lymphocytes.56 Homo- leukocytes, even though it has classically cystinuria heterozygotes have been de­ been utilized as an enzymatic plasma tected employing phytohemagglutinin- membrane marker . 43 Enzymes of the stimulated lymphocytes.20 purine metabolic cycle have been As cell lysis and deproteinizing agents evaluated in normal and leukemic lym­ interfere with quantitative extraction of phocytes. 13 Lymphoid cells from patients free amino acids from leukocytes,23 little with Lesch-Nyhan syndrome are defi­ study of leukocytic amino acid concen­ cient in hypoxanthine-guanine phos- trations has been reported. phoribosyl . 9 The 14 C-formate Moore et al 35 studied the synthesis of to N-formylglycinamide ribonucleotide urea from arginine by unfractionated portion of the purine synthetic pathway leukocytes, and estimated that approxi­ has been shown to be enhanced in lym­ mately one percent of the daily urea pro­ phocytes from patients with primary gout, duction occurs in these cells. Wolfe etal 64 but values overlap rates measured in sec­ have demonstrated the presence in leuko­ ondary hyperuricemia and control cytes of the four urea cycle enzymes (or­ states . 2 7 A “functional folate” assay of nithine transcarbamylase, carbamyl lymphocytes, utilizing 14 C-formate incor­ phosphate synthetase, and argininosucci- poration into serine has been reported to nase plus its ), and point out the util­ detect folate deficiency sensitively, 14 and ity of this tissue in diagnosis of hereditary theoretically should provide a means to hyperammonemic states when a liver evaluate simultaneously several of the biopsy might be quite harmful. In this in­ folate interconversion enzymes. stance, leukocytes demonstrate urea cycle Studies employing leukocytes for de­ enzymes not present in cultured fibro­ tection of homozygote or heterozygote blasts, ornithine transcarbamylase and states of inborn errors of metabolism are carbamyl phosphate synthetase I. Snod­ increasingly being reported, indicating grass50 has recently demonstrated that the vast untapped diagnostic and prognos­ leukocyte ornithine transcarbamylase ac­ tic potential of the numerous “tissue biop­ tivity is not deficient concomitant with de­ sies” circulating in peripheral blood. ficiency of the liver enzyme. References Purine, Pyrimidine and

Folate Metabolism 1. Ar m st r o n g , D., e t a l : Studies in Batten dis­ ease. Arch. Neurol. 30:144-152, 1974. Enzymes for de novo synthesis of 2. Ba um , H., D o d g so n , K. A., and Sp e n c e r , B.: Clin. Chim. Acta 4:453, 1959. pyrimidines48 and purines are most active 3. B eck , w. S.: Occurrence and control of the in immature granulocytes, with little ac­ phosphogluconate oxidation pathway in normal tivity remaining in mature ones. Leuko­ and leukemic leukocytes. J. Biol. Chem. cyte orotidylic decarboxylase is deficient 232:271, 1958. 4. Be r a t is , N. G ., T u r n e r , B. M ., and in orotic aciduria.49 Lymphocyte purine H ir s c h h o r n , K.: Fucosidosis-detection of car­ 5'-nucleotidase deficiency occurs in at rier state in peripheral blood leukocytes. 1. least some patients with primary hypo­ Pediat. 87:1193, 1975. 5. Bla ss, J. P., C ed a r b a u m , S. D., and Kark, R. A. gammaglobulinemia.25 In light of the ob­ P.: Rapid diagnosis of pyruvate and ketogluta- servation that 5'-nucleotidase-positive rate dyhydrogenase deficiencies in platelet- and -negative sub-populations of periph­ enriched preparations from blood. Clin. Chim. Acta 75:21, 1977. eral lymphocytes exist , 44 this defi­ 6. Br o w n , B. I. and Br o w n , D. H .: Lack of a-1, ciency may reflect absence of a popula­ 4-glucan: a-1, 4-glucan 6 glycosyl transferase in 150 BREWSTER

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