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Human serum sialo transferrins in diseases
141
CIinica Chimicu Acta, 165 (1987) 141-145 Elsevier
CCA 03845
Human serum sialo transferrins in diseases H.G. van Eijk a, W.L. van Noort
a, G. de Jong a and J.F. Koster
b
Departments of a Chemical Pathology and b Biochemistry, Medical Faculty, Erasmus Universtty Rotterdam (The NetherlanA) Key worak Dry transferrins; Isoelectric focusing; Immunobiline
Summary
Using isoelectric focusing and crossed immunoelectrophoresis on ready-made Immobiline Dry Plates, pH 5-6, we were able to separate human serum transferrin in subfractions with different sialo acid content. The amount of these subfractions is significant different in sera of patients with diseases like CA, RA, haemochromatosis and in sera of pregnant women.
Introduction
Interest in carbohydrates and the carbohydrate moiety of serum proteins has grown considerably during the last ten years. The functions of the carbohydrate moiety of the glycoproteins have been studied [1,2]. The structural diversity of glycoprotein glycans is wide [2]. Changes in the carbohydrate chain are reported, e.g. for tissue injury, inflammation, rheumatoid arthritis, cancer, pregnancy and liver diseases [3-51. Human transferrin preparations isolated by preparative isoelectric focusing from normal and pathological sera show microheterogeneity which is caused by differences in the carbohydrate chain, mainly in the sialic acid content [6]. It is known that in alcoholics a disialo transferrin fraction is increased [7] and in patients with a demyelinating disease in serum as well as in liquor cerebrospinalis the four sialo transferrin fraction is strongly diminished while the disialo and the asialo transferrin fractions are considerably increased [8]. In this paper, we present the separation of transferrin subfractions in sera of patients on ready-made Immobiline gels with a high separation capacity [9]. Correspondence to: H.G. van Eyk, Department University Rotterdam, The Netherlands.
0009-8981/87/$03.50
of Chemical Pathology, Medical Faculty,
0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Erasmus
142
Materials and methods
Non-heparinized blood was centrifuged for 20 rnin at 4°C at 1200 x g, the serum was stored at - 20” C until use. Normal sera were obtained from healthy volunteers. Other sera were obtained from patients, as follows: (a) cancer, patients with breast carcinoma with metastases, mesothelioma, adeno carcinoma, myelomatosis with K chains; (b) haemochromatosis, patients with idiopathic haemochromatosis with high serum Fe and ferritin; (c) rheumatoid arthritis, patients with acute RA; (d) pregnancy, women at term. In all sera, the transferrin was saturated with Fe, using Fe(II1) citrate. Isoelectric focusing and crossed immunoelectrophoresis using ready-made 10 mmol/l in Immobiline Dry Plates, pH 5-6 (LKB, Stockholm, Sweden) have been described in detail [6,9,10]. All determinations were performed in triplicate. Results
In Fig. 1 we show the result of a separation of a diferric transferrin preparation on Immobiline gels. The fractions are tentatively called asialo, mono-, di-, tri-, tetra-, penta-, hexa- and heptasialo transferrin. Seventy to eighty percent are present as tetrasialo transferrin, a molecule with two biantennary chains. Higher sialo transferrins contain more branched carbohydrate chains. In Fig. 2, examples are given of crossed immunoelectrofocusing patterns of sera from a healthy control, a pregnant woman, a cancer patient, a rheumatoid patient and a haemochromatosis patient. Except for the healthy control, they all showed increased 5 and 6 sialo transferrin fractions. For comparison also the pattern of a child of 6 year with a demyelinating disease [8] is shown, in which the di- and asialo fractions have increased considerably. In Table I, the proportioned contributions of the different sialo transferrins are shown as well as the ratio 5 + 6 + 7 sialo/2 + 3 sialo transferrin. From Fig. 2 and Table I it is clear that in CA, RA, hemochromatosis patients and pregnancy the ratio between 5 + 6 + 7 sialo/2 + 3 sialo transferrin has in-
HUMAN
7
6
5
TRANSFERRINS
4
Rg. 1. Crossed immunoelectrofocusing heptasialo, diferric human transferrin.
3
2
pattern
of human
1
0 transferrin.
0, asialo;
1, mono
sialo
+
7,
8 8 12 5 10 4
Controls a R.A. C.A. Pregnancy Haemochromatosis Demyehnating
0.1 0.5 0.2 -
7
5 16 k1.4 19.5 + 2.6 26 +6 23.4&3 20 f2 6 +4
6
2.1+ 1.4 2.8& 1.0 6.3 +4.0 6.6k2.6 3.9*1.5 0.8 f 0.8
Sialo transferrins
SD
73f 3 73% 3 61+12 61f 5 69k 4 28+ 9
4
a Controls significant different from the patholo~cai sera ( p < 0.05).
n
Patients
Distribution of transferrin subfractions in % f
TABLE I
7.6 It 2.0 4.4f 1.9 5.5*3.3 6.Oirl 6.Oic1.8 7.5f1.7
3 1.2kl.O l.lkO.8 1.5kl.O 2.3*0.5 1.5*0.7 37 k-7
2 3.4f 1.8
1
18&5
0
2.2 iO.8 4.9 &2.3 5.3 +2.4 3.6 50.5 3.6 kl.6 0.12+0.1
3+2(+1+0)
5+6+1 sialo-transfertins
Mean of individual ratios *
Fig. 2. Crossed immunoelectrofocusing patterns of transferrin subfractions (see Fig. 1). A, normal; B, R.A.; C, demyelinating [8]; D, haemochromatosis; E, pregnancy; F, CA (Kahler).
creased significantly, mainly due to the presence of higher sialylated carbohydrate chains on transferrin in the sera of these patients. Discussion
Oligosaccharide moieties with bi-, tri-, or tetraantennary arrangements are involved in the transport of glycoprotein molecules to the target cellular compartments as well as in the initial phase of catabolism. There is a carbohydrate recognition system on the surface of hepatocytes, fibroblasts, lymphocytes, RES cells and probably spermatozoa [ll]. By desialylation, the glycoproteins occur as galactoproteins which are rapidly recognised by galactose-specific membrane receptors on the liver and lymphocytes. Desialylation starts at the site of tissue damage by the action of intracellular sialidase released from lysosomes of broken cells. Desialylated proteins are then transported through the body to their target-organs. The pattern of sialo transferrins is changed in several diseases. One can speculate about the cause of this phenomenon. Is there, aside from changes in the intracellular synthesis of the carbohydrate chains, or the post-secretory degradation of the N-linked oligosaccharides, also a selection of certain antemraxy chains by different cell systems? Do transferrins with a higher branched carbohydrate chain have a higher affinity for liver cells and release more iron to the liver in cases of haemochromatosis or do changes in the carbohydrate structure in pregnancy play a role in the placental iron transport? - questions which require answering. The heterogeneity of serum transferrins, if functional, opens a field for investigations. Acknowledgements
The authors wish to thank Drs. H. Kreeftenberg, for the serum samples of patients.
S. Lamberts and H. Wallenburg
145
References 1 Bezouska K, Taborsky 0, Kubrycht J, Pospisil M, Kocourek J. Carbohydrate-structure-dependent recognition of desialyated serum glycoproteins in the liver and leucocytes. Biochem J 1985;227: 345-354. 2 Berger EG, Buddecke E, Kamerling JP, Kobata A, Vliegenthart JFG. Structure, biosynthesis and function of glycoprotein glycans. Experientia 1982;38:1129-1258. 3 Parekb RB, Dwekm RA, Sutton BJ, et al. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 1985;316:452-457. 4 Poupon RE, Papoz L, Sarmini H, Elinck R. A study of the microheterogeneity of transferrin in cirrhotic patients. Clin Chim Acta 1985;151:245-251. 5 Raynes J. Variations in the relative proportions of microheterogeneous forms of plasma glycoproteins in pregnancy and disease. Biomedicine 1982;36:77-86. 6 Van Eijk HG, Van Noort WL, Dubelaar ML, Van der Heul C. The microheterogeneity of human transferrins in biological fluids. Clin Chim Acta 1983;132:167-171. 7 Stibler H, Sydow 0, Van den Berg S. Quantitative estimation of a normal microheterogeneity of serum transferrin in alcoholics. Pharmacol Biochem behav 1980; 13 suppl.l:47-51. 8 Jaeken J, Van Eijk HG, Van der Heul C, Corbeel L, Beckels R, Eggermont E. Sialic acid-deficient serum and cerebra spinal fluid transferrin in a newly recognized genetic syndrome. Clin Chim Acta 1984;144:245-247. 9 Van Noort WL, Van Eijk HG. Quantitation of subfractions of serum transferrins using crossed immuno focusing with LKB immobihn dry plate. Sci Tools 1987; in press. 10 Van Eijk HG, Van Noort WL, Kroos, MJ, Van der Heul C. The heterogeneity of human transferrin and transferrin preparations in isoelectric focusing gels. Clin Chim Acta 1982;121:2009-2116, 11 Brandlev BK, Schanar RL. Cell surface carbohydrates in cell recognition and response. J Leucocyte Biol 1986;40:97-111.
CIinica Chimicu Acta, 165 (1987) 141-145 Elsevier
CCA 03845
Human serum sialo transferrins in diseases H.G. van Eijk a, W.L. van Noort
a, G. de Jong a and J.F. Koster
b
Departments of a Chemical Pathology and b Biochemistry, Medical Faculty, Erasmus Universtty Rotterdam (The NetherlanA) Key worak Dry transferrins; Isoelectric focusing; Immunobiline
Summary
Using isoelectric focusing and crossed immunoelectrophoresis on ready-made Immobiline Dry Plates, pH 5-6, we were able to separate human serum transferrin in subfractions with different sialo acid content. The amount of these subfractions is significant different in sera of patients with diseases like CA, RA, haemochromatosis and in sera of pregnant women.
Introduction
Interest in carbohydrates and the carbohydrate moiety of serum proteins has grown considerably during the last ten years. The functions of the carbohydrate moiety of the glycoproteins have been studied [1,2]. The structural diversity of glycoprotein glycans is wide [2]. Changes in the carbohydrate chain are reported, e.g. for tissue injury, inflammation, rheumatoid arthritis, cancer, pregnancy and liver diseases [3-51. Human transferrin preparations isolated by preparative isoelectric focusing from normal and pathological sera show microheterogeneity which is caused by differences in the carbohydrate chain, mainly in the sialic acid content [6]. It is known that in alcoholics a disialo transferrin fraction is increased [7] and in patients with a demyelinating disease in serum as well as in liquor cerebrospinalis the four sialo transferrin fraction is strongly diminished while the disialo and the asialo transferrin fractions are considerably increased [8]. In this paper, we present the separation of transferrin subfractions in sera of patients on ready-made Immobiline gels with a high separation capacity [9]. Correspondence to: H.G. van Eyk, Department University Rotterdam, The Netherlands.
0009-8981/87/$03.50
of Chemical Pathology, Medical Faculty,
0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Erasmus
142
Materials and methods
Non-heparinized blood was centrifuged for 20 rnin at 4°C at 1200 x g, the serum was stored at - 20” C until use. Normal sera were obtained from healthy volunteers. Other sera were obtained from patients, as follows: (a) cancer, patients with breast carcinoma with metastases, mesothelioma, adeno carcinoma, myelomatosis with K chains; (b) haemochromatosis, patients with idiopathic haemochromatosis with high serum Fe and ferritin; (c) rheumatoid arthritis, patients with acute RA; (d) pregnancy, women at term. In all sera, the transferrin was saturated with Fe, using Fe(II1) citrate. Isoelectric focusing and crossed immunoelectrophoresis using ready-made 10 mmol/l in Immobiline Dry Plates, pH 5-6 (LKB, Stockholm, Sweden) have been described in detail [6,9,10]. All determinations were performed in triplicate. Results
In Fig. 1 we show the result of a separation of a diferric transferrin preparation on Immobiline gels. The fractions are tentatively called asialo, mono-, di-, tri-, tetra-, penta-, hexa- and heptasialo transferrin. Seventy to eighty percent are present as tetrasialo transferrin, a molecule with two biantennary chains. Higher sialo transferrins contain more branched carbohydrate chains. In Fig. 2, examples are given of crossed immunoelectrofocusing patterns of sera from a healthy control, a pregnant woman, a cancer patient, a rheumatoid patient and a haemochromatosis patient. Except for the healthy control, they all showed increased 5 and 6 sialo transferrin fractions. For comparison also the pattern of a child of 6 year with a demyelinating disease [8] is shown, in which the di- and asialo fractions have increased considerably. In Table I, the proportioned contributions of the different sialo transferrins are shown as well as the ratio 5 + 6 + 7 sialo/2 + 3 sialo transferrin. From Fig. 2 and Table I it is clear that in CA, RA, hemochromatosis patients and pregnancy the ratio between 5 + 6 + 7 sialo/2 + 3 sialo transferrin has in-
HUMAN
7
6
5
TRANSFERRINS
4
Rg. 1. Crossed immunoelectrofocusing heptasialo, diferric human transferrin.
3
2
pattern
of human
1
0 transferrin.
0, asialo;
1, mono
sialo
+
7,
8 8 12 5 10 4
Controls a R.A. C.A. Pregnancy Haemochromatosis Demyehnating
0.1 0.5 0.2 -
7
5 16 k1.4 19.5 + 2.6 26 +6 23.4&3 20 f2 6 +4
6
2.1+ 1.4 2.8& 1.0 6.3 +4.0 6.6k2.6 3.9*1.5 0.8 f 0.8
Sialo transferrins
SD
73f 3 73% 3 61+12 61f 5 69k 4 28+ 9
4
a Controls significant different from the patholo~cai sera ( p < 0.05).
n
Patients
Distribution of transferrin subfractions in % f
TABLE I
7.6 It 2.0 4.4f 1.9 5.5*3.3 6.Oirl 6.Oic1.8 7.5f1.7
3 1.2kl.O l.lkO.8 1.5kl.O 2.3*0.5 1.5*0.7 37 k-7
2 3.4f 1.8
1
18&5
0
2.2 iO.8 4.9 &2.3 5.3 +2.4 3.6 50.5 3.6 kl.6 0.12+0.1
3+2(+1+0)
5+6+1 sialo-transfertins
Mean of individual ratios *
Fig. 2. Crossed immunoelectrofocusing patterns of transferrin subfractions (see Fig. 1). A, normal; B, R.A.; C, demyelinating [8]; D, haemochromatosis; E, pregnancy; F, CA (Kahler).
creased significantly, mainly due to the presence of higher sialylated carbohydrate chains on transferrin in the sera of these patients. Discussion
Oligosaccharide moieties with bi-, tri-, or tetraantennary arrangements are involved in the transport of glycoprotein molecules to the target cellular compartments as well as in the initial phase of catabolism. There is a carbohydrate recognition system on the surface of hepatocytes, fibroblasts, lymphocytes, RES cells and probably spermatozoa [ll]. By desialylation, the glycoproteins occur as galactoproteins which are rapidly recognised by galactose-specific membrane receptors on the liver and lymphocytes. Desialylation starts at the site of tissue damage by the action of intracellular sialidase released from lysosomes of broken cells. Desialylated proteins are then transported through the body to their target-organs. The pattern of sialo transferrins is changed in several diseases. One can speculate about the cause of this phenomenon. Is there, aside from changes in the intracellular synthesis of the carbohydrate chains, or the post-secretory degradation of the N-linked oligosaccharides, also a selection of certain antemraxy chains by different cell systems? Do transferrins with a higher branched carbohydrate chain have a higher affinity for liver cells and release more iron to the liver in cases of haemochromatosis or do changes in the carbohydrate structure in pregnancy play a role in the placental iron transport? - questions which require answering. The heterogeneity of serum transferrins, if functional, opens a field for investigations. Acknowledgements
The authors wish to thank Drs. H. Kreeftenberg, for the serum samples of patients.
S. Lamberts and H. Wallenburg
145
References 1 Bezouska K, Taborsky 0, Kubrycht J, Pospisil M, Kocourek J. Carbohydrate-structure-dependent recognition of desialyated serum glycoproteins in the liver and leucocytes. Biochem J 1985;227: 345-354. 2 Berger EG, Buddecke E, Kamerling JP, Kobata A, Vliegenthart JFG. Structure, biosynthesis and function of glycoprotein glycans. Experientia 1982;38:1129-1258. 3 Parekb RB, Dwekm RA, Sutton BJ, et al. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 1985;316:452-457. 4 Poupon RE, Papoz L, Sarmini H, Elinck R. A study of the microheterogeneity of transferrin in cirrhotic patients. Clin Chim Acta 1985;151:245-251. 5 Raynes J. Variations in the relative proportions of microheterogeneous forms of plasma glycoproteins in pregnancy and disease. Biomedicine 1982;36:77-86. 6 Van Eijk HG, Van Noort WL, Dubelaar ML, Van der Heul C. The microheterogeneity of human transferrins in biological fluids. Clin Chim Acta 1983;132:167-171. 7 Stibler H, Sydow 0, Van den Berg S. Quantitative estimation of a normal microheterogeneity of serum transferrin in alcoholics. Pharmacol Biochem behav 1980; 13 suppl.l:47-51. 8 Jaeken J, Van Eijk HG, Van der Heul C, Corbeel L, Beckels R, Eggermont E. Sialic acid-deficient serum and cerebra spinal fluid transferrin in a newly recognized genetic syndrome. Clin Chim Acta 1984;144:245-247. 9 Van Noort WL, Van Eijk HG. Quantitation of subfractions of serum transferrins using crossed immuno focusing with LKB immobihn dry plate. Sci Tools 1987; in press. 10 Van Eijk HG, Van Noort WL, Kroos, MJ, Van der Heul C. The heterogeneity of human transferrin and transferrin preparations in isoelectric focusing gels. Clin Chim Acta 1982;121:2009-2116, 11 Brandlev BK, Schanar RL. Cell surface carbohydrates in cell recognition and response. J Leucocyte Biol 1986;40:97-111.