- Email: [email protected]
α-lipoprotein heterogeneity in serum of mammals
Journal of Immunological Methods 1 (1972) 375-380. © North-Holland Publishing Company
a-LIPOPROTEIN
HETEROGENEITY
IN S E R U M O F M A M M A L S
Bi)rje W. KARLSSON and Magnus B.N. JOHANSSON Institute of Zoophysiology, University of Lund, Sweden
Received 9 January 1972
Accepted 6 January 1972
Two-dimensional immunoelectrophoresis on the serum of mouse, rat, mongolian gerbil, pig and man indicates heterogeneity in c~-lipoprotein. A pre-c~-lipoproteinwas lbund in pig serum. Traces of a similar fraction could also be detected in human serum. In the serum of the mongolian gerbil and man, two almost parallel lipoprotein precipitates were observed in the a-region. Albumin was also stained with Sudan Black. Thus the quantitative evaluation of a-lipoprotein after zone electrophoresis is uncertain especially when albumin and a-lipoprotein migrate at identical rates as in the serum of pig and man. However, with the two-dimensional immunoelectrophoretic technique, Nbumin and c~-lipoprotein complexes can be evaluated independently of each other.
1. INTRODUCTION Whether a-lipoprotein is a homogeneous fraction in the electrophoretic pattern of serum and whether albumin may interfere with interpretation are controversial matters. The nature of pre-~-lipoprotein in human serum has been the subjecl of much discussion (Dyerberg and Hj~rne, 1971). Whereas some authors believed that this fraction was due to staining of the fatty acids bound to albumin, others considered that it was either an artifact or a true lipoprotein (Postma and Stroes, 1968; Dyerberg and Hj~Crne, 1970). The various results obtained may in part depend upon the different media used for electrophoresis; paper (Lees and Hatch, 1963; Fredrickson et al., 1967), agarose (Rapp and K',dalke, 1968), agarose with albumin (ZiSllner et at., 1969), polyacrylamide (Pratt and Dangerfield, 1969), cellulose acetate (Chin and Blankenhorn, 1968) and starch gel (Scanu, 1966). Moreover, lipoprotein nomenclature is not uniform (Peyer and Rieder, 1970). immunoelectrophoresis of human serum indicates a heterogeneity of the a-lipoprotem fraction (Fredrickson et al., 1967; Scheiffarth et al., 1968; Uriel and Grabar, 1956). Studies now in progress in our laboratory are aimed at investigating the physiological roles of lipoproteins in mammals. In this report some results are presented and discussed on ¢~-lipoproteins in the serum of the rat, the mouse, the mongolian gerbil and the pig. Human serum is used as a reference. The results were obtained by the two-dimensional immunoelectrophoresis of Davies et al. (1971). This tech375
376
B.W. KARLSSON and M.B.N. JOItANSSON
nique gives a better resolution of the e~-lipoprotein complex and a far better separation of albumin and c~-lipoprotein than ordinary immunoelectrophoresis or zone electrophoresis.
2. MATERIALS AND METHODS Blood from adult rat, mouse and gerbil (Meriones unguiculatus) was obtained by decapitation or by bleeding the tip of the tail. Pig blood was collected at slaughter from 5 - 6 months old pigs. Human capillary blood was obtained from eight healthy volunteers. Serum was prepared by the centrifugation of clotted blood. Analyses were performed on fresh serum or on serum samples which had been stored at -20°C for 1 - 1 0 days. Repeated freezing and thawing affected the lipoprotein patterns, as revealed by zone electrophoresis. Antisera to serum from rat, mouse, gerbil and pig were prepared in rabbits weighing 2 . 5 - 3 kg. Freund's adjuvant incomplete (l)ifco Laboratories, Detroit) was mixed with an equal volume of serum and emulsified at low speed with a knife homogenizer. Two injections of this mixture were given at 5 - 6 week intervals. Blood samples were taken every other day during the 8 - 2 0 days following the second injection. Antisera to human serum, human ee-lipoprotein and human/3-1ipoprotein were purchased from Behringwerke AG (Batches nos. T 1892, A 1707T and
1868V). lmmunoelectrophoresis and zone electrophoresis were performed in I 1.5% agarose (l'lndustrie Biologique Fran~aise, S.A.) or in agarose with the addition of 0.26% albumin (Albumin, Fraction V, Bovine, Sigma No A-4503) in HCI sodium veronal buffer (pH 8.6). Two-dimensional immunoelectrophoresis was performed by the modification described by Davies et al. (1970). Details of the technique are found in fig. 1. Fixation after zone electrophoresis was performed in 2% acetic acid for 4 5 - 6 0 rain. Staining of plates from the electrophoretic and the immunological techniques used was usually performed for 2 h r in 0.1% Sudan Black B in 60% ethanol for visualization of lipoproteins. Proteins were stained in 0.1% Amido Black 10 B or in 0.1% Azocarmine 10 B for 10 60 rain. High density (HDL), low density (LDL) and very low density (VLDL) lipoproreins were isolated according to the procedure described by Burstein et al. (1970) for human serum. The details of this procedure and the results will be reported in a future publication.
3. RESULTS With the two-dimensional immunoelectrophoretic technique c~-lipoproteins were localised either ~'ithin or in the vicinity of the albumin fraction for all the five
a-Lipoprotein heterogeneity
377
B A
~iiiiiiiiiiiiiii!iii~ il~iiiiiii!!!iiiiiii~iliiiiiiii!i~i~ii~ !i!::::i;:i~i~i~:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::[~ : :::::::::::::::::::::::::::
iiiiiliiii iiiiiiiiiiiii iiiii i iiiiiii;ili A
A
I~-L
I
i
e-I. .:.:.:.:.: ..................................................-....,-
iiii ii 1iiiiiig!iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii!iii!iiiiiii!iiiiiii
A a-I.
i i
__~
=
•
.
_
.~
,~-I. I i
::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ====================================================
Fig. 1. c~-Lipoprotein (c~-L) and a l b u m i n (A) p a t t e r n s in serum of m o u s e (A), m o n g o l i a n gerbil (B), rat (C), pig (D) and m a n (E,F). T w o - d i m e n s i o n a l i m m u n o e l e c t r o p h o r e s i s in agarose. In A - E antisera to whole sera were used. In F an a n t i s e r u m to h u m a n a-lipoprotein was used. Samples of 5 ~tl of serum dilutions 1 : 8 (1 : 16 for A) were applied at the dark spot in the lower left corner.
378
B.W. KARLSSON and M.B.N. JOHANSSON
species studied (fig. I A- F). The albumin precipitate of the pig, man, rat and gerbil showed detectable lipoprotem staining while the albumin precipitate of mouse serum was not stained. In the serum of rat and mouse the o~-lipoprotein has a greater electrophoretic mobility than albumin (fig. I A,C). If albumin migration is taken as 1, the relative migration rates of the oe-lipoproteins are 1.11 for tire mouse and 1.23 for the rat. For the gerbil the corresponding value is 0.88, for man 0.97 and 0.95 for pig (fig. IB,D,E). The a-lipoprotein complex consists of one homogeneous precipitate in the mouse (fig. 1A) and the rat (fig. IC). Two almost parallel homogeneous precipitates are found for the gerbil (fig. 1B) and man (fig. 1E). In pig serum (fig. 1D) the curvature of the ~-lipoprotein precipitate is asynmtetric. The reason for this ntay be that an additional a-lipoprotein is present, migrating faster than albumin. A similar tendency was observed for human o~-lipoprotein when human serum was electrophorized into gels containing anti-human o~-lipoprotein (fig. 1F). The heterogeneity of the oe-lipoprotein complex in the serum of gerbil was further demonstrated when isolated HDL was tested with antigerbil serum. Three parallel precipitates were observed in these plates. In one series of experiments (fig. 2), serum from newborn unsuckled pigs and pig foetuses were analysed with two-dimensional immunoelectrophoresis using an antiserum to foetal pig serum in the gel. The albumin arc is very small compared with the c~-lipoprotein arc. The asymmetry of the a-lipoprotein arc is evident {,fig. 2). The albumin precipitate was stained with Sudan Black. However, no stain was detected in the ~-foetoprotein precipitate (not recorded in fig. 2).
Fig. 2. Two-dimensional immunoelectrophoresis of serum from newborn piglet, not fed with colostrum. Dilution 1: 1. Note the smaU albumin arc (A) and the heterogeneity in the a-lipoprotein precipitate (a-L). Two sudanophilic precipitates are found in the &lipoprotein region g3-L).
oeLipoprotein heterogeneity
379
4. DISCUSSION The results obtained in this study confirm that c~-lipoprotein is a serum fraction different from albumin. This can be demonstrated without question in the mouse, rat and gerbil, since the a-lipoproteins in the serum of these species migrate as preor postalbumins. For pig and man, on the other hand, the electrophoretic migration rates of c~-lipoprotein and albumin are very similar. However, with the two-dimensional immunoelectrophoretic technique, it was possible to distinguish between albumin and c~-lipoprotein in these two species. The experiments provide evidence for the heterogeneity of the ~-lipoprotein fraction. Thus, a pre-~-lipoprotein precipitate was found in pig serum, though it did not separate completely from the main a-lipoprotein. A similar tendency was found in human serum, but only when anti-~-lipoprotein serum was used. It is possible that these precipitates correspond to the p-lipoproteins described for human sera by Uriel and Grabar (1956) and supposed to be degradation products after storage of the serum. Since the human sera used in the present experiments were fresh, the results may indicate a pre-c~-lipoprotein fraction of very low concentration. Lipoproteins migrating as prealbumins have earlier been detected by immunoelectrophoresis in porcine serum (Karlsson, 1966). Another type of heterogeneity was recorded in sera of the mongolian gerbil and man, where two almost parallel precipitates were recorded in the c~-region. A closer analysis of these precipitates showed that they are slightly displaced from each other. The results may indicate that there is a microheterogeneity in the a-lipoprorein region. A convincing example of the non-interference of albumin with c~-lipoprotein is the serum pattern of the newborn piglet. The albumin level is low at birth, but rises manyfold within the first days of postnatal development (Karlsson, 1970). However, the resulting increase in the albumin precipitate in the immunopattern of piglet's serum is in no way correlated to the developmental changes in the a-lipoprotein precipitates. For all the species concerned, the albumin precipitates were stained more or less intensively with Sudan Black. This was also true for the albumin precipitates in the serum of newborn piglets. There was no lipoprotein staining of the precipitates corresponding to a-foetoprotein with any of the techniques used.
ACKNOWLEDGEMENTS The skilfull technical assistance of Mrs. Marie Adler-Maihofer and the secreterial help of Miss Marianne Andersson are gratefully acknowledged. Financial support was given by the Swedish National Sciences Research Council and by the Royal Physiographic Society of Lund.
380
B.W. KARLSSON and M.B.N. JOHANSSON
REFERENCES Burstein, M., H.R. Scholnick and R. Morfin, 1970, J. Lipid Res. 11,583. Chin, t|.P. and D.H. Blankenhorn, 1968, Clin. Chim. Acta 20, 305. Davies, D.R., E.D. Spurt and J.B. Versey, 1971, Clin. Science 40, 411. Dyerberg, J. and N. !lj~rne, 1970, Clin. Chim. Acta 28, 203. Dyerberg, J. and N. ttjorne, 1971, Clin. Chim. Acta 33,458. Fredrickson, D.S., R.I. Levy and R.S. Lees, 1967, New Engl. J. Med. 276: 34, 94, 148, 215, 273. Karlsson, B.W., 1966, Acta Path. Microbiol. Scand. 67, 83. Karlsson, B.W., 1970, Comp. Biochem. Physiol. 34, 535. Lees, R.S. and F.T. Hatch, 1963, J. Lab. Clin. Med. 61,518. Peyer, Ch. and H.P. Rieder, 1970, Clin. Chim. Acta 30, 295. Postma, T. and J.A.P. Stroes, 1968, Clin. Chim. Acta 22,569. Pratt, J.J. and W.G. Dangerfield, 1969, Clin. Chim. Acta 23, 189. Rapp, W. and W. Kahlke, 1968, Clin. Chim. Acta 19,493. Scanu, A., 1966, J. Lipid Res. 7, 295. Scheiffarth, F., H. G0tz and H. Griehl, 1968, Z. Klin. Chem. Klin. Biochem. 6, 89. Uriel, J. and P. Grabar, 1956, Bull. Soc. Chim. Biol. 38, 1253. Z~511ner, N., W. GriSbner, C. Berger and G. Wolfram, 1969, Z. Klin. Chem. Klin. Biochem. 7, 525.
a-LIPOPROTEIN
HETEROGENEITY
IN S E R U M O F M A M M A L S
Bi)rje W. KARLSSON and Magnus B.N. JOHANSSON Institute of Zoophysiology, University of Lund, Sweden
Received 9 January 1972
Accepted 6 January 1972
Two-dimensional immunoelectrophoresis on the serum of mouse, rat, mongolian gerbil, pig and man indicates heterogeneity in c~-lipoprotein. A pre-c~-lipoproteinwas lbund in pig serum. Traces of a similar fraction could also be detected in human serum. In the serum of the mongolian gerbil and man, two almost parallel lipoprotein precipitates were observed in the a-region. Albumin was also stained with Sudan Black. Thus the quantitative evaluation of a-lipoprotein after zone electrophoresis is uncertain especially when albumin and a-lipoprotein migrate at identical rates as in the serum of pig and man. However, with the two-dimensional immunoelectrophoretic technique, Nbumin and c~-lipoprotein complexes can be evaluated independently of each other.
1. INTRODUCTION Whether a-lipoprotein is a homogeneous fraction in the electrophoretic pattern of serum and whether albumin may interfere with interpretation are controversial matters. The nature of pre-~-lipoprotein in human serum has been the subjecl of much discussion (Dyerberg and Hj~rne, 1971). Whereas some authors believed that this fraction was due to staining of the fatty acids bound to albumin, others considered that it was either an artifact or a true lipoprotein (Postma and Stroes, 1968; Dyerberg and Hj~Crne, 1970). The various results obtained may in part depend upon the different media used for electrophoresis; paper (Lees and Hatch, 1963; Fredrickson et al., 1967), agarose (Rapp and K',dalke, 1968), agarose with albumin (ZiSllner et at., 1969), polyacrylamide (Pratt and Dangerfield, 1969), cellulose acetate (Chin and Blankenhorn, 1968) and starch gel (Scanu, 1966). Moreover, lipoprotein nomenclature is not uniform (Peyer and Rieder, 1970). immunoelectrophoresis of human serum indicates a heterogeneity of the a-lipoprotem fraction (Fredrickson et al., 1967; Scheiffarth et al., 1968; Uriel and Grabar, 1956). Studies now in progress in our laboratory are aimed at investigating the physiological roles of lipoproteins in mammals. In this report some results are presented and discussed on ¢~-lipoproteins in the serum of the rat, the mouse, the mongolian gerbil and the pig. Human serum is used as a reference. The results were obtained by the two-dimensional immunoelectrophoresis of Davies et al. (1971). This tech375
376
B.W. KARLSSON and M.B.N. JOItANSSON
nique gives a better resolution of the e~-lipoprotein complex and a far better separation of albumin and c~-lipoprotein than ordinary immunoelectrophoresis or zone electrophoresis.
2. MATERIALS AND METHODS Blood from adult rat, mouse and gerbil (Meriones unguiculatus) was obtained by decapitation or by bleeding the tip of the tail. Pig blood was collected at slaughter from 5 - 6 months old pigs. Human capillary blood was obtained from eight healthy volunteers. Serum was prepared by the centrifugation of clotted blood. Analyses were performed on fresh serum or on serum samples which had been stored at -20°C for 1 - 1 0 days. Repeated freezing and thawing affected the lipoprotein patterns, as revealed by zone electrophoresis. Antisera to serum from rat, mouse, gerbil and pig were prepared in rabbits weighing 2 . 5 - 3 kg. Freund's adjuvant incomplete (l)ifco Laboratories, Detroit) was mixed with an equal volume of serum and emulsified at low speed with a knife homogenizer. Two injections of this mixture were given at 5 - 6 week intervals. Blood samples were taken every other day during the 8 - 2 0 days following the second injection. Antisera to human serum, human ee-lipoprotein and human/3-1ipoprotein were purchased from Behringwerke AG (Batches nos. T 1892, A 1707T and
1868V). lmmunoelectrophoresis and zone electrophoresis were performed in I 1.5% agarose (l'lndustrie Biologique Fran~aise, S.A.) or in agarose with the addition of 0.26% albumin (Albumin, Fraction V, Bovine, Sigma No A-4503) in HCI sodium veronal buffer (pH 8.6). Two-dimensional immunoelectrophoresis was performed by the modification described by Davies et al. (1970). Details of the technique are found in fig. 1. Fixation after zone electrophoresis was performed in 2% acetic acid for 4 5 - 6 0 rain. Staining of plates from the electrophoretic and the immunological techniques used was usually performed for 2 h r in 0.1% Sudan Black B in 60% ethanol for visualization of lipoproteins. Proteins were stained in 0.1% Amido Black 10 B or in 0.1% Azocarmine 10 B for 10 60 rain. High density (HDL), low density (LDL) and very low density (VLDL) lipoproreins were isolated according to the procedure described by Burstein et al. (1970) for human serum. The details of this procedure and the results will be reported in a future publication.
3. RESULTS With the two-dimensional immunoelectrophoretic technique c~-lipoproteins were localised either ~'ithin or in the vicinity of the albumin fraction for all the five
a-Lipoprotein heterogeneity
377
B A
~iiiiiiiiiiiiiii!iii~ il~iiiiiii!!!iiiiiii~iliiiiiiii!i~i~ii~ !i!::::i;:i~i~i~:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::[~ : :::::::::::::::::::::::::::
iiiiiliiii iiiiiiiiiiiii iiiii i iiiiiii;ili A
A
I~-L
I
i
e-I. .:.:.:.:.: ..................................................-....,-
iiii ii 1iiiiiig!iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii!iii!iiiiiii!iiiiiii
A a-I.
i i
__~
=
•
.
_
.~
,~-I. I i
::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ====================================================
Fig. 1. c~-Lipoprotein (c~-L) and a l b u m i n (A) p a t t e r n s in serum of m o u s e (A), m o n g o l i a n gerbil (B), rat (C), pig (D) and m a n (E,F). T w o - d i m e n s i o n a l i m m u n o e l e c t r o p h o r e s i s in agarose. In A - E antisera to whole sera were used. In F an a n t i s e r u m to h u m a n a-lipoprotein was used. Samples of 5 ~tl of serum dilutions 1 : 8 (1 : 16 for A) were applied at the dark spot in the lower left corner.
378
B.W. KARLSSON and M.B.N. JOHANSSON
species studied (fig. I A- F). The albumin precipitate of the pig, man, rat and gerbil showed detectable lipoprotem staining while the albumin precipitate of mouse serum was not stained. In the serum of rat and mouse the o~-lipoprotein has a greater electrophoretic mobility than albumin (fig. I A,C). If albumin migration is taken as 1, the relative migration rates of the oe-lipoproteins are 1.11 for tire mouse and 1.23 for the rat. For the gerbil the corresponding value is 0.88, for man 0.97 and 0.95 for pig (fig. IB,D,E). The a-lipoprotein complex consists of one homogeneous precipitate in the mouse (fig. 1A) and the rat (fig. IC). Two almost parallel homogeneous precipitates are found for the gerbil (fig. 1B) and man (fig. 1E). In pig serum (fig. 1D) the curvature of the ~-lipoprotein precipitate is asynmtetric. The reason for this ntay be that an additional a-lipoprotein is present, migrating faster than albumin. A similar tendency was observed for human o~-lipoprotein when human serum was electrophorized into gels containing anti-human o~-lipoprotein (fig. 1F). The heterogeneity of the oe-lipoprotein complex in the serum of gerbil was further demonstrated when isolated HDL was tested with antigerbil serum. Three parallel precipitates were observed in these plates. In one series of experiments (fig. 2), serum from newborn unsuckled pigs and pig foetuses were analysed with two-dimensional immunoelectrophoresis using an antiserum to foetal pig serum in the gel. The albumin arc is very small compared with the c~-lipoprotein arc. The asymmetry of the a-lipoprotein arc is evident {,fig. 2). The albumin precipitate was stained with Sudan Black. However, no stain was detected in the ~-foetoprotein precipitate (not recorded in fig. 2).
Fig. 2. Two-dimensional immunoelectrophoresis of serum from newborn piglet, not fed with colostrum. Dilution 1: 1. Note the smaU albumin arc (A) and the heterogeneity in the a-lipoprotein precipitate (a-L). Two sudanophilic precipitates are found in the &lipoprotein region g3-L).
oeLipoprotein heterogeneity
379
4. DISCUSSION The results obtained in this study confirm that c~-lipoprotein is a serum fraction different from albumin. This can be demonstrated without question in the mouse, rat and gerbil, since the a-lipoproteins in the serum of these species migrate as preor postalbumins. For pig and man, on the other hand, the electrophoretic migration rates of c~-lipoprotein and albumin are very similar. However, with the two-dimensional immunoelectrophoretic technique, it was possible to distinguish between albumin and c~-lipoprotein in these two species. The experiments provide evidence for the heterogeneity of the ~-lipoprotein fraction. Thus, a pre-~-lipoprotein precipitate was found in pig serum, though it did not separate completely from the main a-lipoprotein. A similar tendency was found in human serum, but only when anti-~-lipoprotein serum was used. It is possible that these precipitates correspond to the p-lipoproteins described for human sera by Uriel and Grabar (1956) and supposed to be degradation products after storage of the serum. Since the human sera used in the present experiments were fresh, the results may indicate a pre-c~-lipoprotein fraction of very low concentration. Lipoproteins migrating as prealbumins have earlier been detected by immunoelectrophoresis in porcine serum (Karlsson, 1966). Another type of heterogeneity was recorded in sera of the mongolian gerbil and man, where two almost parallel precipitates were recorded in the c~-region. A closer analysis of these precipitates showed that they are slightly displaced from each other. The results may indicate that there is a microheterogeneity in the a-lipoprorein region. A convincing example of the non-interference of albumin with c~-lipoprotein is the serum pattern of the newborn piglet. The albumin level is low at birth, but rises manyfold within the first days of postnatal development (Karlsson, 1970). However, the resulting increase in the albumin precipitate in the immunopattern of piglet's serum is in no way correlated to the developmental changes in the a-lipoprotein precipitates. For all the species concerned, the albumin precipitates were stained more or less intensively with Sudan Black. This was also true for the albumin precipitates in the serum of newborn piglets. There was no lipoprotein staining of the precipitates corresponding to a-foetoprotein with any of the techniques used.
ACKNOWLEDGEMENTS The skilfull technical assistance of Mrs. Marie Adler-Maihofer and the secreterial help of Miss Marianne Andersson are gratefully acknowledged. Financial support was given by the Swedish National Sciences Research Council and by the Royal Physiographic Society of Lund.
380
B.W. KARLSSON and M.B.N. JOHANSSON
REFERENCES Burstein, M., H.R. Scholnick and R. Morfin, 1970, J. Lipid Res. 11,583. Chin, t|.P. and D.H. Blankenhorn, 1968, Clin. Chim. Acta 20, 305. Davies, D.R., E.D. Spurt and J.B. Versey, 1971, Clin. Science 40, 411. Dyerberg, J. and N. !lj~rne, 1970, Clin. Chim. Acta 28, 203. Dyerberg, J. and N. ttjorne, 1971, Clin. Chim. Acta 33,458. Fredrickson, D.S., R.I. Levy and R.S. Lees, 1967, New Engl. J. Med. 276: 34, 94, 148, 215, 273. Karlsson, B.W., 1966, Acta Path. Microbiol. Scand. 67, 83. Karlsson, B.W., 1970, Comp. Biochem. Physiol. 34, 535. Lees, R.S. and F.T. Hatch, 1963, J. Lab. Clin. Med. 61,518. Peyer, Ch. and H.P. Rieder, 1970, Clin. Chim. Acta 30, 295. Postma, T. and J.A.P. Stroes, 1968, Clin. Chim. Acta 22,569. Pratt, J.J. and W.G. Dangerfield, 1969, Clin. Chim. Acta 23, 189. Rapp, W. and W. Kahlke, 1968, Clin. Chim. Acta 19,493. Scanu, A., 1966, J. Lipid Res. 7, 295. Scheiffarth, F., H. G0tz and H. Griehl, 1968, Z. Klin. Chem. Klin. Biochem. 6, 89. Uriel, J. and P. Grabar, 1956, Bull. Soc. Chim. Biol. 38, 1253. Z~511ner, N., W. GriSbner, C. Berger and G. Wolfram, 1969, Z. Klin. Chem. Klin. Biochem. 7, 525.