Gel filtration of urine from Patient ED with acute myebocytic leukemia showed a prominent protein peak with elutionposition corresponding to molecular weights of 20,000 to35,000. The protein (EDC1) was isolated in pure form bysequential gel filtration and ion-exchange chromatography.Molecular weight of purified EDC1 was 27,000; it contained27% carbohydrate and was rich in half-cystine (5% of residues). EDC1 was antigenically and chemically distinct fromthe recognized glycoproteins of normal plasma. With a specific rabbit antiserum and ‘25I-IabeledEDC1, a radioimmunoassay for the glycoprotein was developed. Both noncancemand cancer plasmas contained immunoreactive matenab. In noncancer plasma, all the immunoreactivity waseluted from Sephadex G-75 and G-200 in position corresponding to molecular weights of 60,000 to 100,000 (Peak1). In cancer plasma, an additional peak of immunoreactivity was eluted in the position corresponding to EDC1 (MW.,20,000 to 30,000; Peak 2). Eighty-six % of urines from patients without clinical cancer were nonreactive in radioimmunoassay (<0.1 @gimmunoreactive EDC1 per ml); 11 and3%, respectively, contained immunoreactivity equivalent to0.1 to 0.9 and 1 to 9 @gEDC1 per ml, entirely of Peak 1 type.Ninety-one % of urines from patients with disseminatedcancer contained immunoreactivity equivalent to 10 to9,999 /.LgEDC1 per ml, primarilyof Peak 2 type.
INTRODUCTION
About 15% of patients with disseminated neoplastic disease, during the last 6 months of life, excrete in the urine 1or more proteins, with molecular weight ranges of 10,000 to50,000, which are distinct from the recognized proteins ofnormal plasma (25).
The present investigation focused on a glycoprotein excreted in the urine by Patient ED with acute myelocyticleukemia. Our objectives were to isolate the protein in pureform, to determine its chemical and physical properties, toproduce a specific antiserum, and with this antiserum tostudy the concentration of the protein in body fluids of avariety of patients.
I Supported by USPHS Grants CA12646 and RR00039.
Received November 5, 1975; accepted January 20, 1976.
Patient ED. This 48-year-old Caucasian salesman hadbeen well until 3 weeks before admission, when he began toexperience fatigue, dizziness, and anorexia. He was admitted to Emory University Hospital on July 18, 1974. Positivephysical findings were pallor, an enlarged submandibularlymph node, hepatomegaly, and splenomegaby. Abnormallaboratory findings were hemoglobin, 7.4 g!100 ml; hematocnit, 22%; WBC, 43,000/cu mm with 72% myeloblasts(containing occasional Auer rods); platelets, 84,000/cu mm.Urine protein was reported 1 @.Bone marrow was hypencellular with decreased megakaryocytes, 6% blasts, and 48%promyebocytes. Diagnosis of acute myebobbastic leukemiahaving been substantiated, on Day 12 chemotherapy wasbegun with cytosine arabinoside (225 mg every 12 hr for 10doses), 6-thioguanine (220 mg every 12 hnfor 10 doses), anddaunomycin (22.5 mg every 24 hr for 5 doses). This schedule was repeated at monthly intervals. Marked improvementin peripheral hemogram (WBC, 3,000 to 6,000; 2 to 6%myeboblasts) and bone marrow (bessthan 3% myeboblasts)occurred within 7 days and continued during the remaining3 months of life. During the 3rd month of observation, fever,cough, males,and bilateral interstitial pulmonary infiltratesdeveloped. He died during the 4th month; autopsy, limitedto the chest, showed extensive Pneumocystis Carinii infectioninbothlungs.
MATERIALS AND METHODS
Isolation and Characterization of Protein EDC1
During the 12 days before chemotherapy began, 8 liters ofurine were collected at 0°and stored at —20°.From thisfluid, Protein EDC1 was isolated and characterized withthesetechniques:(a)gelfiltration,in1 N aceticacidorin0.1M ammonium acetate, pH 7.0, On a 2- x 200-cm column of
Sephadex G-75,or a 1-x 100-cm column ofSephadex G200, both columns having been calibrated with purifiedproteins of known molecular weight (15, 25, 26); (b) cationexchange chromatography on a 2- x 30-cm column of camboxymethylcellubose 11 (28); (c) electrophoresis in 7.5%acrybamide gel at pH's 9.0 and 4.3 (7); (d) electnophoresis oncellulose acetate at pH's 9.0 and 4.0 (30); (e) double immunodiffusion at pH 7.2 (5); and (f) immunoelectrophonesis atpH 8.6 (6), against the following antisera (Hyband Division,Travenol Laboratories, Inc. , Costa Mesa, Calif. , and Behr
MAY 1976 1837
Isolation of a Novel Glycoprotein (EDCI) from the Urine of aPatient with Acute Myelocytic Leukemia1
Daniel Rudman, Rajender K. Chawla, Lee J. Hendrickson, W. Ralph Vogler, and Alkis J. Sophianopoulos
Departments of Medicine fD. A., R. C., L. J. H. , W. R. v.j and Biochemistry (A. J. 5.J, Emory University School of Medicine, and Clinical Research Facility,Emory University Hospital, Atlanta, Georgia 30322
7Malignant melanoma(metastatic)9 51 0 2132C8Carcinoma of stomach (metastatic)9 40 2 0241C9Carcinoma of pancreas(metastatic)12 53 0 1152C10Carcinoma of colon (metastatic)12 61 2 1251C11Carcinoma ofbreast(metastatic)14 52 2 1252C12Carcinoma of ovary(metastatic)9 31 1 1132C13Carcinoma of cervix uteri (metastatic)9 32 2 0131C14Carcinoma oflung(metastatic)12 42 2 1 142
D. Rudman et al.
ing Diagnostics, Somerville, N. J.): rabbit antisera to wholehuman serum and rabbit, horse, or goat antisera to 16purified human plasma proteins (a-Iipopnotein, transfenmin,a2-haptoglobin , fibninogen, albumin , -yG-immunoglobulin,/31C!/31A-globulin, a1-antitrypsin , a2-HS-glycopnotein , hemopexin , @32C-gIycopnotein, a1 acid glycopnotein , f3-Iipopnotein, Gc globulins, a2-macrogbobulin, and C-reactive protein); (g) estimation of molecular weight by sedimentationequilibrium (see “Appendix―);(h) analysis of amino acidcomposition (27); (i) determination of content of hexoses(23), hexosamines (31), and sialic acid (35).
Immunochemical Procedures
Antiserum to Protein EDCI isolated from urine of PatientED was prepared by injecting the protein into rabbits according to the method of Campbell et al. (4). This antiserumwas applied in double immunodiffusion to urine and plasmafrom normal volunteers (age 19 to 49) and adult patients(age 18 to 75) on medical and surgical services of EmoryUniversity Hospital. Subjects were arranged according todiagnosis in 32 groups (Table 1). Within each group, mdividuals were numbered consecutively; thus Patient NC 14/6is the 6th patient in Group NC 14, chronic pyelonephritis.Antiserum to EDC1 was also tested in double immunodiffu
sion against 9 purified human plasma proteins (Schwamz/Mann, Orangebumg, N. Y.): albumin, immunoglobulin G,fibminogen, transfennin , a@acid glycoprotein , ceruboplasmin,prealbumin , haptoglobin , and f32C-glycoprotein.
With rabbit antiserum to EDC1, a radioimmunoassay procedure was developed as follows.
Iodlnatlon of Antigen. Ten @gEDC1 in 10 @I0.5 Msodiumphosphate buffer, pH 7.0, were iodinated with 1 mCi Na'251(New England Nuclear, Boston, Mass.; 630 mCi/mb) at 25°for 50 sec in the presence of 10 @lchboramine-T solution(1.0 mg in 1.0 ml 0.05 M phosphate buffer, pH 7.0) according to the method of Hunter (13) as modified by Reichent(22). The reaction was quenched by adding 50 j.d sodiummetabisulfite solution (3.25 mg of 0.05 M sodium phosphatebuffer per ml). ‘25l-EDC1was separated from free iodine on aBio-Rad P-60 column (0.7 x 20 cm), which had been previously equilibrated with 2% ovalbumin in 0.05 M sodiumphosphate buffer, pH 7.0. The column was eluted with thephosphate buffer, and 1-mi fractions were collected intubes containing 1 ml 0.1% ovalbumin. Fraction 3 (Chart 1A)was used as the iodinated antigen in the radioimmunoassay. It was stored at —20°and was stable for up to 5 weeks.Specific activity was determined as described by Reichert(22).
Conditions for Radioimmunoassay. A double antibody
AntiserumDilutionChant 1. A, Bio-Rad P-60 gel chromatography of iodinated antigen. bodinated protein peak appeared at Fraction 3; see text under “Ma
terials and Methods― for experimental details. B, effect of dilution of antiserum on binding of ‘@9-protein.Rabbit antiserum was dilutedfrom 2,500-to 500,000-foldwith 2% normal rabbit serumand tested for binding of ‘251-protein,as described in “Materialsand Methods―under conditions for radioimmunoassay.
Fraction Numbr
radioimmunoassay was performed in 12- x 75-mm disposable polystyrene culture tubes, to which the following meagents were added in sequence: (a) 0.5 ml diluent (1%ovalbumin in 0.05 M phosphate-buffered saline, pH 7.0,containing 1% thimerosal as preservative); (b) 0.2 ml rabbitantiserum, diluted 1:2,500 in phosphate-buffered saline,containing 0.05 M EDTA; (C) 0.2 ml normal rabbit serum,1:2,500, added to a set of 3 tubes to determine nonspecificbinding; (d) 0.1 ml of a solution containing 1 to 1,000 ngEDC1, or 0.1 ml of the urine or plasma under assay in serialdilutions up to 1:1 000,000; (e) after the above mixture hadbeen incubated at 4°for 18 hm,0.1 ml of ‘25l-EDC1diluted tocontain 8,000 to 12,000 cpm. After further incubation for 48hr, 0.2 ml of goat anti-rabbit serum (Clinical Assays, Cambridge, Mass.) was added to each tube. Incubation wascontinued for an additional 48 hr at 4°.One-tenth ml of 1%starch solution was then added to the mixture, and thetubes were centrifuged for 40 mm at 5,000 rpm. Precipitateand supemnatant solutions were counted in an automatic ycounter.
RESULTS
sponding to molecular weights of 20,000 to 35,000 (Chart2A). About 20% was eluted at Kd 0 (Fraction EDA) comesponding to molecular weight of >70,000, and 20% waseluted at Kd 0.0 to 0.1 (Fraction EDB) with an apparentmolecular weight of 40,000 to 60,000. After chemotherapywas begun on Day 12, urine protein declined to 0.1 g/day.
To isolate Fraction EDC, 8 liters of urine excreted duringDays 1 and 12 were dialyzed in 300-mI batches overnight at5°versus H20 and lyophilized. The resulting powder wasdissolved in 50 ml 1 Nacetic acid and chromatognaphed in 1N acetic acid on a 2- x 200-cm column of Sephadex G-75;
the material corresponding to Fraction EDC was recoveredby lyophilization. This material was mepunified 3 times byrepeatedgelfiltrationin1 N aceticacidon a 2- x 200-cmcolumn of Sephadex G-75 until it emerged as single symmetrical peak labeled EDC', with Kd 0.1 to 0.2 (averageyield, 0.6 g!24 hr urine) (Chart 2B). EDC', 100 mg, wasfurther purified by chromatography on carboxymethylcellulose 11 (Chart 2C). The major fraction, EDC1, appeared at acumulative volume of 160 to 180 ml and was recovered insalt-free form by lyophilization followed by chromatographyon a 2- x 200-cm column of Sephadex G-75. EDC1, 396 mg,was obtained from 8 liters of urine.
Isolation of EDCI
During the 1st 12 days of observation, the patient excreted0.6to1.0g urinaryproteinperday.By electrophoretic analysis, composition was reported as 48% albumin,46% a-globulin, 4% j3-gbobulin, and 2% y-globulin. When 5ml urine were examined by gel filtration on Sephadex G-75in 1 N acetic acid, however, 60% of the protein appeared asa symmetrical peak (labeled EDC) with Kd20.1 to 0.2, corre
(V- V0)2 K,,@ where V@ is elution volume of the material under study, V0
is the void volume, and V is the volume of solvent imbibed by the gel.
Characterization of EDCI
The protein moved as a single component in acrylamidegel at pH 9.0 with anodal mobility 1.2 times that of albumin(Fig. 1A). At pH 4.3 in acnylamide gel it moved slowlythrough the stacking gel towards the anode; only 1 component was visible (Fig. 18). On cellulose acetate at pH 9.0,EDC1 moved anodally as 1 component with a mobility about0.7 times that of albumin (Fig. 1C); it migrated as 1 component cathodally on cellulose acetate at pH 4.0. In sedimentation-equilibnium, EDC1 behaved as a single protein with amolecular weight of 27,000 ±500 (details of this analysis
Table 2Amino acid and carbohydratecomposition of EDC1
Amino acid contents are expressed as residues/100 residues,excluding tryptophan (not measured).Valuesshow mean ±SE.for analysesof 4 preparationsof EDC1except for hexoses(averageof 2).
Competition for BindIng between ‘25l-EDCIand the Unlabeled Protein. A typical curve for percentage of inhibition ofbinding of iodinated EDC1 versus concentration of unlabeled protein is shown in Chart 3. The relation was linear inthe concentration mangeof 10 to 300 ng; 70 ng unlabeledantigen caused 50% inhibition in binding. The standarddeviation for each point in the linear portion averaged ±8%between assays and ±4%within assays. The calculation of Afactor according to the method of Odell and Daughaday (18)
D. Rudman et al.
C@ 1@@@/)f7/qf' 0 M@wt@to Kd@@Q00J @203040.5@360?11809 10 o @[email protected] @5t@cii os 0.910
Chart 2. Steps in the isolation of ProteinEDCI . A, gel filtration of 5 ml urine fromPatient ED on 2- x 200-cm column of Sephadex G-75 in 1 N acetic acid. B, chromatography of 100 mg preparation EDC on samecolumn; C, chromatography of 100 mgpreparation EDC' on 2- x 30-cm column ofcarboxymethylcellulose 11; ordinate , transmission of effluent at 280 nm.
@:4 EQC5/
Cumulative Volume of Effluent (ml)
are given in the “Appendix―).Amino acid and carbohydratecontents are given in Table 2. Notable are the presence of27% carbohydrate (12% hexoses, 10% hexosamines, 5%sialic acid) and 5 residues of half-cystine pen 100 residues ofamino acid.
EDC1 did not react in double immunodiffusion with rabbitantiserum to whole human serum on with antiserum to anyof the 16 purified normal human plasma proteins listed in“Materialsand Methods―for which specific antisera wereavailable. In addition, EDC1 did not react with antisera toBJC1 or BJC2, glycoproteins frequently present in leukemicurine (24). By specific radioimmunoassay, EDC1 did notcontain detectable carcinoembryonic antigen or a-fetopnotein.
Development of Immunoassays for EDCI
Periodic injection of EDC1 in Fneund's adjuvant in rabbitsraised an antiserum that produced a single precipitin linewith EDC1 in double immunodiffusion (Fig. 2). With thistechnique 50 @gon more of the glycoprotein per ml could bedetected. In immunoelectrophonesis versus EDC1, the antiserum produced 1 arc with a mobility about 0.6 times that ofalbumin (Fig. 1D). Anti-EDC1 did not react in double immunodiffusion with normal human plasma, with any of the 9available purified human plasma proteins listed under “Matenialsand Methods,―or with glycoproteins BJC1 or BJC2 (24).
EDC1, I'25-EDC1, and antiserum to EDC1 were now usedfor nadioimmunoassay of the glycoprotein.
Binding of ‘251-Protelnby Rabbit Antiserum. Chart lBshows the binding properties of rabbit antiserum towardsEDC1. Maximal binding was 50 to 60% and was observedwith up to 2500-fold dilution of the antiserum. In mostnadioimmunoassays, a 2500-fold diluted antiserum wasused. Normal rabbit serum (diluted 2500-fold) in a parallelassay showed less than 5% binding of ‘251-EDC1.
Chart 3. Relationships between concentration of Protein EDC1 (ng),plasma peak 1 (ng), plasma peak 2 (ng), urine (.cl), or plasma (@.cl)andpercentage of inhibition of binding of ‘25I-EDC1in radioimmunoassay.months
of life under continuing chemotherapy, peripheralWBC was 3,000 to 9,000 (6 to 9% myeboblasts). The daily
radioimmunoassay; curves for percentage of inhibition ver
urine now contained immunoreactivity equivalent to only0.7to9mgEDCl.
All plasmas tested contained material that reacted in the
sus concentration were parallel to the standard curve forEDC1 (Chart 3). The average values ±SE. of immunoreactive EDC1 in plasmas of normals, noncancem patients andcancer patients were 21 ±6 j@g/ml(n = 7), 26 ±7 @g!ml(n
8), and 41 ±10 j.@g!ml(n = 7).To examine the molecular weight of theimmunoreactivematerial
in plasma and urine, we fractionated 1-mbaliquotswasless than 0.1 to 0.2 for the linear portion of the curve,of these fluids from noncancer and cancer patientsonwhich
was used to assay the amount of EDC1 in urine andplasma.columns
ofSephadex G-75 and G-200 at pH 7.0 and assayedeach tube of eluate for madioimmunoreactivity. Theelutionposition
of EDC1 in both columns was alsodetermined.Application
of Immunoassays for EDC1 to Urines andPlasmas of the Clinical Population (Table 1).Representative
results are shown in Charts 4 and 5. Innormal and noncancer patients, immunomeactivematerialwas eluted as 1 fraction from Sephadex G-75 at Kd0(MW.Double
Immunodiffusion. Table 1 shows the incidence ofpositive reactions with random urines from the clinical population. Positive reactions (illustrated in Fig. 2) averaged>70,000),
and as 1 fraction from Sephadex G-200 at Kd 0.3to o.s (M.W. 70,000 to 150,000). This material was labeledPeak 1. Cancer plasmas contained a similar amount ofimmunoreactive material at these K,,values (Peak 1), butin55%
in patients with disseminated neoplastic disease (72%in leukemia, 71% in other hematobogical cancers, 41% innonhematobogical tumors) and 3% in subjects withoutknown neoplasm. No positive reactions were obtained withplasma.
Radioimmunoassay. In urine from 100% of normal subjects and from 86% of patients with nonneoplastic illnesses,immunoneactivity was not detectable in 100 @.tIurine. Thesesamples therefore contained <0.1 @gof EDC1 per ml. In14% of patients with nonneoplastic disease, immunoreactivity was detectable at this level. In such samples, curvesfor percentage of inhibition versus concentration were parallel to the standard curves for EDC1 (Chart 3); accordingly,urine content of “immunoreactive-EDC1―could be calcu
addition a 2nd type of reactive material (Peak 2) was presentwith Kd0.1 to 0.3 in Sephadex G-75 (MW. 20,000 to 50,000)and Kd 0.6 to 0.8 in Sephadex G-200 (M.W. 15,000 to35,000). The elution position of Peak 2 is similar to that ofEDC1 in both columns. Peaks 1 and 2 from chromatographyof cancer plasma on Sephadex G-75 were recovered bydialysis and lyophilization and their standard curves in madioimmunoassay examined (Chart 3). Both types of materialshowed parallelism with EDC1. In 10 plasmas analyzed inthis way on Sephadex G-75 (Table 3), the following resultswere found. In 5 noncancen plasmas, Peaks 1 and 2 contamed 21.2 ±4.8 (average ±S.E.) and <0.1 j.@gimmunoreactive EDC1 per ml, respectively; in plasmas of 5cancerbated
and rangedfrom 0.1to10 jig/mI(11% ofsubjectsinproportion
of immunoreactive umines was highest in parange 0.1 to 0.9 @tg!mband 3% in range 1 to 9 j.@g/mI).The
15). In the population with disseminated cancer, immunoneactivity was detectable in 100 @lurine from 90% ofcases; the curves for percentage of inhibition versus concentration being parallel to that for EDC1 (Chart 3), theimmunoreactivity in these uminescould be quantified as j.@g“immunoreactiveEDC1―per ml. In the cancer population,81% of urines contained >10 j@gEDC1 per ml and 64%contained >100 p.g per ml. The distribution of values within20
30@o
50
@ 70•@80
@ 9°@ lao
°@
@ 0@ 20:
Im no eact ?y Peak 1
Transmission
@ -@ .‘“,@ ‘ ‘ @—
- 0
Ui. 10
. ,
- Immunoreoctiv@ty Peak I
a smssion I0)1.1 a
@-—r-i-@ 0@((@@ , ,@,@, 1-‘‘@‘-
E
:the
range 0.1 to 9999 j.@gimmunomeactive EDC1 per ml urinewas generally similar within the 14 categories of neoplasticdisease (Table 1).
Aliquots of 24-hr urine collections from Patient ED that@°
607°80
00 T ¶ @1@@ 00 200300400500600700800had
been stored at —20°were now analyzed by madioimmunoassay. During Days 1 to 12, before chemotherapybegan,peripheral
WBC was 36,000 to 50,000 (60 to 80% myeloblasts). Patient ED's urine containedimmunoreactivityequivalent
to 200 to 600 mg EDC1 per day. Within 5daysafterchemotherapy was instituted and for the remaining 4
SEPHIADEX G-75
. Immunoreoctivity. Peak 2 - 0
- \A - 0'% Transmiss,on @,
: , T00 200 300 400 500 600 700 800
Cumulotjve Volume of Effluent (ml)
Chart 4. Gel filtration of urine and serum on Sephadex G-75. Above left, 1ml serum from Patient NC 13/6 (chronic glomerulonephritis); above right, 1ml urine from same patient; below left, 1 ml serum from Patient C 2/1(chronic myelocytic leukemia); below right, 1 ml urine from same patient;ordinate, transmission of effluent at 280 nm or content of immunoreactiveEDC1 in effluent (@.cg/ml).
patients, these values were 23.8 ±7.7 and 9.2 ±1.9, mespectiveby.
From 4 patients with chronic renal disease, urine samplescontaining 0.3 to 0.7 @gimmunoneactive-EDC1 pen ml werealso fractionated on Sephadex G-75. All the immunomeactivity was eluted in a position corresponding to plasma Peak 1(Table 3; Chart 4). From 5 patients with cancer, urine sampIes containing 39 to 410 @gimmunoneactiveEDC1 per ml
SEPHADEX G-200
Mu @,@//@2@'/ f/
were similarly analyzed. Over 90% of the immunomeactivityin each sample was in Peak 2 (Table 3; Chart 4).
One mg EDC1 was added to 1 ml normal urine or normalplasma. These samples were then analyzed by gel filtrationOn Sephadex G-75. The increment of immunoreactivity was
recovered in the position of Peak 2.
DISCUSSION
At least 15% of patients with disseminated cancer, includ
ing Patient ED, have low-molecular-weight proteinunia (25).Generally, the low-molecular-weight protein in the urine ofthese patients is a mixture of several components, but inPatient ED's case 1 protein (EDC1) predominated , so that itsisolation was easily accomplished.
EDC1 proved to be a glycopmotein with a molecular weightof 27,000. It is distinguished from the proteins normallypresent in plasma by its antigenic properties. In double
o immunodiffusion and immunoelectrophoresis, EDC1 does
@ not react with an antiserum to normal human plasma. In10@ addition EDC1 differs in amino acid composition, in carbo
hydrate composition, and generally in molecular weight)@ .@ from the recognized plasma gbycopmoteins [For those
@ plasma glycopmotemnsthat have been characterized (29),@ contents of 4 or more amino acids differ by more than 2 S.D.
E from the values of EDC1 shown in Table 3.] and from the.@ several low-molecular-weight proteins usually found in high-@ concentration in urine of patients with renal tubular dys-@, function [bysozyme, /32-microgbobulin, and metinol-binding
:@. protein (20, 21 , 34)]. Finally, EDC1 is antigenically distinct
from the well-characterized cancer-related antigens, carcinoembryonic antigen and a-fetoprotein. Since 1968, besides the latter 2 substances, at least 12 other cancerrelated antigens have been described (2, 8, 10-12, 16, 17,32, 33). Although specific antisera to each of these factorshave been developed, the antigens themselves have notbeen isolated in homogeneous form; their chemical andphysical properties therefore remain unknown. Consequently, we cannot say at present whether EDC1 come
Table 3
C0U,U,
EU,C0
0 0 20 30 40 50 60 70 80
Cumulative Volume of Effluent (ml)
Chart 5. Gel filtration of serum on Sephadex G-200. Above, 1 ml serumfrom Patient NC 13/6 (chronic glomerulonephritis); below, 1 ml serum fromPatient C 2/1 (chronic myelocytic leukemia).
EDC1 and by studying whether incubation of beukemic cellsin vitro with plasma Peak 1 from normal subjects will lead toformation of Peak 2 (presumably EDC1).
Whatever its mode of origin, Peak 2 material (presumablyEDC1) accumulated in the neoplastic plasmas studied to anaverage level of 9 pg/mI. A protein of this molecular weight(27,000) will have a glomenular filtration mate0.2 to 0.7 timesthat of creatinine (1, 3, 20, 21, 34). Contrastingly, Peak 1immunomeactive material, with a molecular weight of 70,000to 100,000 will have a filtration rate <0.001 times that ofcreatinine. These differences in renal handling probablyaccount for the 1,000 times greater renal clearance of Peak2 immunoreactivity than of the Peak 1 material (Table 3).
What clinical applications can be expected for immunoassays based on Protein EDC1? (a) The present studydoes not tell how early in the natural history of neoplasticdisease immunoreactive Peak 2 (presumably EDC1) is excreted in detectable amounts in the urine. If in some typesof tumor it appears in urine during the pmeclinical stage,then analysis of urine for EDC1 by double immunodiffusionor madioimmunoassay could be an inexpensive screeningtest for preclinical cancer. However, positive umineswouldneed further analysis by gel filtration to estimate the moleculamweight of the immunomeactive material. (b) Disappearance of immunomeactivity from the urine of a patient undertreatment for cancer (as Patient ED) may indicate that mostor all tumor cells have been destroyed or removed; reappeamance of immunoreactivity in urine may indicate thatcancer has recurred.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Lewis D. Johnson, Department of Pathology, Emory University School of Medicine, who kindly performed the carbohydrate analyses of EDC1.
REFERENCES
1 . Arturson, G., and Wallenius, G. The Renal Clearance of Dextran ofDifferent Molecular Sizes in Normal Humans. Scand. J. CIin. Lab. Invest., 1: 81-86, 1964.
2. Bhattacharya, M. , and Barlow, J. J. An Immunologic Comparison between Serous Cystadenocarcinoma of the Ovary and Other Human Gynecologic Tumors. Am. J. Obstet. Gynecol., 117: 849-853, 1973.
3. Brewer, D. B. Renal Clearances of Dextrans of Varying MolecularWeights. Proc. Roy. Soc. Med., 44: 561-563, 1951.
4. Campbell, D. H., Garbey, J. S., Cremer, D. E., and Susdorf, D. H.Preparation of Antiserums: Injection of Antigen-Adjuvant Emulsion. In:Methodsin Immunology, Section B-lb, p. 98. New York: W. A. Benjamin,Inc.,1963.
5. Campbell, D. H., Garvey, J. S., Cremer, D. E. , and Susdorf, D. H. GelDiffusion Test. In: Methods in Immunology, Section D-3, p. 143. W. A.Benjamin,Inc.,New York:1963.
6. Campbell, D. H., Garvey, J. S., Cremer, D. E. , and Susdort, D. H.Immunoelectrophoresis. In: Methods in Immunology, Section D-4, p.149. New York: W. A. Benjamin, Inc., 1963.
7. Davis, B. J. Disc Electrophoresis. II. Method and Application to HumanSerum Proteins. Ann. N. Y. Acad. Sci., 121: 404-420, 1964.
8. Edynak, E. M., Old, L. J., Vrana, M., and Lardis, M. P. A Fetal AntigenAssociated with Human Neoplasia. New EngI. J. Med., 286: 1178-1183,1972.
9. Eveleigh, J. W. Glycoproteins Excreted by 5V40-transformed Cells. In: N.G. Anderson, J. H. Coggin, Jr., E. Cole, and J. W. Holleman (eds.),Embryonic and Fetal Antigens in Cancer, Vol. 2. Proceedings of theSecond Conference, pp. 133-146. Oak Ridge, Tenn.: Oak Ridge NationalLaboratory, 1972.
10. Gold, P. Antigenic Reversion in Human Cancer. Ann. Rev. Med., 22: 85-94, 1971.
sponds to, or is rebated to, any of the 12 cancer-rebatedantigens mentioned above. The question can be investigated by determining whether specific antisera to any ofthese factors react with EDC1.
A glycoprotein labeled BJC1 (MW. 29,000) was isolatedin this laboratory in 1974 from the urine of a patient withchronic myebocytic leukemia (24). BJC1 does not crossreact with specific antiserum to EDC1 and vice versa. The 2glycoproteins differ in amino acid and carbohydrate compositions. BJC1 is detectable by double immunodiffusion inthe urine of 40% of patients with nonneoplastic inflammatory disease, and EDC1 is found in less than 5% of suchunines by similar technique. Thus the 2 glycoproteins, despite the similarity in molecular weight, appear to be antigenically, chemically, and physiologically unrelated.
Both noncancemand cancer plasmas contain material thatdisplaces ‘251-EDC1from rabbit antibodies to EDC1 in themadioimmunoassay. In noncancem plasmas, all the immunoreactivity is eluted in position corresponding to a molecularweight of 70,000 to 100,000 (Peak 1). In cancer plasma, anadditional peak of immunomeactivity is eluted as Peak 2 inthe position of EDC1 itself (M.W. 27,000). Little or none ofPeak 1 is excreted by the kidneys, while Peak 2 is clearedrapidly (Table 3). Consequently, 86% of noncancem urinecontains no immunoreactive material; 11% contains 0.1 to0.9 and 3% contains 1 to 9 jig of immunoreactive materialentirely of Peak 1 type per ml. In contrast 81% of cancerumines contain 10 @gof immunoreactive material per ml,primarily of Peak 2 type. Double immunodiffusion was genemallypositive only with cancer unines that contained >501.Lg immunoreactive material per ml by radioimmunoassay.
Peak 2 material of cancer plasma and urine is indistinguishable from EDC1 in elution position on gel filtration andin radioimmunoassay and is probably closely related oridentical to EDC1. The immunomeactive material in Peak 1could include EDC1 covabently linked within its structureand could thus represent a metabolic precursor of EDC1; orit could be metabolically and physiologically distinct fromEDC1 but immunologically cross-reactive with the latterglycoprotemn because of a shared antigenic determinant,e.g., an identical oligosacchamide subunit. It is unlikely thatplasma Peak 1 represents a noncovabently bound, transportform of EDC1 [a complex that would be analogous to thatbetween prealbumin and retinob-binding protein (14, 19)].After ‘251-EDC1was added to either noncancer or cancerplasma, it was still eluted during gel filtration in positioncorresponding to M.W. 20,000 to 35,000 (Peak 2).
How did urinary EDC1 originate in the body of PatientED? When remission was produced by chemotherapy andleukemic cells disappeared from the blood, EDC1 virtuallydisappeared from his urine. This suggests a mole of theleukemic cell in the production ofthis low-molecular-weightglycoprotein . EDC1 could be a component of the glycocalyxof the leukemic (and other types of) neoplastic cell that isreleased into the extracellular fluid (9), or it could be aproduct of the action of leukemic cells on a circulatingprecursor molecule such as the M.W. 60,000 to 100,000immunomeactive plasma component (Peak 1). These possibilities can be investigated by analyzing appropriate extracts of leukemic and other neoplastic cells for content of
11 . Hakkinen, I. P. T. An Immunochemical Method for Detecting Carcinomatous Secretion from Human Gastric Juice. Scand. J. Gastroenterol., 1:28-32, 1966.
12. Halterman, A. H., Leventhal, B. G., and Mann, D. L. An Acute-LeukemiaAntigen: Correlation with Clinical Status. New EngI. J. Med., 287: 1272-1274, 1972.
13. Hunter, W. M. In: D. M. Weir (ed), Handbook of Experimental Immunology, Ed. 2, pp. 17.1-17.36. Oxford, England: Blackwell Scientific Publication, 1973.
14. Kanai, M., Raz, A., and Goodman, D. S. Retinol-binding Protein: TheTransport Protein for Vitamin A in Human Plasma. J. Clin. Invest., 47:2025-2044, 1968.
15. Leach, A. A., and O'Shea, P. C. The Determination of Protein MolecularWeights of up to 225,000 by Gel-Filtration on a Single Column of Sephadcx G-200 at 25°and 40°.J. Chromatog., 17: 245-251, 1965.
16. McNeil, C.. Ladle, J. N., Helmick, YJ.M., Trentelman, E., and Wentz, M.W. An Antiserum to Ovarian Mucinous Cyst Fluid with Colon CancerSpecificity. Cancer Res., 29: 1535-1540, 1969.
17. Metzgar, R. S., Mohanakumar, T., and Miller, D. S. Antigens Specific forHuman Lymphocytic and Myeloid Leukemia Cells: Detection by Nonhuman Primate Antiserums. Science, 178: 986-988, 1972.
18. OdelI, W., and Daughaday, ‘N.H. (ads.), Binding Assays, p. 158. Philadelphia: J. B. Lippincott Co., 1971.
19. Peterson, P. A. Demonstration in Serum of Two Physiological Forms ofthe HumanRetinolBindingProtein.EuropeanJ. Clin. Invest.,1: 437-444, 1971.
20. Peterson, P. A., Evrin, P-E., and Berggard, I. Differentiation of Glomerular, Tubular, and Normal Proteinuria: Determinations of Urinary Excretion of @2-MicrogIobulin,Albumin, and Total Protein. J. Clin. Invest., 48:1189-1198, 1969.
21 . Ravnskov, U. On Renal Handling of Plasma Proteins with Special Reference to a2-Microglobulln, /32-Microglobulin, Lysozyme and Albumin.Scand.J. Urol.Nephrol.,Suppl.20,2-27,1973.
22. Reichert, L. E. Methods in Receptor Research. In: M. Glecker (ed.)Methods in Molecular Biology Series. New York: Marcel Dekker, Inc., inpress.
23. Reinhold, V. N., Dunne, F. T., Wriston, J. C., Schwarz, M., Sarda, L., and
Hirs, C. H. W. The Isolation of Porcine Ribonuclease, a Glycoprotein,from Pancreatic Juice. J. Biol. Chem., 243: 6482-6494, 1968.
24. Rudman, D., Chawla, R. K., Del Rio, A. E., and Hollins, B. Isolationof aNovel Glycoprotein from the Urine of a Patient with Chronic MyelocyticLeukemia. J. Clin. Invest., 53: 868—874,1974.
25. Rudman, D., Del Rio, A. E., Akgun, S., and Frumin, E. Novel Proteinsand Peptides in the Urine of Patients with Advanced Neoplastic Disease.Am. J.Med.,46:174-187,1969.
26. Rudman, D., Del Rio, A. E., Garcia, L. A., Barnett, J., Bixler, T. J., II, andHollins, B. Lipolytic Substances in Bovine Thyroid, Parotid and PinealGlands. Endocrinology, 87: 27-37, 1970.
27. Rudman, D., Del Rio, A. E., Garcia, L. A., Barnett, J., and Howard, C. H.Isolation of Two Lipolytic Pituitary Peptides. Biochemistry, 9: 99-108,1970.
28. Rudman, D., Del Rio, A. E., Hollins, B., Houser, D. H., Keeling, M. E.,Sutin, J., Scott, J. W., Sears, R. A., and Rosenberg, M. Z. MelanotropicLipolytic Peptides in Various Regions of Bovine, Simian and HumanBrains and in Simian and Human Cerebrospinal Fluids. Endocrinology,92: 372-379, 1973.
29. Schultze, H. E., and Heremans, J. F. Molecular Biology of Human Proteins, Vol. 1, p. 173-235. New York: Elsevier Publishing Co., 1966.
30. Smith, I. Chromatographic and Electrophoretic Techniques, Vol. 2, pp.56—90.New York: Interscience Publishers, Inc. , 1960.
31. Spinco Division of Beckman Instruments, Inc. How to Make a Physiological Fluid Analysis with the Model 120C Amino Acid Analyzer. BeckmanProcedures Manual, Bulletin A-TB-034, Palo Alto, Calif., 1966.
32. Stolbach, L. L., Krant, M. J., and Fishman, W. H. Ectopic Production ofan Alkaline Phosphatase lsoenzyme in Patients with Cancer. New EngI.J. Med., 281: 757-762, 1969.
33. von Kleist, S., and Burtin, P. Isolation of a Fetal Antigen from HumanColonic Tumors. Cancer Res., 29: 1961-1964, 1969.
34. Waldmann, T. A., Strober, W., and Mogielnicki, R. P. The Renal Handling of Low Molecular Weight Proteins. II. Disorders of Serum ProteinCatabolism in Patients with Tubular Proteinuria, the Nephrotic Syndrome, or Uremia. J. Clin. Invest., 51: 2162—2174,1972.
35. Warren, L. The Thiobarbituric Acid Assay of Sialic Acids. J. Biol. Chem.,234: 1971-1975, 1959.
Fig. 1. Electrophoretograms of Protein EDC1 (1 mg/mI), purified humanserum albumin (HSA), or normal human serum. A, acrylamide gel, pH 9.0,anode below; B, acrylamide gel, pH 4.3, anode below; C, cellulose acetate,pH 9.0, anode above. Line indicates origin; D, immunoelectrophoresis,pH 8.6, anode above.
Fig. 2. Double immunodiffusion analyses of urine. Center wells contamed rabbit antiserum to Protein EDC1. Peripheral wells contained EDC1(1 mg/mI) or urine from various patients.
APPENDIX: Determination of Molecular Weight of EDC1 by Sedimentation Equilibrium
U.
0C
r@-a2Appendix Chart 1. Plot of In C against r' —a'. Inset, relations between M@
or M@and protein concentration (g/100 ml) along different radial points incell.
and Mzr increase, indicating a small amount of high-moleculam-weight contaminant.
By integrating Equation A between the meniscus andsome point r, it can be shown that C, the protein concentration, depends on r in an exponential fashion, i.e.,
@ —vp)(r2—a2)Ca 2RT
where Cr and Ca refer to the concentration of protein atsome point r on the motorand concentration at meniscus, a,respectively.The slopeof the graph of InC@ VS.r2—a2(Chart 1) can be used to calculate moleculamweight (4). Thegraph is a straight line until high protein concentrations arereached. The molecular weight, as calculated from theslope, is 27,000 ±500.
References to Appendix
1. Cohn, E. J., and Edsall, J. T. Proteins, Amino Acids and Peptides as(B@ DipolarIons,p.370.NewYork:ReinholdPublishingCorp.,1943.
2. Sophianopoulos, A. J., Rhodes, C. K., Holcomb, D. N., and Van Holde, K.E. Physical Studies of Lysozyme. I Characterization. J. Biol. Chem., 237:1107-1112, 1962.
3. Sophianopoulos, A. J. , and Van Holde, K. E. Physical Studies of Muraminidase (Lysozyme). II. pH-dependent Dimerization. J. Biol. Chem., 239:2516-2524, 1964.
4, Van Holde, K. E. Physical Biochemistry, p. 110-113. Englewood Cliffs, N.J.: Prentice Hall, Inc., 1971.
1846 CANCER RESEARCH VOL. 36
Methods
Details for calibration of the Spinco Model E analyticalultracentnifuge and for sedimentation equilibrium havebeen given elsewhere (2, 3). The solution column lengthwas approximately 0.53 cm and at the speed used, 16,000rpm, the ratio of the protein concentration at the bottom ofthe cell (C,J to the concentration at the meniscus (GO)was75. Thus, the concentrations at the meniscus and bottom,respectively, were approximately 0.01 and 0.75 g per 100ml. The temperature was 20°and schlieren optics were usedto facilitate the calculation of both M6.,.and Mzr, the appament statistical molecular weights M@and M@at the variousradial points in the cell. The solvent was 0.1 M potassiumchloride.
Results
The values of Mu.rwere calculated using the fundamentalEquation (4)
@ = Mu.rrA (A)
where C is protein concentration in g/dl, r is distance fromthe center of notation in cm, dC/dr is concentration gradientof protein, A is 1 —(ii po2/RT).@ is partial specific volumeof protein, w is angular velocity of rotor, p is density ofsolution, A is gas constant, and T is absolute temperature.The partial specific volume was calculated from the aminoacid and carbohydrate content by the procedure of Cohnand Edsall (1) and was found to be 0.6875 liter/mole. Theapparent Mzr was calculated by substituting Equation A inthe following
Me,.@ (CMwr)
Chart 1 (inset) shows the values Mr,. and Me,.as functionsof protein concentration, and it is seen that the values ofMw,,and Mzr are nearly equal, indicating that the system ismonodisperse. At higher protein concentrations, both Ma.,.
1976;36:1837-1846. Cancer Res Daniel Rudman, Rajender K. Chawla, Lee J. Hendrickson, et al. Patient with Acute Myelocytic LeukemiaIsolation of a Novel Glycoprotein (EDC1) from the Urine of a
