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The concentration of hyaluronate in amniotic fluid
BIOCHEMICAL
MEDICINE
30,
280-283
(1983)
The Concentration of Hyaluronate in Amniotic Fluid LAURITZ
DAHL,* JOHN J. HOPWOOD,~ ULLA B. G. LAURENT,~: KARIN LILJA,§ AND ANDERS TENGBLAD#
li
*Department of Pediatrics. Institute of Clinical Medicine, University of Tromsp. 9012 Trams@. Norway. tDepartment of Chemical Pathology. The Adelaide Children’s Hospital, North Adelaide, South Australia 5006. Australia. tDepartment c$’ Ophthalmology, University Hospital, S-75185 Uppsala. Suseden, and $lnstitute (
Pu’ovember
!.
I%2
The glycosaminoglycan (mucopolysaccharide) composition of amniotic fluid has been characterized by several authors (l-7). The total polysaccharide concentration has been estimated from hexuronic acid determinations, and values of uranic acid between 0.8 and 60 pg/ml have been reported. Hyaluronic acid, chondroitin 4- and 6-sulfate, dermatan sulfate, and heparan sulfate have been identified by use of electrophoresis, ion-exchange chromatography, and enzymatic methods. The relative polysaccharide composition varies with gestational age, hyaluronate being predominant in the early period. Previous studies on amniotic fluid have been aimed at diagnosing mupolysaccharidoses and therefore at identifying sulfated polysaccharides in these diseases. The nonsulfated hyaluronic acid, however, has received increasing attention in recent years in both cell biology and medicine. It is well known that embryonic tissues, such as umbilical cord (8). contain high concentrations of the polysaccharide. and important roles for hyaluronate in the process of development have been postulated (see. e.g., Ref. (9)). Furthermore, placental tissue contains hyaluronidase (lo13). It is therefore conceivable that hyaluronate turnover is an essential process in fetal development. We have recently described a sensitive radioassay for the determination of hyaluronate (14.15). This technique has been applied to the analysis of amniotic fluid in order to define the normal variations in the concentration of the polysaccharide.
HYALURONATE
MATERIALS
IN AMNIOTIC
FLUID
281
AND METHODS
Amniotic fluid was collected at normal deliveries and by amniocenteses performed in connection with prenatal diagnosis. The gestational ages were usually determined by ultrasound. Some of the samples were from cases with neural tube defects. These were diagnosed by a-fetoprotein determinations and confirmed on the aborted fetuses. The fluid samples were frozen and kept at -20°C until analyzed. After thawing, all fluids were centrifuged at 9000g for 15 min to remove solids. Hyaluronic acid was analyzed by a radioassay (14,15) on appropriate volumes (20-200 ~1). The fluids had not been pretreated by proteolytic digestion and dialysis against assay medium as described for analysis of tissues (15) but were analyzed directly as described previously for aqueous humor (16). This was regarded as safe in view of the high hyaluronate concentration in amniotic fluid (of the same order or higher than in aqueous humor). Control experiments in which pooled amniotic fluid had been dialyzed against assay medium or pretreated proteolytically with papain (17) gave the same results as direct analysis., Treatment with hyaluronidase from Streptomyces hyalurolyticus (Seikagaku Fine Biochemicals, Tokyo, Japan; 2000 TRU/mg protein (18); digestion performed according to Laurent (16) removed the material reacting as hyaluronate completely. Addition of exogenous hyaluronate was recovered quantitatively in the same way as described for aqueous humor (16). RESULTS AND DISCUSSION The analytical data are shown in Fig. 1. The concentration of hyaluronate varies considerably and values between 0.4 and 60 pg/ml were recorded. The concentration range corresponds to what would be expected from other types of analyses (l-7). There is a variation during pregnancy with mean values around 20 pg/ml between the 16th and 20th week and 1 ,ug/ml after the 30th week. There is thus a considerable drop between the 20th and 30th week. The values obtained from cases with neural tube defects did not deviate appreciably from the normal cases although they were usually found in the upper part of the concentration range (Fig. 1). The only exception was a case at Week 32 with a level considerably higher than normal. There was no apparent correlation between hyaluronate and a-fetoprotein levels on the samples taken in midpregnancy. The analyses of cY-fetoprotein were performed in the Department of Clinical Immunology, University Hospital, Uppsala. The volume of amniotic fluid increases during pregnancy (see, e.g., Ref. (19)). With mean values of hyaluronate from Fig. 1 and the mean values of the fluid volume one can calculate the total amount of hyaluronate in amniotic fluid during gestation (Table 1). It is seen that even the total amount is higher in early pregnancy than close to the
282
DAHL ET AL
8---lal-;
20
Gestational
30 age (weeks1
FIG. I. Sodium hyaluronate concentration in human amniotic fluid during pregnancy. Normal cases (e); cases with anencephaly (0); cases with spina bifida ( x ).
term. The half-life of the water in amniotic fluid is 90 min (for references see Milunsky (19)). If hyaluronate is turned over with a similar rate there must be a considerable metabolism of the polysaccharide. Only minute amounts of hyaluronate can pass through the adult kidney due to the very high molecular weight (20). It therefore seems improbable TABLE MEAN
CONCENTRATIONS ___.-
Mean concentration Gestational age t SD (Weeks) ~~dmU ---~ - ~~~~--.-. .~~-~1s 12.8 i 5.2 16 21.4 r 8.8 17 22.7 t 10.8 18 18.3 ~fr 4.9 20 15.8 ” 16.2 23 4.3 t 2.7 24 5.1 2 2.8 34 0.97 2 0.22 35 1.04 + 0.68 36 0.79 k 0.30 37 1.07 f 0.49 38 0.95 k 0.25 39 1.12 ” 0.49 ’ Approximated
I
OF SODIUM HYALURONATE .~ -___
IN HUMAN
AMNIOTIC
FLUID
Number of analyses
Volume of amniotic fluid (ml)
Total amount of Na hyaluronate (mg)
6 23 16 9 4 5 5 4 8 7 11 13 I5
I45 210 230 260 360 510 560 800 800 800 800 800 800
1.9 4.5 5.2 4.7 5.7 2.2 2.9 0.8 0.6 0.8 0.9 0.8 0.9
from literature data cited by Milunsky i 19).
HYALURONATE
IN AMNIOTIC
FLUID
283
that hyaluronate is excreted in the fetal urine as has been proposed for the much smaller sulfated polysaccharide molecules (4). The high concentrations in amniotic fluid in early pregnancy would rather indicate that hyaluronate leaks through cellular linings which become more impermeable with increasing age. SUMMARY
The concentration of sodium hyaluronate has been determined in amniotic fluid by a specific radioassay. It shows a large individual variation. The mean concentration is approximately 20 wg/ml at Weeks 16-20 of the gestational period. It drops to approximately 1 pg/ml at Week 30 and is then constant until the end of the pregnancy. ACKNOWLEDGMENTS This project was supported by the Swedish Medical Research Council (Grant 13x-4) and Gustaf V:s 80-&fond.
REFERENCES I. Matalon, R., Dorfman, A., Nadler, H. L., and Jacobson, C. B., Lancer I, 83 (1970). 2. Danes, B. S., Queenan, J. T., Gadow, E. C., and Cederqvist, L. L., Luncet 1, 947 (1970). 3. Suschke, J., and Kunze, D., Miinch. Med. Wochenschr. 113, 1495 (1971). 4. Matalon, R., and Dorfman, A. in “Antenatal Diagnosis” (A. Dotfman, Ed.), p. 17. Univ. of Chicago Press, Chicago/London, 1972. 5. Duncan, D. M., Logan, R. W., Ferguson-Smith, M. A.. and Hall, F.. Clin. C&m. Acra 45, 73 (1973). 6. Whiteman, P., Lancer 1, 1249 (1973). 7. Lee, T.-Y., and Schafer, I. A., Biochim. Biophys. Acra 354, 264 (1974). 8. Meyer, K., and Palmer, J. W., J. Biol. Chem. 114, 689 (1936). 9. Toole, B. P., Connecr. Tissue Res. IO, 93 (1982). 10. Scarpa, F., Panazzolo, A,, Tanferna, M., and Pavetto, P. F., Boll. Sot. Ital. Biol. Sper. 45, 217 (1969). 11. Rudyuk, M. P., Chem. Absfr. 58, 7193 (1963). 12. Homenyuk, I. P., Pediutr. Akush. Ginekol. 34, 44 (1972). 13. Yamada, M., Hasegawa, E., and Kanamori, M., J. Biochem. Tokyo 81, 485 (1977). 14. Tengblad, A., Biochem. J. 185, 101 (1980). 15. Laurent, U. B. G., and Tengblad, A., Anal. Biochem. 109, 386 (1980). 16. Laurent, U. B. G., Exp. Eye Res. 33, 147 (1981). 17. Scott, J. E., Meth. Biochem. Anal. 8, 145 (1960). 18. Ohya, T., and Kaneko, Y., Biochim. Biophys. Actu 198, 607 (1970). 19. Milunsky, A. in “Genetic Disorders and the Fetus” (A. Milunsky, Ed.), p. 47. Plenum, New York/London, 1979. 20. Fraser, J. R. E., Laurent, T. C., Pertoft, H., and Baxter, E. Biochem. J. 200, 415 (1981).
MEDICINE
30,
280-283
(1983)
The Concentration of Hyaluronate in Amniotic Fluid LAURITZ
DAHL,* JOHN J. HOPWOOD,~ ULLA B. G. LAURENT,~: KARIN LILJA,§ AND ANDERS TENGBLAD#
li
*Department of Pediatrics. Institute of Clinical Medicine, University of Tromsp. 9012 Trams@. Norway. tDepartment of Chemical Pathology. The Adelaide Children’s Hospital, North Adelaide, South Australia 5006. Australia. tDepartment c$’ Ophthalmology, University Hospital, S-75185 Uppsala. Suseden, and $lnstitute (
Pu’ovember
!.
I%2
The glycosaminoglycan (mucopolysaccharide) composition of amniotic fluid has been characterized by several authors (l-7). The total polysaccharide concentration has been estimated from hexuronic acid determinations, and values of uranic acid between 0.8 and 60 pg/ml have been reported. Hyaluronic acid, chondroitin 4- and 6-sulfate, dermatan sulfate, and heparan sulfate have been identified by use of electrophoresis, ion-exchange chromatography, and enzymatic methods. The relative polysaccharide composition varies with gestational age, hyaluronate being predominant in the early period. Previous studies on amniotic fluid have been aimed at diagnosing mupolysaccharidoses and therefore at identifying sulfated polysaccharides in these diseases. The nonsulfated hyaluronic acid, however, has received increasing attention in recent years in both cell biology and medicine. It is well known that embryonic tissues, such as umbilical cord (8). contain high concentrations of the polysaccharide. and important roles for hyaluronate in the process of development have been postulated (see. e.g., Ref. (9)). Furthermore, placental tissue contains hyaluronidase (lo13). It is therefore conceivable that hyaluronate turnover is an essential process in fetal development. We have recently described a sensitive radioassay for the determination of hyaluronate (14.15). This technique has been applied to the analysis of amniotic fluid in order to define the normal variations in the concentration of the polysaccharide.
HYALURONATE
MATERIALS
IN AMNIOTIC
FLUID
281
AND METHODS
Amniotic fluid was collected at normal deliveries and by amniocenteses performed in connection with prenatal diagnosis. The gestational ages were usually determined by ultrasound. Some of the samples were from cases with neural tube defects. These were diagnosed by a-fetoprotein determinations and confirmed on the aborted fetuses. The fluid samples were frozen and kept at -20°C until analyzed. After thawing, all fluids were centrifuged at 9000g for 15 min to remove solids. Hyaluronic acid was analyzed by a radioassay (14,15) on appropriate volumes (20-200 ~1). The fluids had not been pretreated by proteolytic digestion and dialysis against assay medium as described for analysis of tissues (15) but were analyzed directly as described previously for aqueous humor (16). This was regarded as safe in view of the high hyaluronate concentration in amniotic fluid (of the same order or higher than in aqueous humor). Control experiments in which pooled amniotic fluid had been dialyzed against assay medium or pretreated proteolytically with papain (17) gave the same results as direct analysis., Treatment with hyaluronidase from Streptomyces hyalurolyticus (Seikagaku Fine Biochemicals, Tokyo, Japan; 2000 TRU/mg protein (18); digestion performed according to Laurent (16) removed the material reacting as hyaluronate completely. Addition of exogenous hyaluronate was recovered quantitatively in the same way as described for aqueous humor (16). RESULTS AND DISCUSSION The analytical data are shown in Fig. 1. The concentration of hyaluronate varies considerably and values between 0.4 and 60 pg/ml were recorded. The concentration range corresponds to what would be expected from other types of analyses (l-7). There is a variation during pregnancy with mean values around 20 pg/ml between the 16th and 20th week and 1 ,ug/ml after the 30th week. There is thus a considerable drop between the 20th and 30th week. The values obtained from cases with neural tube defects did not deviate appreciably from the normal cases although they were usually found in the upper part of the concentration range (Fig. 1). The only exception was a case at Week 32 with a level considerably higher than normal. There was no apparent correlation between hyaluronate and a-fetoprotein levels on the samples taken in midpregnancy. The analyses of cY-fetoprotein were performed in the Department of Clinical Immunology, University Hospital, Uppsala. The volume of amniotic fluid increases during pregnancy (see, e.g., Ref. (19)). With mean values of hyaluronate from Fig. 1 and the mean values of the fluid volume one can calculate the total amount of hyaluronate in amniotic fluid during gestation (Table 1). It is seen that even the total amount is higher in early pregnancy than close to the
282
DAHL ET AL
8---lal-;
20
Gestational
30 age (weeks1
FIG. I. Sodium hyaluronate concentration in human amniotic fluid during pregnancy. Normal cases (e); cases with anencephaly (0); cases with spina bifida ( x ).
term. The half-life of the water in amniotic fluid is 90 min (for references see Milunsky (19)). If hyaluronate is turned over with a similar rate there must be a considerable metabolism of the polysaccharide. Only minute amounts of hyaluronate can pass through the adult kidney due to the very high molecular weight (20). It therefore seems improbable TABLE MEAN
CONCENTRATIONS ___.-
Mean concentration Gestational age t SD (Weeks) ~~dmU ---~ - ~~~~--.-. .~~-~1s 12.8 i 5.2 16 21.4 r 8.8 17 22.7 t 10.8 18 18.3 ~fr 4.9 20 15.8 ” 16.2 23 4.3 t 2.7 24 5.1 2 2.8 34 0.97 2 0.22 35 1.04 + 0.68 36 0.79 k 0.30 37 1.07 f 0.49 38 0.95 k 0.25 39 1.12 ” 0.49 ’ Approximated
I
OF SODIUM HYALURONATE .~ -___
IN HUMAN
AMNIOTIC
FLUID
Number of analyses
Volume of amniotic fluid (ml)
Total amount of Na hyaluronate (mg)
6 23 16 9 4 5 5 4 8 7 11 13 I5
I45 210 230 260 360 510 560 800 800 800 800 800 800
1.9 4.5 5.2 4.7 5.7 2.2 2.9 0.8 0.6 0.8 0.9 0.8 0.9
from literature data cited by Milunsky i 19).
HYALURONATE
IN AMNIOTIC
FLUID
283
that hyaluronate is excreted in the fetal urine as has been proposed for the much smaller sulfated polysaccharide molecules (4). The high concentrations in amniotic fluid in early pregnancy would rather indicate that hyaluronate leaks through cellular linings which become more impermeable with increasing age. SUMMARY
The concentration of sodium hyaluronate has been determined in amniotic fluid by a specific radioassay. It shows a large individual variation. The mean concentration is approximately 20 wg/ml at Weeks 16-20 of the gestational period. It drops to approximately 1 pg/ml at Week 30 and is then constant until the end of the pregnancy. ACKNOWLEDGMENTS This project was supported by the Swedish Medical Research Council (Grant 13x-4) and Gustaf V:s 80-&fond.
REFERENCES I. Matalon, R., Dorfman, A., Nadler, H. L., and Jacobson, C. B., Lancer I, 83 (1970). 2. Danes, B. S., Queenan, J. T., Gadow, E. C., and Cederqvist, L. L., Luncet 1, 947 (1970). 3. Suschke, J., and Kunze, D., Miinch. Med. Wochenschr. 113, 1495 (1971). 4. Matalon, R., and Dorfman, A. in “Antenatal Diagnosis” (A. Dotfman, Ed.), p. 17. Univ. of Chicago Press, Chicago/London, 1972. 5. Duncan, D. M., Logan, R. W., Ferguson-Smith, M. A.. and Hall, F.. Clin. C&m. Acra 45, 73 (1973). 6. Whiteman, P., Lancer 1, 1249 (1973). 7. Lee, T.-Y., and Schafer, I. A., Biochim. Biophys. Acra 354, 264 (1974). 8. Meyer, K., and Palmer, J. W., J. Biol. Chem. 114, 689 (1936). 9. Toole, B. P., Connecr. Tissue Res. IO, 93 (1982). 10. Scarpa, F., Panazzolo, A,, Tanferna, M., and Pavetto, P. F., Boll. Sot. Ital. Biol. Sper. 45, 217 (1969). 11. Rudyuk, M. P., Chem. Absfr. 58, 7193 (1963). 12. Homenyuk, I. P., Pediutr. Akush. Ginekol. 34, 44 (1972). 13. Yamada, M., Hasegawa, E., and Kanamori, M., J. Biochem. Tokyo 81, 485 (1977). 14. Tengblad, A., Biochem. J. 185, 101 (1980). 15. Laurent, U. B. G., and Tengblad, A., Anal. Biochem. 109, 386 (1980). 16. Laurent, U. B. G., Exp. Eye Res. 33, 147 (1981). 17. Scott, J. E., Meth. Biochem. Anal. 8, 145 (1960). 18. Ohya, T., and Kaneko, Y., Biochim. Biophys. Actu 198, 607 (1970). 19. Milunsky, A. in “Genetic Disorders and the Fetus” (A. Milunsky, Ed.), p. 47. Plenum, New York/London, 1979. 20. Fraser, J. R. E., Laurent, T. C., Pertoft, H., and Baxter, E. Biochem. J. 200, 415 (1981).