The Clinical Biochemistry of 5'-Nucleotidase*

F. WILLIAM SUNDERMAN JR., M.D.

Departments of Laboratory Medicine and Pharmacology, University of Connecticut Medical School, Farmington, CT 06032

ABSTRACT

This review delineates the subcellular distribution, biochemical charac teristics, and metabolic functions of 5'-nucleotidase (5'NT), summarizes the analytical biochemistry of 5'NT, and assesses the clinical significance of5'NT determinations in body fluids, cells, and tissues. Salient aspects of the clinical biochemistry of 5'NT, discussed herein, are as follows: (A) Serum 5'NT activity is generally elevated in hepatobiliary diseases, espe cially with intrahepatic obstruction, but, unlike serum alkaline phospha tase, serum 5'NT activity is not increased in infancy, childhood, preg nancy, or osteoblastic disorders. (B) In cancer patients, elevated serum 5'NT activity does not always indicate hepatobiliary involvement; in some cases, 5'NT may be released into serum from the primary tumor or local metastases. (C) Genetic deficiency of erythrocyte pyrimidine 5'NT activity is a common cause of hereditary non-spherocytic hemolytic anemia. (D) Acquired deficiency of erythrocyte pyrimidine 5'NT activity occurs in patients with P-thalassemia and lead poisoning. (E) 5'NT activity is low in circulating monocytes, increases markedly upon their differentiation to tissue macrophages, and subsequently diminishes during macrophage activation. (F) Lymphocyte ecto-5'NT activity, a plasma membrane marker of cell maturation, is generally low in immunodeficiency states, and undergoes characteristic changes in patients with certain lymphomas and leukemias.

Introduction ribonucleosides and inorganic phos phate. Reis’s enzyme, now commonly In 1934, Reis1 0 9 reported that muscle termed ‘5'-nucleotidase’ (‘5'NT’) and for homogenates contain an enzyme activity mally designated as ‘5'-ribonucleotide that specifically hydrolyzes various phosphohydrolase’ (EC 3.1.3.5), com ribonucleoside-5 ' -monophosphates at prises a group of related enzymes that neutral pH to yield the corresponding are widely distributed in prokaryotic and eukaryotic organisms. Determinations of 5'NT activity in human serum, erythro * Presented at the Fortieth Anniversary Meeting of the Association of Clinical Scientists in Philadel cytes, leukocytes, and tissues have phia, PA, November 1989. become diagnostically important in cer 123 0091-7370/90/0300-0123 $02.00 © Institute for Clinical Science, Inc. 1 2 4 SUNDERMAN JR. tain hepatobiliary, hematological, immu myocyte ecto-5'NT . 1 2 7 In rat kidney, nological, and neoplastic disorders. The ecto-5'NT activity is located in the brush voluminous literature, prior to approxi border of proximal tubular cells and in mately 1970, on the clinical enzymology plasma membranes of cortical interstitial of 5'NT was reviewed by Bodansky and fibroblasts . 8 1 Based on the segregation

S ch w artz , 2 3 Drummond and Yama pattern of ecto-5'NT activity in panels of moto , 4 9 and van der Slik et al. ; 1 4 8 more human/rodent hybrid cells, the genetic recent publications have been surveyed locus of ecto-5'NT resides on human by Belfield 1 1 and Ellis . 5 3 The goals of the chromosome 6 in the region from ql4 to present paper are (a) to summarize the q 2 1 . 2 8 , 2 9 distribution, properties, and functions of 5'NT; (b) to review the analytical bio P r o p e r t ie s chemistry of 5'NT; and (c) to delineate the clinical significance of measurements Since chelators generally inhibit 5'NT of 5'NT activities in body fluids, cells, activity and various divalent metal ions and tissues. restore the activity, 5'NT is probably a metalloprotein; the native constituent Distribution, Properties, and Functions metal has not been identified, but zinc is a likely candidate . 6 1 , 8 6 Based on studies SUBCELLULAR DISTRIBUTION AND of lymphocytes, myocytes, and renal G e n e t ic L o c u s cells, ecto-5'NT is a homodimeric or tetrameric glycoprotein, comprising two

Activity of 5'NT is predominantly or four subunits of 6 8 to 70 kD .80,84,127 located in the plasma membrane of The pH optimum of 5'-NT activity mammalian cells . 7 , 6 0 5'-Nucleotidases ranges from 6.5 to 8.4, depending upon are also present, although in less abun the specific isoenzymes, substrates, dance, in mitochondria , 6 5 m icro - buffers, and reaction condi somes, 5 6 , 1 1 7 Golgi apparatus , 5 6 and cyto tions .68,120,122,129,160 The relative specific so l .143,146,158 Studies of hepatocytes, activities of 5'-adenososine monophos lymphocytes, and placental cells indicate phate (5'-AMP), 5'-uridine monophos that the ectoenzyme, ‘ecto-5'NT’, is phate (5'-UMP), 5'-guanosine mono anchored to the plasma membrane by a phosphate (5'-GMP), 5'-cytidine C-terminal glycolipid moiety that con monophosphate (5'-CMP), and 5'-ino- tains glycosyl phosphotidylinosi- sine monophosphate (5'-IMP) as sub to l .85,135,136,158,159 In rat hepatocytes, strates for ecto-5'NT of rat hepatocytes ecto-5'NT is a short-stalked integral are reported to be 100:81:63:63:59, membrane protein; the catalytic site respectively . 3 3 The relative specific activ faces the extracellular medium and little, ities of 5'-AMP, 5'-CMP, 5'-IMP, and if any, of the enzyme molecule extends 5'-GMP as substrates for ecto-5'NT of to the cytoplasmic surface of the plasma porcine lymphocytes are reported to be m em brane . 7 In contrast, in murine plas 100:65:50:45, respectively . 4 7 The 5'N T macytoma cells, ecto-5'NT is a trans activity of blood serum and plasma membrane protein . 1 5 6 In chicken gizzard membranes of lymphocytes, mammary myocytes, ecto-5'NT activity is stimu cells, glioma cells, and plasmacytoma lated by laminin and inhibited by fibro- cells is strongly inhibited by concanava- n ectin . 4 3 , 1 2 7 These two components of lin A (‘con A ’),32,37,82,126,160 but the cyto the extracellular matrix evidently inter plasmic 5'NT activity of plasmacytoma act with different binding sites of the cells and erythrocytes is unaffected by CLINICAL BIOCHEMISTRY OF 5'-NUCLEOTIDASE 1 2 5 con A . 1 1 2 , 1 6 0 Since con A binds to puri sine production by the 5'NT activity of fied ecto-5'NT in vitro, ecto-5'N T is interstitial fibroblasts helps to regulate believed to be a plasma membrane renal hemodynamics, glomerular filtra receptor for con A . 4 6 -4 7 -8 2 ’ 1 2 3 tion rate, and the release of renin and

Human erythrocytes contain at least erythropoietin . 8 1 The microvillous two cytosolic 5'NT activities: First, there plasma membrane of syncytiotrophoblast is a cytosolic purine 5'NT that avidly in the human placenta is rich in hydrolyzes 5'-IMP and 5'-GMP, but ecto-5'NT activity, which may help to hydrolyzes 5'-AMP and other nucleoside modulate the feto-placental-maternal

5'-monophosphates at much lower microcirculation . 8 9 Human placental ra te s . 2 5 This p u rin e 5'N T activity is ecto-5'NT has FAD-pyrophosphatase inhibited by inorganic phosphate and activity that hydrolyzes flavin adenine strongly stimulated by adenosine tri dinucleotide (FAD) to riboflavin phos phosphate (ATP) and 2,3-diphosphoglyc- phate; whether or not ecto-5'nucleoti erate (2,3-DPG); the activity is higher in dases of other tissues hydrolyze FAD fresh erythrocytes than in outdated remains to be established . 7 9 In hum an c e lls . 2 5 Second, there is a cytosolic erythrocytes, cytosolic purine 5'NT is pyrimidine 5'NT that avidly hydrolyzes secondarily involved in the catabolism of 5'-UMP and 5'-CMP, but is inactive on 5'-AMP, which proceeds initially via purine 5'-ribonucleotides . 1 3 0 , 1 4 4 This deamination by adenosine deaminase to pyrimidine 5'NT activity is deficient in 5'-IMP, which is a preferred substrate patients with a common form of heredi for erythrocyte purine 5'NT . 2 5 , 1 4 6 In tary non-spherocytic hemolytic ane human erythrocytes, cytosolic pyrimi m ia , 1 7 1 4 4 and is inhibited in patients dine 5'NT dephosphorylates pyrimidine with (i-thalassemia4 0 ’ 1 5 1 and lead poison and deoxypyrimidine nucleotides . 1 4 4 in g .30,98’99’145 §everal isoenzymes of Pyrimidine 5'NT activity is high in retic erythrocyte pyrimidine 5'NT have been ulocytes, which contain abundant pyrim identified and characterized .35’36,97’130 idine nucleotides; pyrimidine 5'NT activity diminishes abruptly after F u n c t io n s nascent erythrocytes are released from the bone marrow . 2 0 , 2 1 As discussed sub The primary function of ecto-5'NT is sequently, pathological reduction of the conversion of extracellular nucleo erythrocyte pyrimidine 5'NT activity, as tides (e.g., 5'-AMP), to which cells are a consequence of hereditary enzyme generally impermeable, to the corre deficiency, (3-thalassemia, or lead poi sponding nucleosides (e.g., adenosine), soning, results in hemolytic anemia, which can readily enter most basophilic stippling, and elevated eryth cells .55,132,150 The half-life of adenosine in rocyte concentrations of 5'-CMP, 5'- plasma is brief; adenosine modulates cor UMP, and glutathione (GSH). onary, cerebral, and skeletal blood flow, affects neuronal activity and immune Analytical Biochemistry responses, and influences the metabolic of 5'-Nucleotidase effects of catecholamines . 9 5 , 1 2 5 In lym phocytes, 5'NT activity supplies nucleo C lassification o f 5 '-NT A ssays sides for cellular metabolism and nucleic acid synthesis under conditions where Techniques for the assay of 5'NT activ intracellular de novo synthesis of nucleo ity may be classified in eight major cate tides is lim ited . 1 3 2 In the kidney, adeno gories, as follows: 12 6 SUNDERMAN JR.

Category I comprises 5'NT assays in thine by nucleoside phosphorylase, and which the inorganic phosphate that is the hypoxanthine is oxidized by xanthine released from 5'-nucleotides (usually 5'- oxidase to urate and H 2 0 2. In one such AMP) is measured by a molybdate color technique, the formation of urate is reaction 6.44>66-10°.11().i is, 1 5 2 , 1 5 4 monitored by spectrophotometry at 293

Category II comprises 5'NT assays in nm . 4 5 In another technique, H 2 0 2 for which the nucleoside reaction product mation is monitored at 510 nm using a (adenosine, inosine, uridine) is separated chromogenic system (i.e., 3,5-dichloro- from the substrate by high performance 2-hydroxybenzenesulfonic acid, 4-amin- liquid chromatography and quantitated ophenazone, and peroxidase ) . 1 8 In still by ultraviolet absorptiometry . 1 1 1 3 another technique, which involves five

Category III comprises procedures in coupled enzyme reactions, H 2 0 2 is which 5'NT hydrolyzes 5'~AMP to pro reduced by catalase in the presence of duce adenosine, which is deaminated by ethanol to form acetaldehyde, which is adenosine deaminase (ADA) to yield ino determined by the nicotinamide-adenine sine and ammonia. These coupled enzy dinucleotide phosphate (NADP)-depen- mic reactions are measured kinetically dent aldehyde dehydrogenase reaction, by recording the decrease in absorbance monitoring reduced nicotinamide-aden of 5'-AMP at 265 nm . 1 0 - 1 3 8 3 - 1 1 7 ine dinucleotide phosphate (NADPH)

Category IV procedures are similar to production at 334 nm . 6 4 those of Category III in that 5'-AMP Category VIII comprises radiometric undergoes hydrolysis by the coupled procedures in which 5'-purine or 5'- 5'NT and ADA reactions, but the ammo pyrimidine substrates are labelled with nia that is produced is determined color- 3H or 1 4 C. After a suitable incubation imetrically by the Berthelot indophenol period for the 5'NT reaction, the nucleo reaction. 19>10M48.i6o tide substrate is separated from the Category V comprises procedures in nucleoside product by paper chromatog which the 5'NT and ADA reactions are r a p h y , 5 6 thin-layer chromatogra combined with the NADH-dependent phy ,22,29,6996 ion-exchange chromatogra glutamate dehydrogenase reaction, so p h y , 7 6 high performance liquid that ammonia produced by the ADA chromatography , 4 1 cellulose acetate elec reaction combines with 2 -oxoglutarate to trophoresis , 7 5 adsorption on alu yield L-glutamate. The three coupled mina , 3 3 , 1 2 9 or coprecipitation on barium reactions are measured kinetically by sulfate ;3,48,58,120,122,141 the yield of the recording the decrease in absorbance of labelled product is then quantified by

NADH at 334-340 nm .5,24,59,62,63 Inter liquid scintillation counting. ference by endogenous ammonia in serum can be prevented by treating the I n h ib it io n o f C o m p e t in g serum with an ion-exchange resin prior P hosphohydrolase R e a c t io n s to 5'NT assay. 7 1 Category VI is similar to Category V, To measure 5'NT activity in serum, except that cytidine-5'-monophosphate cell extracts, or tissue homogenates by (5'-CMP) is substituted for 5'-AMP, and any of the cited techniques, it is gener cytidine deaminase (CDA) is substituted ally necessary to correct, inhibit, or for ADA as the second enzyme in the otherwise compensate for non-specific coupled reactions . 1 4 7 hydrolysis of the substrate by alkaline Category VII comprises procedures in phosphatase (AP). There have been six which 5'NT hydrolyses 5'-IMP to form major approaches to solving this prob inosine, which is converted to hypoxan- lem, as follows: CLINICAL BIOCHEMISTRY OF 5'-NUCLEOTIDASE 1 2 7

First, the reaction can be performed little effect on 5'-AMP hydrolysis by in presence and absence of Ni 2 + , a AP 3 3 ’ 1 2 6 or cytosolic 5'NT of erythro non-specific inhibitor of 5'NT activity , 3 cytes . 1 1 2 Therefore, con A is used as a so that 5'NT activity can be calculated differential inhibitor in serum 5'NT or by difference .2,6’31’66’100110’154 The reli ecto-5'NT assays to avoid interference ability of this approach is dubious, from AP and hemolysis , 1 1 9 ’ 1 6 0 and is since AP activity from serum and bone also used in erythrocyte pyrimidine is partially suppressed by Ni2 + . 1 5 5'NT assays to avoid interference from

S econd, nonspecific hydrolysis of serum 5'N T . 1 1 2 5'AMP by AP can be suppressed by Adenosine deaminase in cell and tis adding an excess of a preferential AP sue extracts and plasma membrane substrate, such as (3-glycerophos preparations can interfere in certain p h a te ,3’12,13,14,22’48’76’96 phenyl phos 5'NT assays, owing to conversion of p h a te , 1 5 ’ 1 9 1 0 2 or p-nitrophenyl phos adenosine to inosine . 6 9 To detect this p h ate . 1 3 , 2 4 1 2 0 source of interference, the recovery of Third, compensation for nonspecific added adenosine should be tested hydrolysis of 5'AMP can be partially when 5'NT assays of Categories II and achieved by simultaneous assay of AP VII are employed. activity, with use of an empirical cor rection factor , 4 4 ’ 8 3 1 5 2 or by use of a cor R e c o m m e n d e d A n a l y t ic a l M e t h o d s rection factor derived by simultaneous assay of a control cuvet that contains as Selecting the 5'NT assay that is best a substrate a mixture of 2'-AMP and suited for a specific clinical or research

3'-AM P . 1 0 objective depends upon the specimen F ourth, amino acids (e.g., L-histi- for analysis, the available instrumenta dine, L-glycine) can be added to the tion, the analytical workload, the clinical reaction mixture to suppress the exigency, and the specificity that is hydrolysis of 5'-AMP by AP. 3 1 1 5 required. The following are the present F ifth, a,|3-methyleneadenosine-5'- author’s preferences: diphosphate (MADP), a competitive Routine automated assay of serum inhibitor of 5'NT activity, can be added 5'NT activity as a liver function test in a to the reaction mixture in order to dif hospital laboratory is conveniently per ferentiate 5'NT activity from AP activ formed by a kinetic technique of Cate ity . 1,29,58’76 Zygowitz et al1 6 0 found that gory V, using a centrifugal analyzer, and MADP was unsatisfactory as a differen adding (3-glycerophosphate to the reac tial inhibitor for serum 5'NT assay, tion mixture to suppress nonspecific since it blocked the hydrolysis of 5'- hydrolysis of the substrate by AP. A suit AMP by both AP and 5'NT. Andrew et able reagent kit, based on the method of al, 3 found that MADP (2 mmol/L) did Arkesteijn , 5 is available.* not effectively block 5'NT activity in When serum ecto-5'NT assays are rabbit alveolar macrophages, under performed for clinical research (e.g., for conditions in which 98 percent inhibi drug trials), a procedure of Category IV tion was obtained with NiCl2 ( 1 0 mmol is most reliable, with con A as the differ per L). ential inhibitor, as reported by Zygowitz

Finally, concanavalin A (con A) and et al. 1 6 0 related plant lectins inhibit hydrolysis To assay erythrocyte pyrimidine-5'NT of 5'-AMP by serum 5'NT and by activity for differential diagnosis of ecto-5'NT of cellular plasma mem branes 32,33,37,46,80,82,119,123,126,160 w ith * Sigma Chemical Co., St Louis, MO. 1 2 8 SUNDERMAN JR. hemolyic anemias or evaluation of 3 - purely skeletal disease or in conditions affect ing other tissues such as myositis, muscular thalassemia and lead poisoning, the dystrophy, ulcerative colitis without hepatic radiometric or spectrophotometric involvement, and cardiovascular disease or methods described by Miwa et al. 9 2 are infectious diseases.” recommended; to detect heterozygotic carriers of pyrimidine 5'NT deficiency, Many clinicians have verified these the radiometric high performance liquid generalizations. Based on findings in 236 chromatography (HPLC) technique of de patients, Belfield and Goldberg 1 3 , 1 4

Korte et al. 4 1 , 4 2 is the m ethod of choice. reported serum 5'NT activity to be ele The most convenient way to screen vated in 92 percent of patients with leukocyte samples for diminished 5'NT obstructive jaundice, 70 percent of activity is the colorimetric assay of Perez patients with parenchymal liver disease, et al, 1 0 1 which uses microtitre plates and 81 percent of patients with hepatic an automatic multiscan spectrophotome metastases, and 1 1 percent of patients ter. with bone diseases. In the patients with bone diseases, serum 5'NT activity was Diagnostic Applications highest in cases with osseous metastases of 5'-Nucleotidase Assays of cancer, in whom hepatic metastases could not be excluded. Measurement of S e r u m 5'-NT in H epatobiliary D is e a s e s serum 5'NT activity was more reliable as an indicator of the source of raised serum

In 1954, Dixon and Purdom 4 4 r e AP activity than electrophoretic fraction ported that combined assays of serum ation of serum AP isoenzymes . 1 3 , 1 4 Based 5'NT and alkaline phosphatase (AP) on findings in 184 patients with liver, activities were helpful in differential gall bladder, or bone diseases, Pappo et diagnosis, since serum 5'NT activity is al1 0 0 reported that serum 5'NT activity generally increased in obstructive hepa was seldom increased in bone diseases tobiliary disorders, but not in osseous and was more sensitive than serum AP disorders, whereas serum AP activity is activity as a diagnostic index of cholesta usually increased in both categories of sis. Connell and Dinwoodie 3 4 examined disorders. After these observations were serum 5'NT activity in 114 patients with confirmed by Young1 5 5 and Kowlessar et increased serum AP activity, finding that a l , 7 7 assays of serum 5'NT activity 5'NT activity was increased in most (but became widely adopted in clinical bio not all) patients with hepatobiliary dis chemistry. In 1967, Hill and Sammons6 7 orders and concluding that normal concluded that: serum 5'NT activity did not invariably “Serum 5'-nucleotidase is as valuable as alka signify that bone was the origin of ele line phosphatase as a liver function test. Fur vated serum AP activity. thermore, it is more specific that alkaline Phelan et al1 0 3 performed serial assays phosphatase as it is not significantly raised in bone diseases, it provides confirmatory evi of serum AP and 5'NT activities in an dence of anicteric intrahepatic obstruction and unspecified number of patients with is of value in screening the sera of children acute and chronic liver diseases. Scant under treatment with potentially hepatotoxic drugs.” difference was noted in the responses of the two enzymes in acute infectious hep Reaching a similar conclusion, Bodansky atitis, but in chronic liver diseases, and Schwartz2 3 stated that: including chronic active hepatitis and “Serum 5'-nucleotidase activity has a distinct primary biliary cirrhosis, serum 5'NT value as a biochemical diagnostic aid in the diagnosis of hepatobiliary involvement . . . activity was more markedly and persis The enzyme activity is elevated only rarely in tently elevated than serum AP activ CLINICAL BIOCHEMISTRY OF 5'-NUCLEOTIDASE 1 2 9 ity 1 0 3 EUis et al5 4 compared serum 5'NT S e r u m 5'N T in N e o p l a s t ic D is e a s e s and AP activities in 1,147 patients with suspected hepatobiliary disorders, of Until recently, elevations of serum whom 580 had identifiable primary dis 5'NT activity in patients with cancer eases of the liver or biliary system. The were usually believed to indicate hepa results of the two enzyme assays were tobiliary involvement .74,107,116,124,148 generally correlated, but elevations were Thus, in a series of 98 consecutive more pronounced for serum 5'NT, espe patients admitted to a general oncology cially among patients with biliary dis service, Kim et al7 4 found that elevated eases. Elevated 5'NT activity was serum 5'NT activity gave a predictive slightly more frequent than elevated AP value of 8 6 percent for hepatic metas activity in sera from patients with tases, whereas elevated serum alkaline hepatic metastases; higher proportions of AP, -y-glutamyltranspeptidase, or gluta patients with early hepatitis and portal mate dehydrogenase activities gave pre cirrhosis had elevated activities of 5'NT dictive values of 63, 58, and 58 percent, than AP. 5 4 respectively. Studies during the past Serum 5'NT activity is unequivocally decade suggest that, in some cancer superior to serum AP activity as a diag patients, the primary tumor or its extra- nostic test for obstructive hepatobiliary hepatic metastases may account for diseases in infancy, childhood, and preg increased serum 5'NT activity.39,70,78,111 nancy. 1 6 , 1 1 8 Reference values for serum In patients with breast cancer without 5'-NT activity are lower in infants and demonstrable metastases, serum 5'NT children than in adults, whereas the con activity was offtimes mildly elevated verse applies to serum AP activity . 1 6 prior to mastectomy and returned to Throughout normal pregnancy, serum normal after surgery . 3 9 , 7 0 , 1 1 1 In a group of 5'NT activity remains within the refer 50 patients with head and neck cancers, ence range for non-pregnant women, Lai et al7 8 found that serum 5'NT activity while serum AP activity rises above the was positively correlated with the stage reference range during the last trimester of the tumor and was markedly elevated of pregnancy . 1 1 8 in patients with cervical metastases. As yet, there is no proven explanation After radiotherapy to the head and neck, for the remarkable specificity of tumor regression was attended by grad increased serum 5'NT activity for ual reduction of serum 5'NT activity. Lai obstructive hepatobiliary diseases, in et al7 8 suggested that increased serum view of the ubiquitous occurrence of 5'NT activity in patients with malignant ecto-5'NT on plasma membranes of cells tumors of the head and neck is mediated throughout the body. The present author by release of 5'NT from the solid tumor proposes that two related factors cause into the circulation. In view of such the elevated serum 5'NT activity in reports, the prognostic significance of patients with obstructive hepatobiliary elevated serum 5'NT activity in cancer diseases: first, when bile stasis occurs, patients needs to be reevaluated. biliary glycosidases may cleave the glycosyl-phosphotidylinositol moiety that 5'NT A c t iv it y in P r o st a t ic B io p s ie s anchors ecto-5'NT to the plasma mem brane of bile canalicular cells and peri Rackley et al1 0 5 measured tissue 5'NT portal hepatocytes, and, second, the activity in prostatic carcinomas and detergent action of bile salts may then hyperplastic prostates, biopsied during enable the liberated 5'NT molecules to transurethral resection. In 29 patients enter the circulation. with prostatic cancer, the tissue 5'NT 13 0 SUNDERMAN JR. activity averaged 10.5 ± 10.2 U per mg rent jaundice and basophilic stippling of protein, which was significantly less than erythrocytes . 1 7 , 3 6 , 1 4 4 A simple screening the corresponding mean of 28.3 ± 11.2 test involves measurement of the A260/ in 1 0 patients with benign prostatic A280 absorbance ratio of a protein-free hyperplasia. The 5'NT activity of the erythrocyte extract. 9 1 Purine nucleotides prostatic carcinomas was directly corre (e.g., 5'-AMP) comprise 96 percent of lated with the degree of histological dif the erythrocyte nucleotide pool in nor ferentiation . 1 0 5 Further studies are mal subjects, whereas pyrimidine needed to assess the usefulness of tissue nucleotides (e.g., 5'-CMP, 5'-UMP) 5'NT activity as a prognostic index in comprise more than 50 percent of the patients with prostatic carcinoma. corresponding pool in patients with

pyrimidine 5'NT deficiency . 1 4 1 1 4 2 Purine nucleotides have an absorption maxi E rythrocyte 5 'NT in H eredita ry mum at ~260 nm, while pyrimidine N o n -spherocytic H em o ly tic A n em ia nucleotides have an absorbance maxi mum at ~280 nm. Therefore, the A260/

In 1974, Valentine et al1 4 4 reported A280 ratio of a perchloric acid extract of three kindreds in which hemolytic ane erythrocytes provides a ready index of mia was associated with deficiency of the accumulation of pyrimidine nucleo erythrocyte pyrimidine 5'NT activity. In tid es . 9 1 1 4 1 1 4 2 Erythrocyte pyrimidine addition to the diminished enzyme activ 5'NT deficiency should be confirmed by ity, erythrocytes of affected subjects evi means of colorimetric 9 2 1 1 2 1 4 4 or radio- denced pronounced basophilic stippling metric enzyme assays. 4 1 1 4 1 Erythrocytes and increased contents of 5'-UMP, 5'- from healthy adults have a broad range CMP, and GSH. Numerous such of pyrimidine 5'NT activity, causing dif patients were subsequently reported and ficulty in identifying heterozygotic car the syndrome has become recognized as riers of the enzyme deficiency . 4 1 1 4 2 To a common type of hereditary non-spher detect such carriers, de Korte et al4 1 ocytic hemolytic anemia . 1 7 4 1 ’ 9 1 -9 2 1 3 0 1 4 2 have recommended that erythrocyte

De Korte et al4 2 reported that an affected pyrimidine 5'NT activity should be mea subject had diminished pyrimidine 5'NT sured by a sensitive and specific proce activity in lymphocytes and granulo dure, and interpreted in conjunction cytes, as well as erythrocytes, without with HPLC analysis of erythrocyte apparent derangement of leukocyte ribonucleotide concentrations. functions. Rechavi et al1 0 6 studied two siblings with erythrocyte pyrimidine 5'NT deficiency and chronic hemolytic E rythrocyte P y r im id in e 5 'N T anemia, who both developed an acute in (3-Th a la ssem ia febrile illness with pancytopenia. In each subject, the aplastic crisis was traced to Vives-Corrons et al 1 5 1 found that acute infection with human B19 paro- erythrocyte pyrimidine 5'NT activity virus, suggesting that patients with was markedly diminished in 50 heterozy pyrimidine 5'NT deficiency may have gous carriers of f$-thalassemia. In con enhanced susceptibility to viral induced trast, erythrocyte pyrimidine 5'NT activ pancytopenia . 1 0 6 ity was normal in 38 subjects with In patients with hemolytic anemia hypochromic, microcytic, iron deficiency caused by hereditary pyrimidine 5'NT anemia, and elevated in 50 non-thalasse- deficiency, the diagnosis is usually sug mic subjects with high reticulocyte gested by the presence of anemia, recur counts (32 patients with autoimmune CLINICAL BIOCHEMISTRY OF 5'-NUCLEOTIDASE 131 hemolytic anemia and 18 patients with Erythrocyte pyrimidine 5'NT activity is hereditary spherocytosis). Vives-Corrons inversely correlated with blood lead con et al1 5 1 speculated that patients with (3- centrations in lead-exposed sub thalassemia trait have reduced erythro jects . 3 ’ 3 0 ’ 3 5 Erythrocyte pyrimidine 5'NT cyte pyrimidine 5'NT activity as a result activity is comparable to erythrocyte of peroxidative injury by oxygen free protoporphyrin concentration as a clini radicals, and suggested that the dimin cal correlate of lead poisoning . 3 5 In lead- ished pyrimidine 5'NT activity might be exposed mice, erythrocyte pyrimidine responsible for basophilic stippling of 5'NT activity appears to be less sensitive erythrocytes in such patients. David et than erythrocyte 8 -ALA dehydratase al4 0 measured pyrimidine 5'NT activity activity as a sign of lead toxicity. 1 4 0 Since in 2 1 patients with heterozygous 3 -thal erythrocyte pyrimidine 5'NT activity is assemia and seven children with homo inhibited in vitro by various metals (e.g., zygous 3 -thalassemia, not yet trans copper, zinc, cadmium, lead, mercury, fused. Erythrocyte pyrimidine 5'NT and tin ) 3 6 , 9 3 , 1 4 0 the present author specu activity was significantly diminished in lates that inhibition of pyrimidine 5'NT both groups of thalassemic patients, may contribute to the hemolyic syn compared to healthy controls. No differ dromes that occur in patients with acute ence in absolute values for erythrocyte poisoning by these metals. pyrimidine 5'NT activity was observed between the heterozygotes and homozy 5'N T A c t iv it y o f M o n o c y t e s gotes. David et al4 0 showed that the dim a n d M a c r o p h a g e s inution of erythrocyte pyrimidine 5'NT activity in 3 -thalassemia is an acquired As blood monocytes undergo differen defect, mediated by oxidant damage to tiation to macrophages, ecto-5'NT activ labile sulfhydryl groups on the enzyme. ity increases approximately 35-fold; this Unbalanced globin synthesis causes response can be partially prevented by increased concentration of auto-oxidiz- corticosteroids . 9 6 , 1 0 8 When the macro able free heme in thalassemic erythro phages become ‘activated’ (i.e., develop cytes, which evidently predisposes to enhanced ability to phagocytize microor the production of oxygen free radicals . 4 0 ganisms and exert antimicrobial activity), their ecto-5'NT activity declines, owing to endocytosis of portions of the plasma E r y t h r o c y t e P y r im id in e 5'NT in m em brane .9’38’50’73-90-94’157 Certain xeno- L e a d P o is o n in g biotics affect macrophage ecto-5'NT activity, as exemplified by the marked

In 1975, Paglia et al9 8 discovered that reduction of ecto-5'NT activity in alveo erythrocyte pyrimidine 5'NT activity is lar macrophages from rats with acute significantly diminished in patients with Ni2+ toxicity. 1 2 8 Divalent nickel has long lead poisoning. They proposed that lead- been used as a selective inhibitor in induced deficiency of the enzyme in 5'NT assays, as discussed previously, and maturing erythroid cells is responsible in vitro addition of Ni2+ virtually elimi for basophilic stippling and hemolysis, nates 5'NT activity in extracts of alveolar analogous to the pathogenesis of the macrophages . 3 The present author sug hereditary enzyme deficiency syndrome. gests that Ni2+ binds in vivo to SH- Numerous studies have supported these groups of ecto-5'NT molecules on the inferences and have shown that reduced plasma membrane of macrophages, erythrocyte pyrimidine 5'NT activity is inhibiting the enzyme and triggering an index of lead poisoning . 4 -3 0 ’3 5 ’9 9 1 1 2 1 4 5 macrophage activation . 1 2 8 1 3 2 SUNDERMAN JR.

Ly m p h o c y t e E c t o - 5 'N T in is comparable to B cells and substantially I mmunological D is o r d e r s higher than T helper/inducer (OKT4 + )

cells . 8 8 In peripheral blood of healthy human adults, approximately three-fourths of B E c t o - 5 'N T A c t iv it y in L e u k e m ia s lymphocytes and one-third of T lympho a n d L y m p h o m a s cytes express ecto-5'NT on their sur face. 137,138,139 Ecto-5'NT is considered a Silber et al1 2 1 reported that ecto-5'NT maturation marker for T and B cells, activity is markedly decreased or unde since the enzyme activity is (a) approxi tectable in circulating lymphocytes from mately 10-times higher in peripheral T three-fourths of patients with chronic cells than thymocytes, (b) five to six lymphocytic leukemia (CLL), although times higher in adult peripheral B cells elevated activity does occur in less than than fetal spleen or cord blood B cells, 10 percent of such patients. To deter and (c) virtually absent in non-T, non-B mine if decreased 5'NT activity in CLL

(null) lymphocytes.51,133,138 Bastian et al8 reflects lower enzyme activity per cell or measured ecto-5'NT activity in serial a less-than-normal proportion of specimens of peripheral blood lympho enzyme-containing cells, 5'NT was cytes obtained from 1 1 infants at zero, stained histochemically in isolated lym two, four, and six months of age. phocytes . 1 2 1 Lymphocytes from normal Ecto-5'NT activity of total lymphocytes subjects or patients with CLL contained was lowest at birth and rose to adult distinct 5'NT-positive and 5'NT-nega- levels by six months, reflecting progres tive subpopulations. The leukemic sive expansion of the peripheral B cell patients with lymphocytes that were population. Increased ecto-5'NT activity devoid of ecto-5'NT activity by biochem of peripheral blood B cells of infants pre ical assay had no 5'NT-positive cells by cedes their acquisition of in vitro capac histochemical staining; an exceptional ity for IgG synthesis in response to CLL patient with elevated lymphocyte

Epstein-Barr virus . 8 Boss et al2 7 found ecto-5'NT activity by biochemical assay that ecto-5'NT activity of T and B lym showed an increased percentage of phocytes of healthy adults gradually 5'NT-positive cells by histochemical diminishes with advancing age, parallel staining . 1 2 1 Therefore, selective prolifer ing the normal decline of immune cell ation of 5'NT-positive or 5'NT-negative function in elderly persons. In immuno subpopulations of lymphocytes probably deficiency diseases with arrested lym accounts for the heterogeneity of lymphocyte maturation, ecto-5'NT activity ph ocyte ecto-5'NT activity in CLL is generally low; such diseases include patients. severe combined immuno-defi- Sylwestrowicz et al 1 3 1 m e a su re d c ie n c y , 2 6 , 1 3 4 Wiscott-Aldrich syn ecto-5'NT activity in peripheral blood drome, 1 5 3 common variable immunodefi lymphocytes from healthy controls and c ie n c y , 7 2 , 1 3 8 congenital X-linked in tumor cells from peripheral blood or agammaglobulinemia , 5 2 , 1 3 3 selective IgA bone marrow of patients with various deficiency , 1 5 3 familial reticulo-endothe- leukemias. They found elevated liosis with eosinophilia (Omenn’s syn ecto-5'NT activity in 25 of 27 patients drom e ) , 5 7 and acquired immune defi with common acute lymphoblastic leu ciency syndrome (AIDS ) . 1 1 4 Among T kemia (c-ALL), diminished activity in 10 cell subpopulations, ecto-5'NT activity of 13 patents with thymic acute leukemia of T suppressor/cytotoxic (OKT8 +) cells (Thy-ALL), and variable activity in six CLINICAL BIOCHEMISTRY OF 5'-NUCLEOTIDASE 133 patients with null acute lymphoblastic per day), and a zinc-repletion period ( 1 2 leukemia (null-ALL) and 28 patients weeks, oral Zn intake 30 mg per day). with blast crisis of chronic granulocytic Lymphocyte 5'NT activity declined leu k e m ia . 1 3 1 Massaia et al8 7 stu d ied sharply within four to six weeks on the ecto-5'NT activity in T cell subpopula zinc-restricted diet; the decrease of 5'NT tions of patients with multiple myeloma, activity preceded the eventual reduction finding that the enzyme activity was sig of lymphocyte zinc concentration, sug nificantly decreased in cytotoxic/sup- gesting that lymphocyte 5'NT activity pressor (CD 8 ) lymphocytes of myeloma may indeed be a sensitive indicator of patients, compared to controls. Antibod human zinc deficiency. ies to lymphocyte ecto-5'NT are now available that facilitate the phenotyping of cells separated by flow cytometry, so Conclusions and Speculations there is considerable current interest in the applications of this cell surface marker in hematological diagnosis. Determinations of 5'NT activities in serum, erythrocytes, leukocytes, and tis sue biopsies are becoming increasingly L y m p h o c y t e E c t o - 5 'N T A c t iv it y as an valuable in clinical biochemistry, as I n d e x o f Z in c D e f ic ie n c y knowledge advances concerning the diverse metabolic functions and patho Pilz et al1 0 4 found that human B lym- phoblastoid cells lost more than 90 per physiological alterations of this family of cent of ecto-5'NT activity when cultured enzymes. As yet, information is scanty for 24 hours in a zinc-deficient medium about the amino acid sequences, molec containing dialyzed serum. Supplemen ular domains, subunit assembly, second tation of the zinc-deficient medium with ary structures, membrane integration, Zn2+ (10 fxmol per L) prevented the loss prosthetic groups, active sites, or genetic of ecto-5'NT activity; Zn2+ repletion of regulation of the 5'NT isoenzymes. The zinc-deficient cells resulted in complete interactions of ecto-5'NT with extracel recovery of ecto-5'NT activity within 24 lular laminin and fibronectin, its anchor hours, suggesting that ecto-5'NT can age to plasma membranes via glycosyl persist in the plasma membrane as an phosphotidylinostitol, its role as a concanavalin A receptor, and the characteristic inactive apoenzyme . 1 0 4 Bises et al2 2 found a moderate increase, rather than a increase of ecto-5'NT activity that occurs decrease, of 5'NT activity in lympho during cell maturation, all prompt the cytes from zinc-depleted rats; they con author to speculate that ecto-5'NT is cluded that lymphocyte 5'NT activity important in contact inhibition of cell would not be a reliable test for zinc defi division. As monoclonal antibodies to ciency. On the other hand, Meftah and specific 5'NT molecules (e.g., ecto-5'NT Prasad* sampled peripheral blood lym of bile canalicular cells, ecto-5'NT of B phocytes from human volunteers every lymphocytes, pyrimidine 5'NT of eryth four to six weeks during a baseline rocytes) become available, immuno period (eight weeks, oral Zn intake 12 chemical assays will initially supplement mg per day), a zinc-restricted period (24 and ultimately replace enzymatic assays weeks, oral Zn intake three to five mg for 5'NT. Such developments will enhance the specificity, sensitivity, avail ability, and convenience of 5'NT deter * Personal communication, October 1989. minations for clinical diagnosis. 134 SUNDERMAN JR.

Acknowledgments 13. B e l f i e l d , A. and G o l d b e r g , D. M.: Applica tion of a continuous spectrophotometric assay This work was supported by a grant from North for 5'nucleotidase activity in normal subjects east Utilities. The author is grateful to Ananda S. and patients with bone disease. Clin. Chem. Prasad, M.D., Sidney M. Hopfer, Ph.D., and Eva 15:931-939, 1969. Horak, Ph.D. for helpful suggestions. 14. B e l f ie l d , A. and G o l d b e r g , D. M.: Activa tion of serum 5'nucleotidase by magnesium References ions and its diagnostic applications. J. Clin. Path. 22:144-151, 1969. 1. Ab d -E lfa tta h , A. S. and W e c h s l e r , A. S.: 15. Be l f ie l d , A. and G o l d b e r g , D. 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