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The free amino acids of amniotic fluid during pregnancy of the rhesus monkey
The free amino acids of amniotic fluid during pregnancy of the rhesus monkey GEORGE
R.
KERR,
ALFRED
L.
KENNAN,
Madison,
M.D. M.D.
Wisconsin
The free amino acids of amniotic fluid were analyzed during pregnancy of the rhesus monkey. Samples of amniotic fluid and of fetal and maternal blood were obtained at cesarean section delivery at exactly SO, 75, 100, 125, and 150 days of gestational age. The total value of 19 amino acids in amniotic fluid decreased sequentially with advancing gestational age; the same trend was seen for most of the individual amino acids. Taurine was unique in showing the opposite trend, and demonstrated increasing levels at each subsequent stage of pregnancy. The free amino acids of amniotic fluid did not ref?ect the levels in the serum of either fetal or maternal blood.
AMNIOTIC FLUID COIIStitUteS an important but inadequately studied aspect of pregnancy, although it is apparent that this fluid must provide an optimal physical and bioche:mical environment for development of the fetus. The recent ability to sample this fluid throughout gestation has demonstrated the value of amniotic fluid analysis in defining fetal acid-base metabolism,l fetal maturity, 2, 3 the state of isoimmunization processes,4 and the presence of hereditary and genetic disorders5; studies into more important roles of amniotic fluid in assuring normal fetal growth and development may now be possible. It is not possible to perform certain investigations in normal human pregnancies, and many of the studies necessary to fully
define the functions of amniotic fluid will have to be conducted in experimental animals. Fortunately, several other primate species demonstrate many of the changes which occur in human pregnancy, and these animals may be used to study the dynamics of amniotic fluid with some assurance that the resulting data will be pertinent to those which occur in the human subject. A major biochemical aspect of fetal growth involves the synthesis of structural and functional proteins from the free amino acids provided via the placental circulation or ingested with amniotic fluid. As a prerequisite to investigations into the nutritive role of amniotic fluid, this communication reports the levels of the free amino acids which occur in amniotic fluid during normal pregnancy in the rhesus monkey.
From the Departments of Pediatrics and of Gynecology and Obstetrics and the Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin. Supported in part by grant FR-00167 from the National Institutes of- Health to the Wisconsin Regional Primate Research Center; and by grant AM-3313 from the United States Public Health Service, training grant No. I-T-HD-104-01 from the National Institute of Child Health and Human Develofiment, and a grant from the Ford Foundation.
Moferialr
and
methods
Female rhesus monkeys (Macaca mulatta) were maintained in individual cages under standard conditions of light, temperature, and humidity, and were fed a commercial diet* with daily supplements of milk,+ *Purina Missouri. fsimilac,
363
Monkey
Chow,
Ross Laboratories,
Ralston
Purina
Columbus,
Co., Ohio.
St. Louis,
364 Kerr and Kennan
vitamins, and seasonal fruit. Menstrual cycles and changes in weight and clinical health were recorded, and the exact date of conception was established for all pregnancies.” Full-term gestation in this species is 168 -t 4 days. Pregnancy was interrupted at exactly 50, 75, 100, 125, and 150 days of gestational age by cesarean section delivery performed with the female under local anesthesia and immobilized in a restraining device. Oral feeding was discontinued 8 hours prior to the procedure: and premeditation or other anesthetic, agents were not utilized. Maternal hydration was maintained during surgery by a slow intravenous infusion of 5 per cent glucose in 0.2 per cent sodium chloride. All animals tolerated the procedure without evident distress. Amniotic fluid was aspirated through the lower uterine segment without manipulation of the uterus or placenta, and without contamination with either maternal or placental blood. Samples of umbilical vein blood and maternal venous blood were then collected,
Table I. The free amino
acids of amniotic per 100 ml.) *
(in milligrams
and the fetus delivered for ;I variety of other studies. Amniotic fluid \vas centrifuged to remove cellular elements and debris, and frozen at --20’ C. until analyzed; most samples were analyzed within one I\-eek. The total protein value I\-as measured by a micro-Kjeldahl method,’ and, after acidification and highspeed centrifugations the free amino acids Lvere quantitated by the method of Spackman, Stein, and Moore,” with the use of a Reckman/Spinco amino acid analyzer. A standard of ,B-2-thienylalanine was incorporated with each analysis; recovery of the standard in this series of investigations was 98.3 & 5.9 per cent (mean + 1 S. D.) . Values will not be reported for cystine and tryptophan because of their instability during storage,lO and errors introduced by albumin binding of tryptophan.s Glutamine decreases and glutamic acid increases during storage of plasma’“; although glutamine is also metabolized to compounds other than glutamic acid, these two amino acids will be
fluid
during
pregnancy
Gestational Amino
acid
50
Taurine Aspartic acid + threonine Serine Glutamine + glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine 3-CH3-histidine Arginine Total of 19 amino acids Amniotic fluid Maternal serum$ Umbilical vein serum ‘Mean
f
+Numbers $The
value
days
(4)f
/
75
days
(5)
in M.
age
1 1’00 days
(5)
1 125
in
adult
days
(5)
1 1.50 days
(5)
1.45 1.57 1.12 6.52
+ t t +
0.45 0.45 0.42 1.64
1.97 1.40 0.67 7.77
1 + 2 t
0.27 0.13 0.19 0.57
2.12 1.31 0.64 6.73
t ?I 2 t
0.36 0.23 0.32 1.29
2.18 0.86 0.53 5.14
C k ? +
0.30 0.15 0.19 0.67
2.33 0.71 0.36 3.39
k rt t +
0.71 0.10 0.15 0.63
2.96 1.95 3.45 2.54 0.66 0.87 2.16 1.35 1.34 3.95 1.76 0.75 1.48
t + + 5 + + ” ++-c + + +-
0.58 0.33 0.59 0.89 0.14 0.32 0.73 0.36 0.60 0.26 0.43 0.20 0.44
4.05 2.23 2.59 2.14 0.52 0.75 1.43 0.73 1.04 2.84 1.93 1.79 0.88
ct: 2 -I 2 t r + 5 t t + + t
0.71 0.31 0.29 0.28 0.09 0.13 0.27 0.15 0.11 0.79 0.30 0.27 0.34
3.62 2.33 2.40 1.39 0.42 0.46 0.89 0.63 0.73 2.30 1.60 1.40 0.41
t t 2 2 t t t 2 If: t + r 2
0.70 0.42 0.26 0.19 0.05 0.07 0.21 0.12 0.21 0.61 0.26 0.30 0.14
2.91 2.10 1.68 0.75 0.31 0.28 0.47 0.45 0.52 2.44 0.96 1.33 0.25
t ?I 2 2 5 t t ” 2 2 rt + +
0.62 0.16 0.25 0.12 0.05 0.07 0.13 0.07 0.13 0.65 0.19 0.28 0.11
1.96 1.68 1.22 0.66 0.23 0.25 0.40 0.55 0.51 1.43 0.58 0.91 0.13--
t 1 ” 5 ” k r k -I 2 k r -c
0.77 0.13 0.22 0.17 0.03 0.04 0.07 0.19 0.14 0.36 0.08 0.20 0.07
35.88 2 7.37 44.40 t 7.56 (inadequate volume)
34.01 32.50 66.55
t 3.08 2 3.91 5 4.23
29.38 34.43 57.75
21 2.53 r 4.75 t 3.51
23.16 37.0'8 60.01
1 SD. in parentheses
ndatta
indicate
nonpregnant
the
number
female
of
monkeys
animals
studied
is 52.28
+ 10.44 mg.
at
each
gestational
per
100 ml.‘*
age.
t 2.55 + 4.91 t 3.82
17.30 35.64 56.70
L 1.68 5 6.82 in 8.82
Volume Number
105 3
reported as one value. Aspartic acid and threonine occasionally overlap on chromatographs and their levels will also be reported as a single value. Poor resolution was occasional.ly seen for arginine and 3-CH,-histidine; the values reported for these amino acids should be accepted with reservation. The values for 19 amino acids were combined at each gestational age as an indication of the changes in the total amino acid content of amniotic fluid which occur coincident to pregnancy in this species. Rewlts The free amino acids of amniotic fluid at each gestational age are presented in Table I. Although a relatively wide range of values was present for each amino acid at each age of gestation, several patterns of change were apparent. The total value of the 19 amino acids decreased sequentially with advancing gestational age; the same trend was seen for most of the individual amino acids. Taurine, reflecting sulfur amino acid metabolism and bile acid conjugation, was unique in showing the opposite trend, however, and the mean values for glutamine plus glutamic acid, proline, glycine, histidine, and 3-CH,-histidine were lower in the 50 day sample than at 75 days of gestational age; thereafter these amino acids also showed a progressive decrease. The total value of the 19 amino acids in maternal and umbilical vein sera are also indicated in Table I. The value in fetal serum was consistently elevated over those in both maternal serum and amniotic fluid. The value in maternal serum showed a rapid decrease early in pregnancy which stabilized by 75 days od gestational age. At this point the value in amniotic fluid was comparable to that in maternal serum; thereafter it was always below the value in both maternal and fetal sera. Comment Despite numerous investigations and speculations, the source of amniotic fluid has not been firmly established.4* 111 l2 The amniotic fluid of early gestation has been considered to represent a dialysate or transudate of ma-
Amniotic
fluid acids in pregnancy
365
ternal plasma, or a secretion from the amniotic epitheliumll; later in pregnancy the fetal respiratory tract, intestinal tract, umbilical cord, and salivary glands may also contribute to this fluid. Once renal function has been established, fetal micturition becomes the major source of amniotic fluid.13 As the source of amniotic fluid is complex, so also is its route of disposal. Fetal swallowing is one obvious route and polyhydramnios frequently accompanies esophageal or intestinal atresia.13 A limited number of investigations have been performed in relation to the amino acid content of amniotic fluid. With regard to the levels early in gestation, McKay and associatesl* reported that the amino nitrogen content of human hydatidiform mole fluid was elevated over that of maternal plasma. Lesinski and associates15 also noted high levels of amino acids in the blastocyst fluid of rabbits between the fifth and seventh days after mating: shortly thereafter a notable drop occurred in the level of most of the individual amino acids. Several studies have reported the amino acids of amniotic fluid during later gestation. WirtschafteP investigated amniotic fluid throughout the last eight days of pregnancy in the rat, and noted that amino acid levels rose with increasing gestation. He also noted that several amino acids were found at higher levels in amniotic fluid than in maternal blood, indicating a mechanism other than simple dialysis. In a subsequent study Wirtschafterl? also reported the free amino acids of human amniotic fluid at full-term pregnancy and noted a species difference; whereas the amino acid levels of rat amniotic fluid were often elevated over the maternal level, the opposite result was consistently seen in human pregnancies. Although a considerable amount of information has been reported with regard to the transplacental levels and transport of amino acids in this speciesl*p I9 and in the human,?O much less is available concerning the relationships between the free amino acids of amniotic fluid and those of fetal or maternal plasma. Bonsnes,?l in a recent review on the composition of amniotic fluid, concluded
366
Kerr
and
Kennan
that the levels of free amino acids at fullterm pregnancy were generally comparable to those found in maternal plasma. The evidence for this conclusion was partly derived from the report of Orlandi and colleagues,‘” who noted that values for several amino acids in amniotic fluid were elevated over maternal levels. Unfortunately, the samples of amniotic fluid and maternal plasma in this study were not obtained from the same patients; their conclusions are therefore open to question. The reports of Stass? and Wirtschafterlr indicated that the combined levels of amino acids in human amniotic fluid at full-term pregnancy varied from 9 to 25 mg. per 100 ml., and in the latter study the amniotic fluid values were consistently below those in maternal serum. The values at late gestation in the rhesus monkey would be in accord with these latter studies. The free amino acids of maternal and fetal blood during pregnancy in M. mulatta have been reported previously.l* All maternal amino acids except taurine, lysine, 3-CH,histidine, and arginine were lower at fullterm pregnancy than in nonpregnant adult females. This pattern was present by 75 days of gestation and did not show a significant change thereafter. Several amino acids, most notably glutamine and glutamic acid, histidine, methionine, and valine, were elevated in fetal blood during early pregnancy, and fell as gestation proceeded. 3-CH,-histidine and glycine showed the opposite trend and reached their maximum value at full-term pregnancy. The other amino acids of fetal blood did not show significant changes throughout gestation. Umbilical cord levels for each amino acid were invariably elevated over maternal values, and still further elevated over the levels in amniotic fluid. Most of the individual amino acids of amniotic fluid showed a gradual and progressive decrease as pregnancy progressed, in
Seeds, A. E., Kock, H. C., Myers, Stolte, L. A. M., and Hellegers, A. J. OBST. & GYNEC. 97: 67, 1967.
The surgical assistance of Drs. Harry A. Waisman and James R. Allen, and the technical assistance of Guenther Scheffler, James A. Campbell, and Arthur Helmuth are gratefully acknowledged.
2. Pitkin,
REFERENCES
1.
distinction to the rapid reduction seen irr maternal strum. The maternal Icvrl at late gestation was alvzays higher than the amniotic fluid level; at early pregnancy, however, the amniotic fluid levels were higher in the cases of proline, lysine, histidine, and 3-CH,-histidine, and for several other amino acids the levels were approximately equal. Only with the progress& reduction in amniotic fluid levels, possibly reflecting a maturation of renal tubular function,“’ did they all eventually fall belokv the maternal levels. The notable exception of taurine to the pattern of change shown for the other amino acids is intriguing, and probably reflects those factors which produce elevated levels of taurine in postnatal urine, and in the umbilical vein blood of both human?: and subhumar? primates. It is apparent, however, that the changes in the amino acid levels of amniotic fluid are not simply a reflection of the changes which occur in either fetal or maternal serum. The observation that amniotic fluid is swallowed by the fetusZ7 suggests that it may have a role in fetal nutrition. Sugawa and co-workers’” have reported that the amino acids of amniotic fluid in rodents may be utilized for protein synthesized by both fetus and placenta. The nutritive role of this fluid in fetal nitrogen metabolism would be related to the volume ingested. The rate of transfer of water between the pregnant rhesus monkey and fetus has been reported at 1 L. per hour.?” A major proportion of this volume may involve fluid swallowed by the fetus, and the amino acid content of this fluid may play a greater role in fetal nitrogen metabolism than has previously been considered.
R. E., E.: AM.
OBST.
3.
Gordon, 30: 652,
R. M., and &
GYNEC.
H., and 1967.
Zwirek, 1135, Brosens, I.: 98:
S. 1967. Obst.
J.: AM. J. & Gynec.
Volume Number
4.
5. 6. 7. 8. 9. 10. 11.
12.
13. 14.
15.
16.
105 3
Lilev, A. W.: In Carey, H. M., editor: Modern trends in human reproductive physiology, London. 1963. Butterworth & Co.. , Ltd.. , vol. I, pp. 227-244. Fuchs, F.: Clin. Obst. & Gynec. 9: 565, 1966. Ponce de Lugo, C. G.: Biol. Neonat. 6: 104, 1964. Miller, G. L., and Miller, E. E.: Anal. Chem. 20: 481, 1948. Gerritsen, T., Rheberg, M. L., and Waisman, H. A.: Anal. Biochem. 11: 460, 1965. Spackman, D. H., Stein, W. H.; and Moore, S.: Anal. Chem. 30: 1190. 1958. Dickinson, J. C., Rosenblum, H., and Hamilton, P. B::-Pediatrics 36: 2,. 1965. As&i. N. S.. Dilts. P. V.. Plentl. A. A.. Kirschbaum, T. H.; and Gross, S J.: Zn Assali, N. S., editor: Biology of gestation, New York, 1968, Academic Press, Inc., vol. I, pp. 273-289. Seeds, A. E.: In Barnes, A. C.. editor: Intrauterine development, .Philaddlphia, 1968, Lea & Febiaer. KID. 129-142. Jeffcoate, Ty N.-k., and Scott, J. S.: Canad. M. A. J. 80: 77, 1959. McKay, D. G., Roby, C. C., Hertig, A. T., and Richardson, M. V.: AM. J. OBST. & GYNEC. 69: 722, 1955. Lesinski, J., Jajszczak, St., Bentyn, K., and Janczarski, I.: AM. J. OBST. & GYNEC. 99: 280, 1967. Wirtschafter, Z. T.: Aol. J. OBST. & GYNEC. 75: 718. 1958.
Amniotic
17. 18. 19.
20.
21. 22. 23. 24.
25. 26. 27. 28. 29.
fluid
acids
in pregnancy
367
Wirtschafter, Z. T.: AM. .I. OBST. & GYNEC. 76: 1219, 1958. Kerr. G. R.: Pediat. Res. 2: 493. 1968. Kelly, W. T., Hutchinson, D. L.; Friedman, E. A., and Plentl, A. A.: AM. J. OBST. & GYNEC. 89: 776, 1964. Page, E. W., Glendenning, M. B., Margolis, A., and Harper, H. A.: AM. J. OBST. & GYNEC. 73: 589, 1957. Bonsnes. R. W.: Clin. Obst. & Gvnec. 9: 440, 1966. Orlandi, Cl., Torsello, R. V., and Bottiglioni, F.: AttualitB ostet. e ginec. 4: 871. 1958. Stassi, D.: Monititore-Ostet. Ginec. 33: 683, 1962. McCance, R. A., and Widdowson, E. M.: In Munro, H. N., and Allison, J. B., editors: Mammalian protein metabolism, New York, 1964, Academic Press, Inc., vol. II, pp. 225241. Woolf, L. I., and Norman, A. P.: J. Pediat. 50: 271, 1957. Kerr, G. R.: Pediat. Res. 2: 187, 1968. Pritchard, J. A.: Obst. & Gynec. 25: 289, 1965. Sugawa, T., Nakamura, K., and Ozaki, M.: J. Jap. Obst. & Gynec. Sot. 10: 1, 1963. Friedman, E. A., Gray, M. J., Hutchinson, D. L., and Plentl, A. A.: J. Clin. Invest. 38: 961, 1959.
R.
KERR,
ALFRED
L.
KENNAN,
Madison,
M.D. M.D.
Wisconsin
The free amino acids of amniotic fluid were analyzed during pregnancy of the rhesus monkey. Samples of amniotic fluid and of fetal and maternal blood were obtained at cesarean section delivery at exactly SO, 75, 100, 125, and 150 days of gestational age. The total value of 19 amino acids in amniotic fluid decreased sequentially with advancing gestational age; the same trend was seen for most of the individual amino acids. Taurine was unique in showing the opposite trend, and demonstrated increasing levels at each subsequent stage of pregnancy. The free amino acids of amniotic fluid did not ref?ect the levels in the serum of either fetal or maternal blood.
AMNIOTIC FLUID COIIStitUteS an important but inadequately studied aspect of pregnancy, although it is apparent that this fluid must provide an optimal physical and bioche:mical environment for development of the fetus. The recent ability to sample this fluid throughout gestation has demonstrated the value of amniotic fluid analysis in defining fetal acid-base metabolism,l fetal maturity, 2, 3 the state of isoimmunization processes,4 and the presence of hereditary and genetic disorders5; studies into more important roles of amniotic fluid in assuring normal fetal growth and development may now be possible. It is not possible to perform certain investigations in normal human pregnancies, and many of the studies necessary to fully
define the functions of amniotic fluid will have to be conducted in experimental animals. Fortunately, several other primate species demonstrate many of the changes which occur in human pregnancy, and these animals may be used to study the dynamics of amniotic fluid with some assurance that the resulting data will be pertinent to those which occur in the human subject. A major biochemical aspect of fetal growth involves the synthesis of structural and functional proteins from the free amino acids provided via the placental circulation or ingested with amniotic fluid. As a prerequisite to investigations into the nutritive role of amniotic fluid, this communication reports the levels of the free amino acids which occur in amniotic fluid during normal pregnancy in the rhesus monkey.
From the Departments of Pediatrics and of Gynecology and Obstetrics and the Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin. Supported in part by grant FR-00167 from the National Institutes of- Health to the Wisconsin Regional Primate Research Center; and by grant AM-3313 from the United States Public Health Service, training grant No. I-T-HD-104-01 from the National Institute of Child Health and Human Develofiment, and a grant from the Ford Foundation.
Moferialr
and
methods
Female rhesus monkeys (Macaca mulatta) were maintained in individual cages under standard conditions of light, temperature, and humidity, and were fed a commercial diet* with daily supplements of milk,+ *Purina Missouri. fsimilac,
363
Monkey
Chow,
Ross Laboratories,
Ralston
Purina
Columbus,
Co., Ohio.
St. Louis,
364 Kerr and Kennan
vitamins, and seasonal fruit. Menstrual cycles and changes in weight and clinical health were recorded, and the exact date of conception was established for all pregnancies.” Full-term gestation in this species is 168 -t 4 days. Pregnancy was interrupted at exactly 50, 75, 100, 125, and 150 days of gestational age by cesarean section delivery performed with the female under local anesthesia and immobilized in a restraining device. Oral feeding was discontinued 8 hours prior to the procedure: and premeditation or other anesthetic, agents were not utilized. Maternal hydration was maintained during surgery by a slow intravenous infusion of 5 per cent glucose in 0.2 per cent sodium chloride. All animals tolerated the procedure without evident distress. Amniotic fluid was aspirated through the lower uterine segment without manipulation of the uterus or placenta, and without contamination with either maternal or placental blood. Samples of umbilical vein blood and maternal venous blood were then collected,
Table I. The free amino
acids of amniotic per 100 ml.) *
(in milligrams
and the fetus delivered for ;I variety of other studies. Amniotic fluid \vas centrifuged to remove cellular elements and debris, and frozen at --20’ C. until analyzed; most samples were analyzed within one I\-eek. The total protein value I\-as measured by a micro-Kjeldahl method,’ and, after acidification and highspeed centrifugations the free amino acids Lvere quantitated by the method of Spackman, Stein, and Moore,” with the use of a Reckman/Spinco amino acid analyzer. A standard of ,B-2-thienylalanine was incorporated with each analysis; recovery of the standard in this series of investigations was 98.3 & 5.9 per cent (mean + 1 S. D.) . Values will not be reported for cystine and tryptophan because of their instability during storage,lO and errors introduced by albumin binding of tryptophan.s Glutamine decreases and glutamic acid increases during storage of plasma’“; although glutamine is also metabolized to compounds other than glutamic acid, these two amino acids will be
fluid
during
pregnancy
Gestational Amino
acid
50
Taurine Aspartic acid + threonine Serine Glutamine + glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine 3-CH3-histidine Arginine Total of 19 amino acids Amniotic fluid Maternal serum$ Umbilical vein serum ‘Mean
f
+Numbers $The
value
days
(4)f
/
75
days
(5)
in M.
age
1 1’00 days
(5)
1 125
in
adult
days
(5)
1 1.50 days
(5)
1.45 1.57 1.12 6.52
+ t t +
0.45 0.45 0.42 1.64
1.97 1.40 0.67 7.77
1 + 2 t
0.27 0.13 0.19 0.57
2.12 1.31 0.64 6.73
t ?I 2 t
0.36 0.23 0.32 1.29
2.18 0.86 0.53 5.14
C k ? +
0.30 0.15 0.19 0.67
2.33 0.71 0.36 3.39
k rt t +
0.71 0.10 0.15 0.63
2.96 1.95 3.45 2.54 0.66 0.87 2.16 1.35 1.34 3.95 1.76 0.75 1.48
t + + 5 + + ” ++-c + + +-
0.58 0.33 0.59 0.89 0.14 0.32 0.73 0.36 0.60 0.26 0.43 0.20 0.44
4.05 2.23 2.59 2.14 0.52 0.75 1.43 0.73 1.04 2.84 1.93 1.79 0.88
ct: 2 -I 2 t r + 5 t t + + t
0.71 0.31 0.29 0.28 0.09 0.13 0.27 0.15 0.11 0.79 0.30 0.27 0.34
3.62 2.33 2.40 1.39 0.42 0.46 0.89 0.63 0.73 2.30 1.60 1.40 0.41
t t 2 2 t t t 2 If: t + r 2
0.70 0.42 0.26 0.19 0.05 0.07 0.21 0.12 0.21 0.61 0.26 0.30 0.14
2.91 2.10 1.68 0.75 0.31 0.28 0.47 0.45 0.52 2.44 0.96 1.33 0.25
t ?I 2 2 5 t t ” 2 2 rt + +
0.62 0.16 0.25 0.12 0.05 0.07 0.13 0.07 0.13 0.65 0.19 0.28 0.11
1.96 1.68 1.22 0.66 0.23 0.25 0.40 0.55 0.51 1.43 0.58 0.91 0.13--
t 1 ” 5 ” k r k -I 2 k r -c
0.77 0.13 0.22 0.17 0.03 0.04 0.07 0.19 0.14 0.36 0.08 0.20 0.07
35.88 2 7.37 44.40 t 7.56 (inadequate volume)
34.01 32.50 66.55
t 3.08 2 3.91 5 4.23
29.38 34.43 57.75
21 2.53 r 4.75 t 3.51
23.16 37.0'8 60.01
1 SD. in parentheses
ndatta
indicate
nonpregnant
the
number
female
of
monkeys
animals
studied
is 52.28
+ 10.44 mg.
at
each
gestational
per
100 ml.‘*
age.
t 2.55 + 4.91 t 3.82
17.30 35.64 56.70
L 1.68 5 6.82 in 8.82
Volume Number
105 3
reported as one value. Aspartic acid and threonine occasionally overlap on chromatographs and their levels will also be reported as a single value. Poor resolution was occasional.ly seen for arginine and 3-CH,-histidine; the values reported for these amino acids should be accepted with reservation. The values for 19 amino acids were combined at each gestational age as an indication of the changes in the total amino acid content of amniotic fluid which occur coincident to pregnancy in this species. Rewlts The free amino acids of amniotic fluid at each gestational age are presented in Table I. Although a relatively wide range of values was present for each amino acid at each age of gestation, several patterns of change were apparent. The total value of the 19 amino acids decreased sequentially with advancing gestational age; the same trend was seen for most of the individual amino acids. Taurine, reflecting sulfur amino acid metabolism and bile acid conjugation, was unique in showing the opposite trend, however, and the mean values for glutamine plus glutamic acid, proline, glycine, histidine, and 3-CH,-histidine were lower in the 50 day sample than at 75 days of gestational age; thereafter these amino acids also showed a progressive decrease. The total value of the 19 amino acids in maternal and umbilical vein sera are also indicated in Table I. The value in fetal serum was consistently elevated over those in both maternal serum and amniotic fluid. The value in maternal serum showed a rapid decrease early in pregnancy which stabilized by 75 days od gestational age. At this point the value in amniotic fluid was comparable to that in maternal serum; thereafter it was always below the value in both maternal and fetal sera. Comment Despite numerous investigations and speculations, the source of amniotic fluid has not been firmly established.4* 111 l2 The amniotic fluid of early gestation has been considered to represent a dialysate or transudate of ma-
Amniotic
fluid acids in pregnancy
365
ternal plasma, or a secretion from the amniotic epitheliumll; later in pregnancy the fetal respiratory tract, intestinal tract, umbilical cord, and salivary glands may also contribute to this fluid. Once renal function has been established, fetal micturition becomes the major source of amniotic fluid.13 As the source of amniotic fluid is complex, so also is its route of disposal. Fetal swallowing is one obvious route and polyhydramnios frequently accompanies esophageal or intestinal atresia.13 A limited number of investigations have been performed in relation to the amino acid content of amniotic fluid. With regard to the levels early in gestation, McKay and associatesl* reported that the amino nitrogen content of human hydatidiform mole fluid was elevated over that of maternal plasma. Lesinski and associates15 also noted high levels of amino acids in the blastocyst fluid of rabbits between the fifth and seventh days after mating: shortly thereafter a notable drop occurred in the level of most of the individual amino acids. Several studies have reported the amino acids of amniotic fluid during later gestation. WirtschafteP investigated amniotic fluid throughout the last eight days of pregnancy in the rat, and noted that amino acid levels rose with increasing gestation. He also noted that several amino acids were found at higher levels in amniotic fluid than in maternal blood, indicating a mechanism other than simple dialysis. In a subsequent study Wirtschafterl? also reported the free amino acids of human amniotic fluid at full-term pregnancy and noted a species difference; whereas the amino acid levels of rat amniotic fluid were often elevated over the maternal level, the opposite result was consistently seen in human pregnancies. Although a considerable amount of information has been reported with regard to the transplacental levels and transport of amino acids in this speciesl*p I9 and in the human,?O much less is available concerning the relationships between the free amino acids of amniotic fluid and those of fetal or maternal plasma. Bonsnes,?l in a recent review on the composition of amniotic fluid, concluded
366
Kerr
and
Kennan
that the levels of free amino acids at fullterm pregnancy were generally comparable to those found in maternal plasma. The evidence for this conclusion was partly derived from the report of Orlandi and colleagues,‘” who noted that values for several amino acids in amniotic fluid were elevated over maternal levels. Unfortunately, the samples of amniotic fluid and maternal plasma in this study were not obtained from the same patients; their conclusions are therefore open to question. The reports of Stass? and Wirtschafterlr indicated that the combined levels of amino acids in human amniotic fluid at full-term pregnancy varied from 9 to 25 mg. per 100 ml., and in the latter study the amniotic fluid values were consistently below those in maternal serum. The values at late gestation in the rhesus monkey would be in accord with these latter studies. The free amino acids of maternal and fetal blood during pregnancy in M. mulatta have been reported previously.l* All maternal amino acids except taurine, lysine, 3-CH,histidine, and arginine were lower at fullterm pregnancy than in nonpregnant adult females. This pattern was present by 75 days of gestation and did not show a significant change thereafter. Several amino acids, most notably glutamine and glutamic acid, histidine, methionine, and valine, were elevated in fetal blood during early pregnancy, and fell as gestation proceeded. 3-CH,-histidine and glycine showed the opposite trend and reached their maximum value at full-term pregnancy. The other amino acids of fetal blood did not show significant changes throughout gestation. Umbilical cord levels for each amino acid were invariably elevated over maternal values, and still further elevated over the levels in amniotic fluid. Most of the individual amino acids of amniotic fluid showed a gradual and progressive decrease as pregnancy progressed, in
Seeds, A. E., Kock, H. C., Myers, Stolte, L. A. M., and Hellegers, A. J. OBST. & GYNEC. 97: 67, 1967.
The surgical assistance of Drs. Harry A. Waisman and James R. Allen, and the technical assistance of Guenther Scheffler, James A. Campbell, and Arthur Helmuth are gratefully acknowledged.
2. Pitkin,
REFERENCES
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distinction to the rapid reduction seen irr maternal strum. The maternal Icvrl at late gestation was alvzays higher than the amniotic fluid level; at early pregnancy, however, the amniotic fluid levels were higher in the cases of proline, lysine, histidine, and 3-CH,-histidine, and for several other amino acids the levels were approximately equal. Only with the progress& reduction in amniotic fluid levels, possibly reflecting a maturation of renal tubular function,“’ did they all eventually fall belokv the maternal levels. The notable exception of taurine to the pattern of change shown for the other amino acids is intriguing, and probably reflects those factors which produce elevated levels of taurine in postnatal urine, and in the umbilical vein blood of both human?: and subhumar? primates. It is apparent, however, that the changes in the amino acid levels of amniotic fluid are not simply a reflection of the changes which occur in either fetal or maternal serum. The observation that amniotic fluid is swallowed by the fetusZ7 suggests that it may have a role in fetal nutrition. Sugawa and co-workers’” have reported that the amino acids of amniotic fluid in rodents may be utilized for protein synthesized by both fetus and placenta. The nutritive role of this fluid in fetal nitrogen metabolism would be related to the volume ingested. The rate of transfer of water between the pregnant rhesus monkey and fetus has been reported at 1 L. per hour.?” A major proportion of this volume may involve fluid swallowed by the fetus, and the amino acid content of this fluid may play a greater role in fetal nitrogen metabolism than has previously been considered.
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