ADONIS 003655139100057M
Scand J Clin Lab Invest 1991; 51: 411-415
Tetranectin in amniotic fluid, maternal serum and fetal fluids
Scand J Clin Lab Invest Downloaded from informahealthcare.com by ThULB Jena on 01/14/15 For personal use only.
E. V. S. HOGDALL," C . K. HQ)GDALL,t I. CLEMMENSEN,$ B. N0RGAARD-PEDERSEN" & A. G. R. LOFT" *Department of Clinical Biochemistry, Statens Seruminstitut. ?Department of Obstetrics and Gynecology, Rigshospitalet, ?Department of Obstetrics and Gynaecology, Hillerad County Hospital, $Department of Clinical Microbiology, Statens Seruminstitut, University of Copenhagen and SDakopatts NS, Glostrup, Denmark
H ~ g d a l EVS, l Hagdall CK, Clemmensen I , N~rgaard-PedersenB, Loft AGR. Tetranectin in amniotic fluid, maternal serum and fetal fluids. Scand J Clin Lab Invest 1991; 51: 411-415.. The concentration of the newly discovered protein tetranectin has been measured in different fetal and maternal compartments. In amniotic fluid a significant, positive correlation between the tetranectin concentration and gestational age was found (a mean of 0.2 mg I-' at week 14 to a mean of 0.5 mg I-' at week 21). In maternal serum a slight negative correlation was found between tetranectin concentration and gestational week (a mean of 6.17 mg 1-' at week 14 to a mean of 5.79 mg I-' at week 21). In-term cord blood collected at delivery a mean level of 6.0 mg I-' was found, and no difference was found between arterial- and venous-blood tetranectin concentration. In fetal serum the overall mean level was 2.6 mg 1-' and a significant positive correlation between tetranectin concentration and gestational age was found. The mean level was 1.1 mg 1-' in fetal cerebrospinal fluid and no correlation to gestational age was found. Fetal tetranectin may, therefore, be correlated to fetal maturation.
Key words: amniotic fluid; fetus; pregnancy; tetranectin Estrid V . S. H@gdall,Department of Clinical Biochemistry, Statens Seruminstitut, Artillerivej 5 , 2300 Copenhagen S Denmark.
The newly discovered plasma protein tetranectin (TN) [l] has been characterized as four noncovalently linked, identical peptide chains of 181 amino acids with a total M,of 20 000 [2]. TN is present in exocrine and endocrine glands as well as in secreting surface epithelia and mesenchyme, suggesting that TN, in conjunction with proteoglycans, may have a function in the packaging of granules or as a participant in exocytocic secretion of certain hormones [3, 41.
The plasma levels of TN are lower in the third than in the second trimester of pregnancy and during use of oral contraceptives, suggesting that the concentrations are affected by sex steroid hormone metabolism [5].Furthermore, the presence of TN in human embryonal fibroblasts [4,5], placenta [4] and cord blood [6] may indicate a role in embryogenesis and fetal growth. The purpose of the present study is to determine the normal levels of T N in amniotic
41 1
412
E. V. S. H@gdall et al.
fluid, maternal serum, cord blood, fetal serum and fetal cerebrospinal fluid.
MATERIALS AND METHODS
Scand J Clin Lab Invest Downloaded from informahealthcare.com by ThULB Jena on 01/14/15 For personal use only.
Study population.
Amniotic fluid samples were collected from 160 patients subjected to amniocentesis for chromosomal analysis. Only clear samples without visible blood contamination were used. Amniotic fluid samples were centrifuged at 2000 g for 10 min and stored at -20°C for 2 to 5 years before assay. Only samples from pregnancies with normal outcome were included in the study. Maternal serum samples were collected in 1988 from 121 pregnant women who had blood drawn for serum a-fetoprotein determination. All serum samples were prepared in the similar manner at one laboratory. Blood was left for clotting for 1 h at room temperature. After centrifugation at 2000 g for 10 min the serum samples were stored at -20 "C. Only samples from uncomplicated pregnancies with delivery of healthy infants were included. Arterial and venous cord blood from 16 deliveries were obtained immediately after birth. Serum samples were prepared and stored in a similar manner as described above. Eleven females had prostaglandin-induced abortions. Immediately after passing of the fetus, blood was obtained by cardiac puncture and squeezing of the umbilical cord. Serum samples were prepared and stored from 1987 in the similar manner as described for maternal serum. Cerebrospinal fluid was collected by puncture of the cisterna magna. Only clear cerebrospinal fluid with no visible blood was included in the study. Cerebrospinal fluid was stored for 2 years at -20 "C. The study was approved by the regional Ethical Committees in Fredericksborg and Copenhagen, Denmark.
METHODS Tetranectin was quantitated using an avidinbiotin enzyme-linked immunosorbent assay (ELISA) [6, 71. Specific rabbit immunoglobulin
against human tetranectin A-371 (IgG concentration 2.5 g I-', Dakopatts Ltd., Glostrup, Denmark) were diluted 1:250. Biotinylated affinity-purified antibody was diluted 1:1500 and for detection system peroxidase-conjugated streptavidin P-397 (Dakopatts Ltd.) diluted 1:5000 were used. The enzyme reaction was carried out by addition of 100 pl of the OPD colour reagent solution (12 mlO.1 moll-' citric acid-phosphate buffer, pH 5.0, and 8 mg 1.2phenylenediamine dihydrochloride (Dakopatts Ltd.) were mixed and 5 pl 30% hydrogen peroxide was added. After incubation the reaction was stopped by addition of 100 pl H2S04,1 mol I-'. Standard (10.6 mg I-') serially diluted from 1:800 to 1:25600 and a serum control diluted 1:800 and 1:3200 were incubated in duplicates on each plate. Duplicate samples diluted 1:lOO for amniotic fluid and 1:800 for serum and cerebrospinal fluid were assayed. The plates were read on an EAR 340 FW SLT Easy Reader (SLT-LabInstruments, GroediglSalzburg, Austria) using the dualwavelength mode at 492 nm and 620 nm, and the difference in absorbance at these wavelengths. Intra-assay CV at 3.1 mg was 3.5% (n=15), whereas the interassay CV was 6.9% (n=15). For statistical analysis the Mann-Whitney two-sample unpaired rank-sum test and Wilcoxon-Pratt paired rank-sum test were used. Tests of correlation were by the Spearman rank test. A value of p t 0 . 0 5 was considered statistically significant.
-'
RESULTS A significant, positive, linear correlation was observed between amniotic fluid TN concentration and gestational age (given as meankSD throughout) between weeks 14 (0.2250.09 mg I-') and 21 (0.5850.22 mg I-') (r=0.63; p
Scand J Clin Lab Invest 1991; 51: 411-415
Tetranectin in amniotic fluid, maternal serum and fetal fluids
Scand J Clin Lab Invest Downloaded from informahealthcare.com by ThULB Jena on 01/14/15 For personal use only.
E. V. S. HOGDALL," C . K. HQ)GDALL,t I. CLEMMENSEN,$ B. N0RGAARD-PEDERSEN" & A. G. R. LOFT" *Department of Clinical Biochemistry, Statens Seruminstitut. ?Department of Obstetrics and Gynecology, Rigshospitalet, ?Department of Obstetrics and Gynaecology, Hillerad County Hospital, $Department of Clinical Microbiology, Statens Seruminstitut, University of Copenhagen and SDakopatts NS, Glostrup, Denmark
H ~ g d a l EVS, l Hagdall CK, Clemmensen I , N~rgaard-PedersenB, Loft AGR. Tetranectin in amniotic fluid, maternal serum and fetal fluids. Scand J Clin Lab Invest 1991; 51: 411-415.. The concentration of the newly discovered protein tetranectin has been measured in different fetal and maternal compartments. In amniotic fluid a significant, positive correlation between the tetranectin concentration and gestational age was found (a mean of 0.2 mg I-' at week 14 to a mean of 0.5 mg I-' at week 21). In maternal serum a slight negative correlation was found between tetranectin concentration and gestational week (a mean of 6.17 mg 1-' at week 14 to a mean of 5.79 mg I-' at week 21). In-term cord blood collected at delivery a mean level of 6.0 mg I-' was found, and no difference was found between arterial- and venous-blood tetranectin concentration. In fetal serum the overall mean level was 2.6 mg 1-' and a significant positive correlation between tetranectin concentration and gestational age was found. The mean level was 1.1 mg 1-' in fetal cerebrospinal fluid and no correlation to gestational age was found. Fetal tetranectin may, therefore, be correlated to fetal maturation.
Key words: amniotic fluid; fetus; pregnancy; tetranectin Estrid V . S. H@gdall,Department of Clinical Biochemistry, Statens Seruminstitut, Artillerivej 5 , 2300 Copenhagen S Denmark.
The newly discovered plasma protein tetranectin (TN) [l] has been characterized as four noncovalently linked, identical peptide chains of 181 amino acids with a total M,of 20 000 [2]. TN is present in exocrine and endocrine glands as well as in secreting surface epithelia and mesenchyme, suggesting that TN, in conjunction with proteoglycans, may have a function in the packaging of granules or as a participant in exocytocic secretion of certain hormones [3, 41.
The plasma levels of TN are lower in the third than in the second trimester of pregnancy and during use of oral contraceptives, suggesting that the concentrations are affected by sex steroid hormone metabolism [5].Furthermore, the presence of TN in human embryonal fibroblasts [4,5], placenta [4] and cord blood [6] may indicate a role in embryogenesis and fetal growth. The purpose of the present study is to determine the normal levels of T N in amniotic
41 1
412
E. V. S. H@gdall et al.
fluid, maternal serum, cord blood, fetal serum and fetal cerebrospinal fluid.
MATERIALS AND METHODS
Scand J Clin Lab Invest Downloaded from informahealthcare.com by ThULB Jena on 01/14/15 For personal use only.
Study population.
Amniotic fluid samples were collected from 160 patients subjected to amniocentesis for chromosomal analysis. Only clear samples without visible blood contamination were used. Amniotic fluid samples were centrifuged at 2000 g for 10 min and stored at -20°C for 2 to 5 years before assay. Only samples from pregnancies with normal outcome were included in the study. Maternal serum samples were collected in 1988 from 121 pregnant women who had blood drawn for serum a-fetoprotein determination. All serum samples were prepared in the similar manner at one laboratory. Blood was left for clotting for 1 h at room temperature. After centrifugation at 2000 g for 10 min the serum samples were stored at -20 "C. Only samples from uncomplicated pregnancies with delivery of healthy infants were included. Arterial and venous cord blood from 16 deliveries were obtained immediately after birth. Serum samples were prepared and stored in a similar manner as described above. Eleven females had prostaglandin-induced abortions. Immediately after passing of the fetus, blood was obtained by cardiac puncture and squeezing of the umbilical cord. Serum samples were prepared and stored from 1987 in the similar manner as described for maternal serum. Cerebrospinal fluid was collected by puncture of the cisterna magna. Only clear cerebrospinal fluid with no visible blood was included in the study. Cerebrospinal fluid was stored for 2 years at -20 "C. The study was approved by the regional Ethical Committees in Fredericksborg and Copenhagen, Denmark.
METHODS Tetranectin was quantitated using an avidinbiotin enzyme-linked immunosorbent assay (ELISA) [6, 71. Specific rabbit immunoglobulin
against human tetranectin A-371 (IgG concentration 2.5 g I-', Dakopatts Ltd., Glostrup, Denmark) were diluted 1:250. Biotinylated affinity-purified antibody was diluted 1:1500 and for detection system peroxidase-conjugated streptavidin P-397 (Dakopatts Ltd.) diluted 1:5000 were used. The enzyme reaction was carried out by addition of 100 pl of the OPD colour reagent solution (12 mlO.1 moll-' citric acid-phosphate buffer, pH 5.0, and 8 mg 1.2phenylenediamine dihydrochloride (Dakopatts Ltd.) were mixed and 5 pl 30% hydrogen peroxide was added. After incubation the reaction was stopped by addition of 100 pl H2S04,1 mol I-'. Standard (10.6 mg I-') serially diluted from 1:800 to 1:25600 and a serum control diluted 1:800 and 1:3200 were incubated in duplicates on each plate. Duplicate samples diluted 1:lOO for amniotic fluid and 1:800 for serum and cerebrospinal fluid were assayed. The plates were read on an EAR 340 FW SLT Easy Reader (SLT-LabInstruments, GroediglSalzburg, Austria) using the dualwavelength mode at 492 nm and 620 nm, and the difference in absorbance at these wavelengths. Intra-assay CV at 3.1 mg was 3.5% (n=15), whereas the interassay CV was 6.9% (n=15). For statistical analysis the Mann-Whitney two-sample unpaired rank-sum test and Wilcoxon-Pratt paired rank-sum test were used. Tests of correlation were by the Spearman rank test. A value of p t 0 . 0 5 was considered statistically significant.
-'
RESULTS A significant, positive, linear correlation was observed between amniotic fluid TN concentration and gestational age (given as meankSD throughout) between weeks 14 (0.2250.09 mg I-') and 21 (0.5850.22 mg I-') (r=0.63; p
![[Variations of the concentrations of certain glycoproteins in maternal serum, fetal serum and amniotic fluid during pregnancy (author's transl)].](/assets/img/hold.png)
