Eliwrophoresis 1992, 13, 269-274 269 Purification of alginates by free flow electrophoresis

Original papers

Ulrich Zimmermann’) Gerd Klock’ Konrad Federlin’ Kurt Hannig’ Matthias Kowalski’ Reinhard G. Bretzel’ Andrea Horcher’ Heike Entenmann’ LJlrike Sieber’ Tobias Zekorn‘

’Institut f ~ r Biotechn Universitat Wiirzburg ’Medizinische Klinik Universitat GieRen

Production of mitogen-contamination free alginates with variable ratios of mannuronic acid to guluronic acid by free flow electrophoresis

Commercial alginates consisting of variable homopolymeric regions of p-D-man- nuronic acid and a-L-guluronic acid, interspaced with regions of alternating blocks, are potent stimulators of macrophages and lymphocytes. Therefore, in- flammatory reactions and fibrotic overgrowth of the beads result if Langerhans islets are encapsulated in raw alginate hydrogel beads (cross-linked with divalent cations). The result is random failure of the islets some time after transplantation. Analysis of raw alginates by using free flow electrophoresis demonstrated that commercial alginates contained at least 10-20 fractions (characterized by differ- ent electrophoretic mobilities) which showed mitogenic activity. These fractions could be quantitatively separated from the alginic acids by free flow electrophore- sis on a preparative scale. The purified alginates cross-linked with Ca2+ ions exhib- ited no mitogenic reactions as proved by an in vitro assay. In addition, examina- tion of purified Ba”a1ginate beads implanted intraperitoneally in rats or mice for three weeks showed no fibrotic overgrowth in contrast to implants made from un- purified alginate.

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1 Introduction

Alginates (extracted from seaweeds) are anionic poly- saccharides composed of homopolymeric regions of p - ~ - mannuronic (M) and a-L-guluronic (G) acids (called “M-blocks” and “G-blocks”) interspaced with regions of mixed sequence (“MG-blocks”). Alginates have hydrogel- forming properties as di- or trivalent cations (e . g. Ca’+, Ba” or Fe3+) bind to the G-blocks [I] and - in the case of Ba2+ ions - also to the M-blocks (unpublished data). Entrap- ment of living cells in alginate gels can be accomplished under mild conditions, and is therefore widely used for the immobilization of microorganisms as well as plant and mammalian cells [l-51. For the production of metabolites, immobilized cells are preferred to free cells in biotechnol- ogy and biomedicine because they are easier to handle and the gel matrix also provides the cells with a protective bar- rier against infection and mechanical stress. In addition, processes of cell aging or senescence can be considerably delayed in immobilized cells compared to freely suspended cells [6].

In recent years several laboratories have examined alginate gel beads for the transplantation of islets of Langerhans. Such insulin-producing tissue is required to treat diabetes mellitus [7-lo]. For this application the alginate gels must meet several requirements. These are mechanical and chemical stability, defined (and narrow) pore size distribu- tion, and the absence of any contamination with toxic, pyro- genic and immunogenic compounds. The first two require- ments are fulfilled when alginates are cross-linked with Ba’+ions [11,12]. In contrast to cross-linkage by Ca’+ions, Ba2+ions lead to gel matrices which are chemically stable

Correspondence: Prof. Dr. Ulrich Zimmermann, Universitat Wiirzburg, Lehrstuhl fur Biotechnologie, Am Hubland,W-8700 Wiirzburg, Germany

Abbreviations: G, guluronic acid; LPS, lipopolysaccharide M, mannu- ronic acid

under both in vitro and in vivo conditions. Such gels cannot be dissolved by citrate, phosphate or even ethylenediami- notetraacetate (EDTA) at physiological pH (unpublished data). However, until now uncharacterized commercial algi- nates (containing phenolic and other toxic, mitogenic com- pounds [1,4]) were used for immobilization of islets in Ca“ or Ba”cross1inked alginates. The presence of these conta- minants was presumably the primary reason for processes which induced fibrotic overgrowth of the beads. This re- sulted in the random failure of the islets some time after im- plantation because this barrier inhibits the diffusional sup- ply of the encapsulated islet with oxygen and nutrients.

In this communication we will show that the numerous mi- togenic contaminants associated with commercial algina- tes can be detected and quantitatively removed by using free flow electrophoretic mobilities of these compounds in relation to the electrophoretic mobility of the highly charged G-, M- and MG-blocks. It is demonstrated that algi- nate beads made up of electrophoretically purified algina- tes did not induce fibroticovergrowth after intraperitoneal implantation in mice or rats. This result was achieved with various commercially available alginates containing differ- ent amounts of mannuronic and glucuronic acids.

2 Methods and materials

2.1 Alginates

The following commercial sodium alginates of the Kelco company (Hamburg, Germany) were analyzed: Manugel GHB (51 O/o guluronate), Manucol DH (16% guluronate), Kelcogel AV (14% guluronate), Manugel DJB (54% guluro- nate). In addition, the sodium alginate (No. 9180) of the Roth company (Karlsruhe, Germany) was investigated. The sodium alginates were dissolved in distilled water (2 O/o

w/v) and successively filtered through membrane filters (Sartorius, Gottingen, Germany) of decreasing pore radii

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270 IJ. Zimmermann c la l . Elecrrophorr~is 1992. 13. 269-274

(3.0 pm, 0.8 pm, 0.45 pm and 0.2 pm). For some experi- ments, 1 mL of the filtered alginate solutions was dialyzed overnight against 5 L of distilled water. The dialysis tubing (Visking size 5/24, Medicell, London, UK) retained mole- cules with a molecular mass greater than 12000-14000.

2.2 Free flow electrophoresis

We used an ACE 710 free flow electrophoresis apparatus manufactured by Hirschmann Projektentwicklung (Neu- ried, Germany). The instrument and the separation tech- nique are described in detail elsewhere [13,14]; only the op- erational parameters are given here. The dimensions of the separation chamber were 30 X 130 X 0.3 mm and it was op- erated at a (chamber buffer) temperature of 22°C. The chamber buffer contained 15 mM triethanolamine, 7.1 mM potassium acetate,216 mM glycine (pH 7.2), 11 mM glucose, 0.2 mM ethylenediaminotetraacetate, 100 units mL-’ peni- cillin, and 100 pg mL-’ streptomycin (Biochrom, Berlin, Germany). The conductivity was 1.9 mS cm-’. The flow rate of the buffer was 2.5 mL h-’.The sample was injected at 350 pL h-’.The field strength was 100 V cm-’ (corresponding to a current of 65 mA). Pherograms were recorded at a wave- length of 228 nm. The electrophoretic mobility (cmzV-’ s-’) was calibrated by using the mobility of human red blood cells (1.74 X 10 V-I s-l) and dextran (0 X lo-‘ cm2 V-’ s-I) as reference points. The eluate from the separation cham- ber was continuously collected in 35 fractions. Each frac- tion represented a band of electrophoretic mobility of 0.2 X

cm2 V-’ s-’. For analytical separations, 10 pL of a 2Yo solution of raw alginate material were injected into the separation chamber. For preparative separations, the free flow electrophoresis apparatus was operated continuously for up to 60 h. The separation profile remained completely stable during this time.

2.3 Assay of mitogenic activity

After electrophoretic separation, 10 pL of each fraction was evaporated overnight in a 96-well plate (Greiner, Nurtin- gen, Germany) under sterile conditions. Splenocytes were prepared from male Balb-c mice (8-10 weeks old) as de- scribed elsewhere [15]. The cells were suspended at a sus- pension density of 1 X 10‘ mL-’ in complete growth med- ium (CGM) consisting of RPMI 1640 medium supple- mented with 10 O/o fetal calf serum (Boehringer, Mannheim, Germany), 2 mM L-glutamine, 2 mM sodium pyruvate, non- essential amino acids (1 X, Boehringer, Mannheim, Ger- many), 50 p~ of 2-mercaptoethanol, 100 units mL-’ penicil- lin and 100 pg mL-’ streptomycin (Biochrom). One hun- dred pL of this suspension were added to the plates coated with the dried material from the electrophoretic separation. Since the CGM medium contains 5.5 mM Ca2+ (as in “mito- genic” experiments of other authors, see [16,17], alginate gels formed in those wells that contained alginate fractions. In all wells the cells were grown for 5 days at 37°C in a 5% C0,-supplemented atmosphere. Then, 5 pL of a 0.5% Try- pan Blue solution were added to 45 pL of the cell suspen- sion and the dead (i. c. stained) as well as the intact spleno- cytes per well were counted by using a Neubauer chamber. Separate experiments in which the “mitogenic”effect of the electrophoretically separated fractions was studied by in- corporation of [“Clthymidine into splenocytes yielded identical results to the assay described above. We therefore

used that assay throughout the experiments described below because of its simplicity. Parallel to the mitogenic assay, 2.5 mL of each fraction were filled into 24-well plates and dried overnight. Then, 1 mL of a solution of BaCl, (150 mM) in distilled water was added. If alginic acids were pres- ent in the fractions, this resulted in the formation of an in- soluble gelatinous Ba”a1ginate precipitate. The alginate content of the fractions was determined gravimetrically.

2.4 Intraperitoneal implantation of Ba2+alginate beads

The method of forming Ba2’alginate beads was described elsewhere in detail [ l l] . Briefly, electrophoretically purified sodium alginate (Manugel GHB, Kelco); 2% solution in 0.9% NaCL) was injected through the central channel (0.5 mm in diameter) of a homemade nozzle and dropped into a 20 mM BaCI, solution. This resulted in the formation of beads with a mean diameter of approximately 600 pm. After several washings in 0.9% NaCI, the beads were incu- bated for 2 days in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco Europe, Germany) to mimic the conditions after transplantation. The beads were im- planted by medial incision under ether anaesthesia into the peritoneal cavity of NMRI-mice (n=3) and Lewis rats (n=3). The animals were purchased from Charles River Wiga (Sulzfeld, Germany). In the control experiments, three recipients from each species received the same amount of capsules made from raw sodium alginate. After three weeks the animals were sacrificed, and implanted beads were removed from the peritoneal cavity by lavage and peritoneal biopsy. The beads were immobilized in a fi- brin clot, Bouin-fixed, and paraffin-embedded. Serial sec- tions were stained with hematoxylin-eosin and examined under the microscope.

3 235 4 45

Electrophoretic Mobility

(prn.crn.V-’.sec-l) FLgure I . Typical pherograms of commercial alginates subjected to free flow electrophoresis.The commercial sodium alginates were dissolved in distilled water (2%1 w/v) and membrane-filtered before electrophoretic separation. For separation conditions, see Section 2.2. The pherograms were recorded at a wavelength of228 nm. (a) Manugel GHB,(b) Manucol DH, (c) alginic acid “Rolh’’, (d) Kelkogel AV.

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3.1 Electrophoresis of crude alginates

When murine splenocytes were cultured in tissue culture wells coated with unpurified commercial Ca''a1ginate gels, a strong activation of the splenocytes was observed, inde- pendent of the M/G content of the alginate used. The total mitogenic activity of 100 pg mL-' raw alginate (10 pg per well) was of the same order of magnitude as observed with 1 pg mL-' lipopolysaccharide (LPS), which is known to be a strong bacterial mitogen (see below). Electrophoretic separations of solutions of crude sodium alginate from dif- ferent sources revealed complex pherograms, indicating significant impurities with absorption at 228 nm. Figure 1 shows typical pherograms of different commercial algi- nates. Two main peaks were recorded at an electrophoretic mobility of 3.9 X cm' V-' s-', but several other minor peaks towards lower electropho- retic mobility could also be seen in some material (e .g . Fig. lc). However, the intensity of these peaks was often close to the noise level. Pherograms of the fractions representing

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3.2 Mitogenic activity

By means of the mitogenic assay, at least 10 to 20 fractions could be identified, which induced strong proliferation of the splenocytes. The number and distribution of these mi- togenic fractions varied with the source of the alginate and (in some cases) within different batches of the same prod- uct. Fractions representing a mobility of 0.2 to 1.9 X cm2 V-' s-' contained substances with a high mitogenic ef- fect on the splenocytes. These fractions were found in all commercial alginates. Pure LPS isolated from Streptococ- cus thyphosa (Sigma, Taufkirchen, Germany) exhibited an electrophoretic mobilityof 1.0 f 0.5 X cmzV-' s-' (data not shown). This suggests that a part of these mitogenic

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compounds may be LPS or LPS-related. Strong mitogenic activity was also detected for fractions corresponding to electrophoretic mobilities of -1.8 to 0 X cm2 V-’ s-’. The finding of mitogenic fractions with “negative” electro- phoretic mobility indicated that the corresponding com- pounds must be positively charged. Gravimetric analysis of these mitogenic fractions gave no indication of the pres- ence of alginate.

3.3 Dialysis of alginates

All commercial alginates contained mitogenic contamina- tions with an electrophoretic mobility similar to the mobil- ity of the Ca*+-precipitable material. The corresponding electrophoretic mobilities were between 3.3 X cm2 V-’ s-’ (sodium alginate from Roth, Karlsruhe, Germany) and 3.7 X cm2 V-’ s-’ (Manugel GHB). These mitogenic contaminations disappeared completely when the pooled fractions containing the alginic acids were dialyzed against distilled water overnight and then re-analysed by free flow electrophoresis. None of the fractions that were analyzed after the second electrophoretic treatment contained com- pounds with mitogenic activity (Fig. 3).

In the following set of experiments we investigated algin- ates from different sources after 24 h of dialysis against dis- tilled water before electrophoretic separation. It is evident from Fig. 4 that after this treatment the pattern of mito- genic activity in the electrophoretically separated fractions remained comparable to that of the raw material, although the amount of mitogenic activity in some of the fractions of the dialysed alginate preparations was slightly reduced. It is also clear from the figure that the fractions with a similar electrophoretic mobility as the alginic acids still induced

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Figure 4. Alginate content (closed circles) and mitogenic activity (open circles) in the 35 fractions collected after electrophoretic separation of pre-dialyzed commercial alginate (Manugel GHB). It is evident that a 24 h dialysis of the alginates before electrophoretic separation only removed the mitogenic contaminants to a limited extent.

Figure3. Free flow electrophoresis analysis ofthe electrophoreticallypu- rified alginate fractions of Fig. 2a (Manugel GHB, electrophoretic mobil- ity of 3.9 k 0.4 X cm2 V-’ s-‘) after 24h dialysis against distilled water. The closed circles represent the alginate content, the open circles the mitogenic activity of the 35 fractions collected after separation. It is clear that post-dialysis resulted in the quantitative removal of the remain- ing residues of mitogenic compounds. Similar results were obtained when the electrophoretically purified alginates of Fig 2 b-d were sub- jected to post-dialysis. tion procedure).

Figure 5. (a) Beads prepared from raw alginate were removed from the peritoneal cavity of rats by lavage three weeks after implantation. The beads are surrounded by thick layers (50-100 pm) offibrotic tissue (fibrin clot embedding, hematoxilin-eosin staining, magnification X 100). (b) Beads made from electrophoretically purified alginate were free of any fibrotic tissue (magnification X 100,irregularities in shape due to fixa-

Ekcrrophoresis 1992. I S , 269-214 Purification of alginates by free flow electrophoresis 273

proliferation of lymphocytes. This shows that post-dialysis is more efficient than pre-dialysis for complete removal of these compounds.

3.4 Fibrotic or inflammatory reaction

The quantitative removal of mitogenic contaminants from electrophoretically purified alginates could be also verified by implantation of Ba2'-cross-linked, empty alginate beads into mice and rats. The control experiments using raw ma- terial showed that formation of a layer of fibrotic tissue around each bead occurred as expected. In rats the fibrotic layer was more intense (Fig. 5a) than in the case of mice (data not shown). Biopsies from the peritoneum of the con- trol animals of both species also showed adherent capsules and fibrotic overgrowth (Fig. 6a) which was more pro- nounced than around beads removed by peritoneal lavage. In contrast, beads made from electrophoretically purified alginate showed no visible fibrotic or inflammatory reac- tion in mice nor in rats (Fig. 5b). Even if the beads were lo- cated close to the peritoneum viscerale (Fig. 6b) there was no fibrotic foreign-body reaction.

Figure 6. Biopsies from the peritoneum of rats three weeks after implan- tation demonstrated adherent beads. (a) Barium alginate beads from raw alginate induced a severe foreign body reaction. (b) Beads made from electrophoretically purified alginate were areactively located in the peri- toneum.

4 Discussion

Commercial alginate material is apparently strongly con- taminated with various mitogenic compounds. Most of the mitogenic compounds in the raw material could be separ- ated in one step from the alginates by using free flow elec- trophoresis. Mitogenic contamination exhibiting similar electrophoretic mobilities (3.3 - 3.7 X cm2 V-' s-') to the polymeric alginic acids (3.9 X cm2 V-' s-') could be removed either partly by pre-dialysis of the raw material or completely by post-dialysis.

The mitogenic substances were not identified because this was beyond the scope of this investigation. However, it is likely that at least some of these contaminants were oligo- mers of the mannuronic or guluronic acids. This is sug- gested by the charge (i. e. the electrophoretic mobility) of these molecules and by their rapid removal during post- dialysis.

Soon-Shiong et.al . [16] and Otterlei et al. I171 have also shown that alginates contain mitogenic compounds. These authors showed that raw alginates stimulated monocytes to produce high levels of cytokines including interleukin-1, in- terleukin-6 and tumor necrosis factor (TNF-a) [16,17]. These authors also examined the ability of "purified" algin- ates with different ratios of mannuronic acid to guluronic acid, and of isolated M-blocks to stimulate cytokine pro- duction from monocytes. They showed that activation of the monocytes depended strongly on the concentration of mannuronic acid. Therefore, these authors arrived at the conclusion that isolated M-blocks and MG-blocks were po- tent stimulators for the production of cytokines and that these polymers were the active components in alginates.

The conclusions of these authors are in contrast to the find- ings reported here. Neither mannuronate-high (Mannucol DH, Kelcogel AV) nor mannuronate-low (Manugel GHB, Manugel DJB) alginate gels induced a foreign-body reac- tion in the mitogenic assay and in the implantation experi- ment, provided that the alginate was purified electropho- retically and post-dialyzed before cross-linkage with Caz+ or Ba2+ions. In the light of our results it is therefore quite li- kely that the "purified"materia1 used by Soon-Shiong et al. [16] and Otterlei et al. [17] was still contaminated with some mitogenic compounds. However, since these authors did not publish their protocol for purfication of raw alginates, it is not possible at the present to speculate about the possi- ble nature (and charge) of these contaminants in relation to the work presented here.

It is also interesting to note that the absence of fibrotic over- growth on implanted, empty beads (made from electropho- retically purified alginate) rules out the possibility of che- lated barium being responsible for the induction of the for- eign-body reactions as observed with raw material. In con- trast,Darqui et al. [18] and Waterfall et al. [19] reported a tre- mendous foreign-body reaction of alginate after polylysine treatment. It is also well known that under certain circum- stances polylysine is strongly cytotoxic [20].Apart from the high mechanical and chemical stability of Ba" alginate cap- sules, this is another reason for prefering this technique to Ca2+ alginate/polylysine capsules [7].

274 u. Zirnrnermann ern/ . ~ / c c r , n i ~ h ~ l i - ~ s r s 1992. 13, 260-274

To sum up, the possibility of quantitative removal of mito- genic compounds by free flow electrophoresis is promising and supports efforts in the direction of the development of techniques for immunoisolated transplantation of pan- creatic islets on the basis of alginate microcapsules. There is apparently no need to use an alginate with a low M and a high G content, as suggested by Soon-Shiong et a/. [16] and Otterlie et al. [17]. This is an important conclusion because the highest diffusion rates of proteins, indicating the most open structure,are found in beads made from high-G algin- ates (cross-linked with Ca2+ ions 14,211). On the other hand, microcapsules with optimum properties for islet transplan- tation should at least exclude substances with molecular weights larger than M, 10000 in order to avoid adverse side effects which may arise from external proteins (cytokines, complement, antibodies, etc.) or from the release of intra- cellular proteins from damaged islets. It is clear that this re- quirement demands the use of alginates containing some mannuronic acid. Note that the results presented here show that electrophoresis is a promising tool for prepara- tive applications, which have so far been exclusively the do- main of chromatographic techniques. Further important contributions to this (and related) field(s) are also expected from the novel invention of the multicompartment electro- lyzerwith isoelectric membranes [22] and from the possibil- ity of removal of pyrogenes from protein preparations by the use of electrophoresis [23].

This work was supported by grants of the BMFT(OlQV88630 and FKZ 07024806) to U.Z. and K.I?

131 Brodelius, P. and Vandamme, E. J., in: Rehm, H. J . and Reed, G. (Eds.) Biotechnology Verlag Chemie, Weinheim 1987, Vol. 7a , pp.

[4] Skjak, Braek, G. and Martinsen,A., in: Guiry,M.D. and Blunden,G. (Eds.), Seaweed Resources in Europe: Uses and Potential John Wiley, NewYork 1991, pp. 219-257.

[ 5 ] Vorlop, K. D., Klein, J . and Steinert, H. J., Ann. NYAcarl. Sci. 1987,

[6] Schnabl,H.,Youngman,R. J.andZimmermann,U.,Planta 1983,158,

[7] Lim, F. and Sun, A,, Science 1982,210,908-910. [ S ] Fritschy, W. M., Stubbe, J. H.,Wolter,G. H. J. and van Schilfgarde,T.,

Diabetologia 1991, 34, 542-547. [9] Weber. C., Krekun, S., Koschitzky, T., Zabinski, S., D’Agati, V.,

Hardy, M. and Reemtsma, K., Transplant. Proc 1991,23,764-766. [lo] Mazaheri, R., Atkinson, P., Stiller, C., Dupre, J.,Vose, J. and O’Shea,

G., Transplantation 1991, 51, 750-754. [11] Zekorn,T., Horcher, A., Siebers,U., Schnettler, R., Hering, B., Zim-

mermann, U., Bretzel, R. G. and Federlin, K., Diabetologia 1992, in press.

[12] Zekorn,T., Sieber, U . , Horcher,A., Schnettler,R.,Klock, G.,Bretzel, R. G.. Zimmermann, U. and Federlin, K., Transplant. Proc. 1992, in press.

[131 Kowalski, M., Hannig, K., Klock, G., Gessner, P., Zimmermann, U., Neil, G. A. and Sammons, D. W., BioTechniques 1990, 9, 322-341.

[141 Zeiler, K., Loser, R., Pascher, G. and Hannig, K., Hoppe-Seylers 2. Physiol. Chem. 1975, 356, 1225-1244.

[151 Bohmer, v. H., J. Immunol. 1974, 112, 70-78. [I61 Soon-Shiong,P.,Otterlei,M.,Skjak-Braek,G.,Smidsrod,O.,Heintz,

R., Lanza, R. P. and Espevik, T., Transplant. Proc. 1991,23,758-759. [171 Otterlei, M., Ostgaard, K., Skjak-Braek, G., Smidsrod, O., Soon-

Shiong, P. and Espevik,T., J. Immunotherapy 1991, 10, 286-291. [I81 Darqui,S., Chicheportiche,D., Capron, F.,Boitard,C. and Reach,G.,

in: Federlin, K., Bretzel, R. G. and Hering, B. J. (Eds.), Methods in Is- lets Transplantation Research. Horm. Metabol. Res. SUDDI. Ser. 1990.

405-464.

502,339-342.

3 92-3 97.

.. 25,209-213.

logia 1990, 33, 181A.

Targeted Drugs John Wiley, New York 1983, pp. 89-112.

Received December 9, 1991 [19] Waterfall, M., Cole,D., Chicheportiche, D. and Baird, J. D., Diabeto-

[20j Arnold,L. J . , Dugan,A. and Kaplan,N. O., in: Goldberg,E. P. (Ed.),

[21] Klein, J., Stock, .I. and Vorlop, K. D., Eur. J . Microb. Biotechnol. 1983,

[22] Righetti, P. G., Wenisch, E., Jungbauer,A., Katinger, H. and Faupel,

[23] Lucas, J., Faupel, M. and Goecking, C., Elecrrophoresis 1990, 11,

5 References 18,86-91.

[ l ] Smithrod, 0. and Skjak-Braek, G., Trends Biotechnol. 1990,8,71-78. 121 Schnabl,H.and Zimmermann,U.,in: Bajay,Y.P.S.(Ed.),PlantPruto-

plasrs and Genetic Engineering Springer Verlag, Berlin 1989, vol. I , M., J . Chromatogr. 1991, 500,681-696.

pp. 63-96. 981-982.