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Preparation of Carcinoembryonic Antigen (CEA) Containing Significantly Increased Amounts of Galactose and Galactosamine a
a
a
J. I. Joung , H. Rochman , B. Levin & J. A. Cifonelli
a
a
Department of Pathology , University of Chicago , Chicago, Illinois, 60637 Published online: 06 Dec 2006.
To cite this article: J. I. Joung , H. Rochman , B. Levin & J. A. Cifonelli (1975) Preparation of Carcinoembryonic Antigen (CEA) Containing Significantly Increased Amounts of Galactose and Galactosamine, Preparative Biochemistry, 5:4, 359-374, DOI: 10.1080/00327487508061583 To link to this article: http://dx.doi.org/10.1080/00327487508061583
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and
Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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PREPARATIVE BIOCHEMISTRY, 5 ( 4 ) , 359-374 (1975)
PREPARATION OF CARCINOEMBRYONIC ANTIGEN (CEA) CONTAINING SIGNIFICANTLY INCREASED AMOUNTS
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O F GALACTOSE AND GALACTOSAMINE
J. I. Joung. H. Rochman, B. Levin and J. A. Cifonelli Department of Pathology, University of Chicago, Chicago, Illinois 60637
Abstract CEA was prepared by combined isoelectric precipitation, ultrafiltration and column chromatography under controlled conditions of pH. The resulting immunologically active materials were higher i n carbohydrate (85-87%), N-acetyl galactosamine (10-11.570'0)and galactose (28-32700) content than that previously reported.
Differences i n amino
acid yield were also noted; the concentrations of aspartate, serine, glycine and alanine being higher and that of lysine, histidine, arginine, proline, valine, isoleucine, leucine and tyrosine were lower than that reported for CEA prepared by previous methods.
The tumor tissues
for CEA extraction were obtained from two Group 0 Rh positive deceased.
Neither preparation showed Group A or B activity a s
measured by hemagglutination inhibition.
It is suggested that the method
of purification influences the carbohydrate and amino acid yields. 359 Copyright 0 1975 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
360
JOUNG ET AL.
Introduction Onco-fetal antigens a r e widely employed i n experimental studies. More recently, their usefulness i n a clinical context has been explored.
A major difficulty for the investigator is i n obtaining sufficient suitably purified material
1
.
CEA is present in low concentrations
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(< 10 mg/kg) in secondaries to the l i v e r originating from a carcinoma
of the colon.
These secondaries a r e commonly the source for obtaining
CEA. The isolation and purification procedure normally adopted is that described by Krupey g
2 g., or a
modification.
Their method involves a
preliminary extraction with perchloric acid of glycoprotein-rich material from malignant tissue and the product is progressively purified using column chromatography.
I n the present studies, CEA was found to be stable over a wide range of pH and therefore the method of purification described below incorporates isoelectric precipitation a t various hydrogen ion concentrations.
Further, employing a modified hollow fiber ultrafiltration
system the removal of compounds less than 5 0 , 0 0 0 m. w. and concentration of large preparatory volumes could be achieved.
These procedures
allowed for a considerable reduction in purification time and the resulting material contained CEA of high specific activity.
Analysis of CEA
prepared by this method showed it to contain galactose and N-acetyl galactosamine i n concentrations higher than that previously reported and the significance of these findings a r e discussed.
CEA CONTAINING GALACTOSE AND GALACTOSAMINE
361
Materials and Methods Preparation of CEA. ing to the method of Krupey
CEA from Tumor III was prepared accord2
&.
The method for preparing CEA from tumors Hunt and T65 was
that a s described below.
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Following autopsy, liver metastases from a primary cancer of
the colon were dissected free from normal liver tissue and stored a t -2OOC.
F o r isolation and purification of CEA the tissue was thawed and
sliced. The sliced tissue (2 kg wet weight) was made up to a volume of 0
4 liters with deionized water and a homogenate prepared (at 4 C) using
a Waring blender.
Perchloric acid extraction, with an equal volume of 0
1.5 M HC104, was then carried out over a 20 min. period at 4 C. The mixture was then centrifuged and the precipitate discarded.
Immediately
thereafter NaOH was added to adjust the hydrogen ion concentration of the resulting solution to pH 4.
An aliquot was removed and the pH a t
which a maximum precipitate formed was determined; this varied from pH 4 to 10 i n different experiments.
The pH of the extract was then
adjusted accordingly and the precipitate discarded.
The c l e a r solution
was introduced into an Amicon (Amicon Corporation, Massachusetts, Model CDS- 10) hollow fiber system and concentrated ten-fold at a pressure of 1 Kg/cm2; by this procedure compounds of less than
50,000 m. w. were excluded from the concentrate.
Assay of the con-
centrated solution showed only a minimal loss (< 570)in CEA activity. The concentrated solution was then dialyzed against deionized water
JOUNG ET AL.
362
(3 days at 4OC), the precipitate discarded and the supernatant lyophilized.
The lyophilized material (4 g) was dissolved in 45 ml of 0.05 M phosphatebuffered saline, pH 7.2. A t this pH a precipitate frequently formed which was discarded.
The supernatant was subjected to pressure filtra-
tion employing an Amicon (Amicon TCF 10) thin channel ultrafiltration
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system (pore size, 0.2
p;
2 pressure, 3 Kg/cm ) and the filtrate
(130-150 ml) applied to a Sepharose 4B column (10x 100 cm) equilibrated
with 0.05 M phosphate-buffered saline pH 4. 5, at 4OC. Ascending chromatography was then carried out and 20 ml fractions were collected using a fraction collector. Eluates were examined for CEA activity by radioimmunoassay and the protein content monitored at 280 mp. Fractions containing high CEA activity were pooled. Fractions with low CEA activity or a high protein content were combined for rechromatography. The CEA-rich fraction was ultrafiltered and concentrated (x 10) using an Amicon thin channel ultrafiltration system (XM50, pressure 2 3 Kg/cm 1, dialyzed at 4OC for 3 days, and then lyophilized.
The
lyophilized material (250 mg) was dissolved in 10 ml of phosphatebuffered saline (pH 4. 5), and subjected to gel filtration by application to a Sephadex G-200 column (5 x 100 cm). The conditions for chromatography were as described above. Eluates were assayed for CEA activity an4 protein concentration, and fractions containing high CEA activity were pooled, dialyzed and lyophilized.
The lyophilized material (20-30 mg
was dissolved in 2 ml phosphate-buffered saline (pH 7. 5). centrifuged and any precipitate discarded. This material was rechromatographed (2-3 times) until no further improvement in separation could be achieved.
363
CEA CONTAINING GALACTOSE AND GALACTOSAMINE
Measurement of CEA? The procedure for measuring CEA was similar to the double antibody radioimmune assay described by Laurence
--
5
4
et al., which is a modification of the triple isotope methods of Egan z t &
CEA and its antisera of defined immunological activity were used a s reference materials.
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Hansen
These preparations were gifts f r o m Todd
&.3
and from
.
3
Chemical analysis of CEA. Neutral sugar components were determined with a Technicon Carbohydrate Analyzer using a borate column. Hydrolysates were prepared by heating 0.2 1 M HC1 for 3 hours at 100°C.
- 0. 3 mg of sample in 1 rnl of
A f t e r addition of 10 ml of H20 to the
hydrolysate, rhamnose (50 pg) was added a s an internal standard. Deacidification was accomplished by adding Dowex 3 (carbonate) in portions until a pH of approximately 2.0 was attained.
The solution was then
passed over a Dowex 50 (H+) column (1.0 x 30 c m ) to remove hexosamines and peptides and 10 ml of methanol was added to the effluent before 0
concentrating t o a small volume a t 30 C in wcuo.
This latter
procedure was repeated several times after fresh additions of methanol and the solution finally taken to dryness.
The residue was dissolved
in 1.0 ml of H 2 0 and 0 . 4 ml was used f o r sugar analysis. Hexosamines were estimated by the Elson-Morgan procedure
or during amino acid autoanalysis a s reported elsewhere 6
.
Results Monitoring of eluates from chromatography columns showed the presence of a single peak of CEA activity.
The partition coefficients
364
JOUNG ET At.
found with Sepharose 4B and Sephadex G-200 were 0.65 0. 08
- 0.74 and
- 0. 15 respectively. There are no accepted criteria for determining the presence of
.
CEA except by immunological comparison with described preparations of other investigators. CEA prepared from tumors Hunt, T65 and III
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showed immunological identity with such known CEA preparations.
When
examined by double immunodiffusion in agar gel (Figs. 1 81 2 ) a single line of complete identity with known CEA preparations was obtained. Also, the standard inhibition curves obtained by radioimmunoassay for the prepared CEA over the range of 0
-
100 ng per ml w e r e similar in
all respects to that of known CEA (Fig. 3). Compared to a known standard the relative immunological activities of the prepared materials a r e listed in Table I .
However, these figures of activities a r e probably
under-estimates since no corrections were made for the water content of preparations T65. Hunt and III; one lyophilized sample when subjected to differential thermal balance analysis revealed a moisture content of 28%. Analysis of the CEA preparations for amino acids a r e given in Table 11. The amino acids present in highest concentration for all preparations were aspartic and glutamic acids, threonine and serine. The yields of individual amino acids present in CEA materials prepared by the above described method were similar (compare CEA from tumors Hunt and T65). However, when compared with those prepared by previously described methods (see Table II) striking differences were noted; i n particular, aspartate, serine, glycine and alanine were present
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CEA CONTAINING GALACTOSE AND GAJACTOSAMINE
365
FIGURE 1 Immunodiffusion in an agar gel buffered with 0. 1 M phosphate-saline (containing EDTA, 0. 4 m g l m l ) , pH 7. 2. The c e n t e r well contained Hansen anti-CEA s e r u m (10 pl) undiluted. The contents of the peripheral wells w e r e a s follows: a. Todd CEA, 5 pg ( d r y weight); b. T65 CEA, 20pg; c. I11 CEA, 8 p g ; and d. Hunt CEA, 5 p g .
in higher concentration in CEA prepared by the present procedure whereas lysine, histidine, arginine, proline, valine, isoleucine, leucine and tyrosine w e r e lower.
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366
JOUNG ET AL.
FIGURE 2 Immunodiffusion in an agar gel buffered with 0. 1 M phosphate-saline (containing EDTA, 0 . 4 mg/ml), pH 7. 2. The center well contains Todd anti-CEA serum 5 pl (1:2 dilution). The contents of the peripheral wells were as follows: A. Hansen CEA, 2 pg (dry weight); B. Todd CEA, 2 pg; C. London CEA (a CEA preparation from hepatic metastases of a carcinoma of the colon), 2 pg: D. Hunt CEA, 2 pg; and E. I11 CEA, 2 pg.
Sugar and amino sugar analyses of CEA from tumors Hunt and T65 a r e shown in Table KII a s a r e the values for CEA preparations and blood Group A substance reported by others.
The higher carbohydrate
(85-870/0), N-acetyl galactosamine (10-110/0) and galactose (28-32%)
367
CEA CONTAINING GALACTOSE AND GALACTOSAMINE
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80
60
40
20
0
I
1
I
FIGURE 3 Standard inhibition curve of the CEA radioimmune system. The percentage of free (unbound) radioactive labelled "9 CEA (ordinate) plotted against the ratios of unlabelled standards to their respective radioactive labelled 125 I CEA (abscissa). Anti-CEA serum (Ace 17 from Todd 3, in a final dilution of l:lZ, 800 was allowed to react with Hansen CEA. A ; Todd CEA, 0; and Hunt CEA, U
.
content of CEA from tumors Hunt and T65 distinguish them from all other CEA preparations listed i n Table 111. In contrast, the carbohydrate content of CEA material from Tumor III was within the range of that reported for CEA prepared by similar procedures.
368
JOUNG ET AL.
TABLE I Immunological Activity (measured by radioimmunoassay) of Various CEA Preparations CEA Preparation
Activity p e r unit d r y weight
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Todd
100
III
45
Hunt
67
T65
20
Discussion
The purified CEA demonstrated complete immunological identity with known CEA preparations (Figs. 1 and 2).
The amino terminal amino
acid sequencing of the first twelve residues of Hunt CEA agreed with previously reported results. l 3 Also, the partition coefficients of CEA during purification of Sephadex and Sepharose columns were similar ‘to those calculated from the data of other studies.
”
These findings
indicate that the present preparations contained CEA similar to that of other investigators. In preliminary experiments, it was found that the CEA materials decreased in immunological activity following storage for a few days under marked acidic (pH 1 2 ) conditions.
Previous
methods used for purifying CEA involve longer periods of contact with perchloric acid so that the preparatory materials become exposed to a low pH and an oxidative environment.
It is known that prolonged periods
CEA CONTAINING GALACTOSE AND CALACTOSAMINE
369
T AB LE IT A m i n o Acid An al y s i s ( m o l e s
70)of Var i o us CEA P r e p a r a t i o n s
CEA P r e p a r a t i o n s
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Amino Acid
CEA
CEA
Banjo is"% "
C EA Turberville
111
-
Hunt
-
T65
Lysine
2.8
2. 5
3. 4
3. 5
0. 9
0. 6
Histidine
2. 1
1. 3
2. 2
2.0
0. 7
1.0
3. 8
3.2
1. 8
0. 6
11. 8
20. 1
22. 6
Arginine Aspartic acid T h r eonine
3. 8
3. 4
14. 0
15. 9
16. 6
8. 1
9. 3
10. 6
12.3
11.2
10. 7
Serine
10. 0
11.4
11. 7
12. 5
21. 1
19. 0
Glutamic a c i d
10. 4
11. 0
9. 9
10.4
11. 6
10. 3
Proline
7. 7
8. 0
8. 0
9. 1
4. 3
Glycine
5. 6
5. 5
5. 7
7. 1
13. 2
14. 9
Alanine
6. 2
5. 9
6. 6
6. 7
8. 5
8. 7
Valine
ND
5. 5
3. 4
2. 8
3. 1
7. 5
6. 8
Methionine
0. 2
ND
0
ND
ND
ND
Isoleucine
4.9
5. 1
5. 6
3. 6
1. 6
1. 6
Leucine
9. 3
9. 3
8. 9
7. 2
1. 7
1. 4
T y r o sine
5. 4
2. 5
4. 0
2. 5
0
0
Phenylalanine
ND
2. 1
2. 5
2.5
0
2. 6
Cysteine
ND
ND
0. 1
ND
ND
ND
Cystine
1. 9
ND: Not d e t e r m i n e d. 111, Hunt and T65: S e e M a t e r i a l s and Methods.
370
JOUNG ET AL.
TABLE III Sugar and Amino Sugars in Various CEA Preparations and Human Blood Group A
12 AHC
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23.1
33.2
20. 2 21.9 1.6
82.2
16.8
III, Hunt and T65: See Materials and Methods. of acid treatment results in hydrolysis of most glycoproteins.
Further-
more, perchloric acid at the concentration used readily oxidizes biological materials. 22 To minimize these effects in the present experiments, conditions were chosen so that the pH was more rigidly controlled and the treatment with perchloric acid was of brief duration. Analyses of CEA materials for amino acids and carbohydrate suggest
371
CEA CONTAINING GALACTOSE AND GALACTOSAMINE
that the method of purification probably w a s a major factor contributing to the compositional differences found when comparing the various CEA preparations.
Amino acid analysis (Table 11) showed that the concentra-
tions of aspartate, serine, glycine and alanine were higher, and that of lysine, histidine, arginine, proline, valine, isoleucine, leucine and
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tyrosine were lower for CEA from tumors Hunt and T65 than that of CEA prepared by previous methods. hydrate analysis was performed.
Similar results were noted when carboThe yields of galactose, N-acetyl
galactosamine and total carbohydrate (Table III) were much higher for materials prepared by the present procedure.
These findings suggest
that prolonged perchlorate treatment may have contributed to the low yields of carbohydrate found by others studying CEA. 1 , 8 , 9 The levels of galactose, N-acetyl glucosamine and to a l e s s e r extent N-acetyl galactosamine found in the present preparations a r e similar to what could be expected for blood group A substance (see Table 111). However, the striking difference in mannose content between CEA and that of blood group A substance distinguishes them at the bio-
chemical level a t least i n this respect.
Despite the similarities of CEA
and blood group A substance, no blood group activity (A o r B) could be detected by hemagglutination inhibition for either of the CEA preparations (Hunt or T65) up to a concentration of 1 mg/ml.
These CEA materials
studied were isolated from Group 0 Rh positive deceased.
These
findings make it difficult to support the concept that C EA is a modified blood group substance as proposed by Simmons and Perlman.
12
372
JOUNG ET AL.
Banjo
2.8 and Gold and Gold"
i n early studies found weak
Group A activity in their CEA preparations but were unable to explain such activity since their preparations showed low levels of N-acetyl galactosamineal- a necessary amino sugar for the reaction of Group A with Anti-A. "' l8 Holburn
2?tt9 reported the blood group activity
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of their CEA preparations to be similar to the blood group types of their patients.
In the present experiments no blood group activity was
detected in either CEA preparation but these were isolated from Group 0 Rh positive deceased.
Denk
2 : '
found the respective blood group
activity on colonic cancer cells and their hepatic metastases of Group A and B patients, but the results with Group 0 patients were inconsistent. These findings suggest that CEA exhibiting blood group activity is only found when isolated from Group A o r Group B materials.
It would
therefore seem that the blood group activity which has been observed to be associated with CEA is caused by contamination. In view of the similar physicochemical properties among many glycoproteins and considering the inherent limitations of the preparation process, the selection of suitable raw materials for isolation may be as important as the extraction procedures employed.
Blood group
activity is only an example demonstrating the heterogenity among variously prepared CEA materials.
CEA CONTAINING GALACTOSE AN0 GALACTOSAMINE
373
References W. D. Terry, P. A. Henkart, J. E. Coligan and C . W. Todd,
Transplant Rev.,
E, 100-129 (1974).
J. Krupey, T. Wilson, S. 0. Freedman and P. Gold, Immunochem-
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istry,
9,
617-622 (1972).
Todd CEA and antisera were gifts from C. W. Todd, Department of Immunology, City of Hope National Medical Center, Duarte, California 91010. Hansen CEA and antisera were gifts from H. J. Hansen, Department of Biochemical Nutrition, HoffmanLaRoche, Inc., Nutley, New Jersey 07110.
4.
D. J. R. Laurence, U. Stevens, €2. Bettelheim, D. Darcy, C. Leese, C. Turberville, P. Alexander, E. W. Johns and A. M.
Neville, Brit. Med. J . ,
5.
M. L. Egan, J. T. Lautenschleger, J. E. Coligan and C. W. Todd, Immunochemistry,
6.
289-299 ( 1972 ).
3, 2817-2820 (1965).
J. E. Coligan, P. A. Henkart, C. W. Todd and W. D. Terry, Immunochemistry,
8.
2,
V. Lindahl, J. A. Cifonelli, B. Lindahl and L. Roden, J. Biol. Chem.,
7.
3, 605-609 (1972).
g, 591-599 (19731.
C. Banjo, P. Gold, S. 0. Freedman and J. Krupey, Nature,
238.
183-185 (1972). 9.
C. Turberville, D. A. Darcy, D. J. R. Laurence, E. C. Johns and A. M. Neville. Immunochemistry,
lo, 841-843 (1973).
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374 10.
H. Z. Kupchik, N. Z a m c h e k a n d C . A. SaravLs, J. Nat. Cancer Inst.,
11.
g,1741-1749
(1973).
C. Banjo, J. Shuster and P. Gold, Cancer Res., 34, 2114-2121 (1974).
12.
D. A. R. Simmons and P. Perlmann, Cancer Res., 33, 313-322
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(1973). 13.
J. I. Joung, R. Hsu, H. Rochman and 6. A. Cifonelli, (Abst. ),
34, 3542 (1975).
Fed. Proc., 14.
J. P. Mach and G. Pusztaszeri, Immunochemistry,
2,
1031- 1034
(1972). 15.
H. Z. Kupchik and N. Zamchek, Gastroenterology, 63, 95-101 (1972).
16.
J. M. Gold and P. Gold, Cancer Res.,
17.
I. A. F. L. Cheese and W. T. J. Morgan, Nature, 191, 149-150
33,
2821-2824 (1973).
(1961). 18.
T. Feizi, E. A. Kabat, G. Vicari, B. Anderson and L. Marsh, J. Immunol.,
19.
106,1578-1592
(1971).
A. M. Holburn, J. P. Mach, D. MacDonald and M. Newlands,
Immunology,
26,
831-843 (1974).
20. H. Denk, G. Tappeiner and J. H. Holzner, Europ. J. Cancer,
10, 487-490
(1974).
21. J. M. Gold, C. Banjo, S. 0. Freedman and P. Gold, J. Immunol.,
-
111, 18'72-1879 (1973). 22. J. C. Schmacher and
R. D. Stewart, i n "Kirk-Othmer Encyclopedia
of Chemical Technology, I ' Vol. 5, A. Standen ed. , Interscience Publishers, New York, 1964, PP. 61-84.
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Preparation of Carcinoembryonic Antigen (CEA) Containing Significantly Increased Amounts of Galactose and Galactosamine a
a
a
J. I. Joung , H. Rochman , B. Levin & J. A. Cifonelli
a
a
Department of Pathology , University of Chicago , Chicago, Illinois, 60637 Published online: 06 Dec 2006.
To cite this article: J. I. Joung , H. Rochman , B. Levin & J. A. Cifonelli (1975) Preparation of Carcinoembryonic Antigen (CEA) Containing Significantly Increased Amounts of Galactose and Galactosamine, Preparative Biochemistry, 5:4, 359-374, DOI: 10.1080/00327487508061583 To link to this article: http://dx.doi.org/10.1080/00327487508061583
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and
Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Columbia University] at 02:20 13 November 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-andconditions
PREPARATIVE BIOCHEMISTRY, 5 ( 4 ) , 359-374 (1975)
PREPARATION OF CARCINOEMBRYONIC ANTIGEN (CEA) CONTAINING SIGNIFICANTLY INCREASED AMOUNTS
Downloaded by [Columbia University] at 02:20 13 November 2014
O F GALACTOSE AND GALACTOSAMINE
J. I. Joung. H. Rochman, B. Levin and J. A. Cifonelli Department of Pathology, University of Chicago, Chicago, Illinois 60637
Abstract CEA was prepared by combined isoelectric precipitation, ultrafiltration and column chromatography under controlled conditions of pH. The resulting immunologically active materials were higher i n carbohydrate (85-87%), N-acetyl galactosamine (10-11.570'0)and galactose (28-32700) content than that previously reported.
Differences i n amino
acid yield were also noted; the concentrations of aspartate, serine, glycine and alanine being higher and that of lysine, histidine, arginine, proline, valine, isoleucine, leucine and tyrosine were lower than that reported for CEA prepared by previous methods.
The tumor tissues
for CEA extraction were obtained from two Group 0 Rh positive deceased.
Neither preparation showed Group A or B activity a s
measured by hemagglutination inhibition.
It is suggested that the method
of purification influences the carbohydrate and amino acid yields. 359 Copyright 0 1975 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
360
JOUNG ET AL.
Introduction Onco-fetal antigens a r e widely employed i n experimental studies. More recently, their usefulness i n a clinical context has been explored.
A major difficulty for the investigator is i n obtaining sufficient suitably purified material
1
.
CEA is present in low concentrations
Downloaded by [Columbia University] at 02:20 13 November 2014
(< 10 mg/kg) in secondaries to the l i v e r originating from a carcinoma
of the colon.
These secondaries a r e commonly the source for obtaining
CEA. The isolation and purification procedure normally adopted is that described by Krupey g
2 g., or a
modification.
Their method involves a
preliminary extraction with perchloric acid of glycoprotein-rich material from malignant tissue and the product is progressively purified using column chromatography.
I n the present studies, CEA was found to be stable over a wide range of pH and therefore the method of purification described below incorporates isoelectric precipitation a t various hydrogen ion concentrations.
Further, employing a modified hollow fiber ultrafiltration
system the removal of compounds less than 5 0 , 0 0 0 m. w. and concentration of large preparatory volumes could be achieved.
These procedures
allowed for a considerable reduction in purification time and the resulting material contained CEA of high specific activity.
Analysis of CEA
prepared by this method showed it to contain galactose and N-acetyl galactosamine i n concentrations higher than that previously reported and the significance of these findings a r e discussed.
CEA CONTAINING GALACTOSE AND GALACTOSAMINE
361
Materials and Methods Preparation of CEA. ing to the method of Krupey
CEA from Tumor III was prepared accord2
&.
The method for preparing CEA from tumors Hunt and T65 was
that a s described below.
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Following autopsy, liver metastases from a primary cancer of
the colon were dissected free from normal liver tissue and stored a t -2OOC.
F o r isolation and purification of CEA the tissue was thawed and
sliced. The sliced tissue (2 kg wet weight) was made up to a volume of 0
4 liters with deionized water and a homogenate prepared (at 4 C) using
a Waring blender.
Perchloric acid extraction, with an equal volume of 0
1.5 M HC104, was then carried out over a 20 min. period at 4 C. The mixture was then centrifuged and the precipitate discarded.
Immediately
thereafter NaOH was added to adjust the hydrogen ion concentration of the resulting solution to pH 4.
An aliquot was removed and the pH a t
which a maximum precipitate formed was determined; this varied from pH 4 to 10 i n different experiments.
The pH of the extract was then
adjusted accordingly and the precipitate discarded.
The c l e a r solution
was introduced into an Amicon (Amicon Corporation, Massachusetts, Model CDS- 10) hollow fiber system and concentrated ten-fold at a pressure of 1 Kg/cm2; by this procedure compounds of less than
50,000 m. w. were excluded from the concentrate.
Assay of the con-
centrated solution showed only a minimal loss (< 570)in CEA activity. The concentrated solution was then dialyzed against deionized water
JOUNG ET AL.
362
(3 days at 4OC), the precipitate discarded and the supernatant lyophilized.
The lyophilized material (4 g) was dissolved in 45 ml of 0.05 M phosphatebuffered saline, pH 7.2. A t this pH a precipitate frequently formed which was discarded.
The supernatant was subjected to pressure filtra-
tion employing an Amicon (Amicon TCF 10) thin channel ultrafiltration
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system (pore size, 0.2
p;
2 pressure, 3 Kg/cm ) and the filtrate
(130-150 ml) applied to a Sepharose 4B column (10x 100 cm) equilibrated
with 0.05 M phosphate-buffered saline pH 4. 5, at 4OC. Ascending chromatography was then carried out and 20 ml fractions were collected using a fraction collector. Eluates were examined for CEA activity by radioimmunoassay and the protein content monitored at 280 mp. Fractions containing high CEA activity were pooled. Fractions with low CEA activity or a high protein content were combined for rechromatography. The CEA-rich fraction was ultrafiltered and concentrated (x 10) using an Amicon thin channel ultrafiltration system (XM50, pressure 2 3 Kg/cm 1, dialyzed at 4OC for 3 days, and then lyophilized.
The
lyophilized material (250 mg) was dissolved in 10 ml of phosphatebuffered saline (pH 4. 5), and subjected to gel filtration by application to a Sephadex G-200 column (5 x 100 cm). The conditions for chromatography were as described above. Eluates were assayed for CEA activity an4 protein concentration, and fractions containing high CEA activity were pooled, dialyzed and lyophilized.
The lyophilized material (20-30 mg
was dissolved in 2 ml phosphate-buffered saline (pH 7. 5). centrifuged and any precipitate discarded. This material was rechromatographed (2-3 times) until no further improvement in separation could be achieved.
363
CEA CONTAINING GALACTOSE AND GALACTOSAMINE
Measurement of CEA? The procedure for measuring CEA was similar to the double antibody radioimmune assay described by Laurence
--
5
4
et al., which is a modification of the triple isotope methods of Egan z t &
CEA and its antisera of defined immunological activity were used a s reference materials.
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Hansen
These preparations were gifts f r o m Todd
&.3
and from
.
3
Chemical analysis of CEA. Neutral sugar components were determined with a Technicon Carbohydrate Analyzer using a borate column. Hydrolysates were prepared by heating 0.2 1 M HC1 for 3 hours at 100°C.
- 0. 3 mg of sample in 1 rnl of
A f t e r addition of 10 ml of H20 to the
hydrolysate, rhamnose (50 pg) was added a s an internal standard. Deacidification was accomplished by adding Dowex 3 (carbonate) in portions until a pH of approximately 2.0 was attained.
The solution was then
passed over a Dowex 50 (H+) column (1.0 x 30 c m ) to remove hexosamines and peptides and 10 ml of methanol was added to the effluent before 0
concentrating t o a small volume a t 30 C in wcuo.
This latter
procedure was repeated several times after fresh additions of methanol and the solution finally taken to dryness.
The residue was dissolved
in 1.0 ml of H 2 0 and 0 . 4 ml was used f o r sugar analysis. Hexosamines were estimated by the Elson-Morgan procedure
or during amino acid autoanalysis a s reported elsewhere 6
.
Results Monitoring of eluates from chromatography columns showed the presence of a single peak of CEA activity.
The partition coefficients
364
JOUNG ET At.
found with Sepharose 4B and Sephadex G-200 were 0.65 0. 08
- 0.74 and
- 0. 15 respectively. There are no accepted criteria for determining the presence of
.
CEA except by immunological comparison with described preparations of other investigators. CEA prepared from tumors Hunt, T65 and III
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showed immunological identity with such known CEA preparations.
When
examined by double immunodiffusion in agar gel (Figs. 1 81 2 ) a single line of complete identity with known CEA preparations was obtained. Also, the standard inhibition curves obtained by radioimmunoassay for the prepared CEA over the range of 0
-
100 ng per ml w e r e similar in
all respects to that of known CEA (Fig. 3). Compared to a known standard the relative immunological activities of the prepared materials a r e listed in Table I .
However, these figures of activities a r e probably
under-estimates since no corrections were made for the water content of preparations T65. Hunt and III; one lyophilized sample when subjected to differential thermal balance analysis revealed a moisture content of 28%. Analysis of the CEA preparations for amino acids a r e given in Table 11. The amino acids present in highest concentration for all preparations were aspartic and glutamic acids, threonine and serine. The yields of individual amino acids present in CEA materials prepared by the above described method were similar (compare CEA from tumors Hunt and T65). However, when compared with those prepared by previously described methods (see Table II) striking differences were noted; i n particular, aspartate, serine, glycine and alanine were present
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CEA CONTAINING GALACTOSE AND GAJACTOSAMINE
365
FIGURE 1 Immunodiffusion in an agar gel buffered with 0. 1 M phosphate-saline (containing EDTA, 0. 4 m g l m l ) , pH 7. 2. The c e n t e r well contained Hansen anti-CEA s e r u m (10 pl) undiluted. The contents of the peripheral wells w e r e a s follows: a. Todd CEA, 5 pg ( d r y weight); b. T65 CEA, 20pg; c. I11 CEA, 8 p g ; and d. Hunt CEA, 5 p g .
in higher concentration in CEA prepared by the present procedure whereas lysine, histidine, arginine, proline, valine, isoleucine, leucine and tyrosine w e r e lower.
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366
JOUNG ET AL.
FIGURE 2 Immunodiffusion in an agar gel buffered with 0. 1 M phosphate-saline (containing EDTA, 0 . 4 mg/ml), pH 7. 2. The center well contains Todd anti-CEA serum 5 pl (1:2 dilution). The contents of the peripheral wells were as follows: A. Hansen CEA, 2 pg (dry weight); B. Todd CEA, 2 pg; C. London CEA (a CEA preparation from hepatic metastases of a carcinoma of the colon), 2 pg: D. Hunt CEA, 2 pg; and E. I11 CEA, 2 pg.
Sugar and amino sugar analyses of CEA from tumors Hunt and T65 a r e shown in Table KII a s a r e the values for CEA preparations and blood Group A substance reported by others.
The higher carbohydrate
(85-870/0), N-acetyl galactosamine (10-110/0) and galactose (28-32%)
367
CEA CONTAINING GALACTOSE AND GALACTOSAMINE
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80
60
40
20
0
I
1
I
FIGURE 3 Standard inhibition curve of the CEA radioimmune system. The percentage of free (unbound) radioactive labelled "9 CEA (ordinate) plotted against the ratios of unlabelled standards to their respective radioactive labelled 125 I CEA (abscissa). Anti-CEA serum (Ace 17 from Todd 3, in a final dilution of l:lZ, 800 was allowed to react with Hansen CEA. A ; Todd CEA, 0; and Hunt CEA, U
.
content of CEA from tumors Hunt and T65 distinguish them from all other CEA preparations listed i n Table 111. In contrast, the carbohydrate content of CEA material from Tumor III was within the range of that reported for CEA prepared by similar procedures.
368
JOUNG ET AL.
TABLE I Immunological Activity (measured by radioimmunoassay) of Various CEA Preparations CEA Preparation
Activity p e r unit d r y weight
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Todd
100
III
45
Hunt
67
T65
20
Discussion
The purified CEA demonstrated complete immunological identity with known CEA preparations (Figs. 1 and 2).
The amino terminal amino
acid sequencing of the first twelve residues of Hunt CEA agreed with previously reported results. l 3 Also, the partition coefficients of CEA during purification of Sephadex and Sepharose columns were similar ‘to those calculated from the data of other studies.
”
These findings
indicate that the present preparations contained CEA similar to that of other investigators. In preliminary experiments, it was found that the CEA materials decreased in immunological activity following storage for a few days under marked acidic (pH 1 2 ) conditions.
Previous
methods used for purifying CEA involve longer periods of contact with perchloric acid so that the preparatory materials become exposed to a low pH and an oxidative environment.
It is known that prolonged periods
CEA CONTAINING GALACTOSE AND CALACTOSAMINE
369
T AB LE IT A m i n o Acid An al y s i s ( m o l e s
70)of Var i o us CEA P r e p a r a t i o n s
CEA P r e p a r a t i o n s
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Amino Acid
CEA
CEA
Banjo is"% "
C EA Turberville
111
-
Hunt
-
T65
Lysine
2.8
2. 5
3. 4
3. 5
0. 9
0. 6
Histidine
2. 1
1. 3
2. 2
2.0
0. 7
1.0
3. 8
3.2
1. 8
0. 6
11. 8
20. 1
22. 6
Arginine Aspartic acid T h r eonine
3. 8
3. 4
14. 0
15. 9
16. 6
8. 1
9. 3
10. 6
12.3
11.2
10. 7
Serine
10. 0
11.4
11. 7
12. 5
21. 1
19. 0
Glutamic a c i d
10. 4
11. 0
9. 9
10.4
11. 6
10. 3
Proline
7. 7
8. 0
8. 0
9. 1
4. 3
Glycine
5. 6
5. 5
5. 7
7. 1
13. 2
14. 9
Alanine
6. 2
5. 9
6. 6
6. 7
8. 5
8. 7
Valine
ND
5. 5
3. 4
2. 8
3. 1
7. 5
6. 8
Methionine
0. 2
ND
0
ND
ND
ND
Isoleucine
4.9
5. 1
5. 6
3. 6
1. 6
1. 6
Leucine
9. 3
9. 3
8. 9
7. 2
1. 7
1. 4
T y r o sine
5. 4
2. 5
4. 0
2. 5
0
0
Phenylalanine
ND
2. 1
2. 5
2.5
0
2. 6
Cysteine
ND
ND
0. 1
ND
ND
ND
Cystine
1. 9
ND: Not d e t e r m i n e d. 111, Hunt and T65: S e e M a t e r i a l s and Methods.
370
JOUNG ET AL.
TABLE III Sugar and Amino Sugars in Various CEA Preparations and Human Blood Group A
12 AHC
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23.1
33.2
20. 2 21.9 1.6
82.2
16.8
III, Hunt and T65: See Materials and Methods. of acid treatment results in hydrolysis of most glycoproteins.
Further-
more, perchloric acid at the concentration used readily oxidizes biological materials. 22 To minimize these effects in the present experiments, conditions were chosen so that the pH was more rigidly controlled and the treatment with perchloric acid was of brief duration. Analyses of CEA materials for amino acids and carbohydrate suggest
371
CEA CONTAINING GALACTOSE AND GALACTOSAMINE
that the method of purification probably w a s a major factor contributing to the compositional differences found when comparing the various CEA preparations.
Amino acid analysis (Table 11) showed that the concentra-
tions of aspartate, serine, glycine and alanine were higher, and that of lysine, histidine, arginine, proline, valine, isoleucine, leucine and
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tyrosine were lower for CEA from tumors Hunt and T65 than that of CEA prepared by previous methods. hydrate analysis was performed.
Similar results were noted when carboThe yields of galactose, N-acetyl
galactosamine and total carbohydrate (Table III) were much higher for materials prepared by the present procedure.
These findings suggest
that prolonged perchlorate treatment may have contributed to the low yields of carbohydrate found by others studying CEA. 1 , 8 , 9 The levels of galactose, N-acetyl glucosamine and to a l e s s e r extent N-acetyl galactosamine found in the present preparations a r e similar to what could be expected for blood group A substance (see Table 111). However, the striking difference in mannose content between CEA and that of blood group A substance distinguishes them at the bio-
chemical level a t least i n this respect.
Despite the similarities of CEA
and blood group A substance, no blood group activity (A o r B) could be detected by hemagglutination inhibition for either of the CEA preparations (Hunt or T65) up to a concentration of 1 mg/ml.
These CEA materials
studied were isolated from Group 0 Rh positive deceased.
These
findings make it difficult to support the concept that C EA is a modified blood group substance as proposed by Simmons and Perlman.
12
372
JOUNG ET AL.
Banjo
2.8 and Gold and Gold"
i n early studies found weak
Group A activity in their CEA preparations but were unable to explain such activity since their preparations showed low levels of N-acetyl galactosamineal- a necessary amino sugar for the reaction of Group A with Anti-A. "' l8 Holburn
2?tt9 reported the blood group activity
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of their CEA preparations to be similar to the blood group types of their patients.
In the present experiments no blood group activity was
detected in either CEA preparation but these were isolated from Group 0 Rh positive deceased.
Denk
2 : '
found the respective blood group
activity on colonic cancer cells and their hepatic metastases of Group A and B patients, but the results with Group 0 patients were inconsistent. These findings suggest that CEA exhibiting blood group activity is only found when isolated from Group A o r Group B materials.
It would
therefore seem that the blood group activity which has been observed to be associated with CEA is caused by contamination. In view of the similar physicochemical properties among many glycoproteins and considering the inherent limitations of the preparation process, the selection of suitable raw materials for isolation may be as important as the extraction procedures employed.
Blood group
activity is only an example demonstrating the heterogenity among variously prepared CEA materials.
CEA CONTAINING GALACTOSE AN0 GALACTOSAMINE
373
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J. Krupey, T. Wilson, S. 0. Freedman and P. Gold, Immunochem-
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istry,
9,
617-622 (1972).
Todd CEA and antisera were gifts from C. W. Todd, Department of Immunology, City of Hope National Medical Center, Duarte, California 91010. Hansen CEA and antisera were gifts from H. J. Hansen, Department of Biochemical Nutrition, HoffmanLaRoche, Inc., Nutley, New Jersey 07110.
4.
D. J. R. Laurence, U. Stevens, €2. Bettelheim, D. Darcy, C. Leese, C. Turberville, P. Alexander, E. W. Johns and A. M.
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R. D. Stewart, i n "Kirk-Othmer Encyclopedia
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![[Carcinoembryonic antigen (CEA) (author's transl)].](/assets/img/hold.png)
