- Email: [email protected]
Quantitative amino acids in amniotic fluid and maternal plasma in early and late pregnancy
Quantitative amino acids in amniotic fluid and maternal plasma in early and late pregnancy Preliminary
DAVID
report
W.
J.
REID,
M.D.
DONALD
J.
CAMPBELL,
LUELLA
Y.
YAKYMYSHYN,
Edmonton,
Alberta,
PH.D. B.Sc.
Canada
Twenty-one amino acids were quantitatively determined in amniotic fluid and maternal plasma of 27 normal women at 2 periods of normal gestation. The plasma levels of the same 21 amino acids were also predetermined for a grouQ of normal nonpregnant adults. Significant differences were found for levels of plasma amino acids in both the 7 to 18 week and 36 to 40 week gestational groups (u compared to the nonpregnant group. The free amino acid levels of amniotic @uid did not reflect maternal plasma levels. At 7 to 18 weeks, IO amino acids were significantly higher in amniotic fluid than maternal plasma, while at term the reverse was found, 15 amino acids being lower in amniotic fluid than maternal plasma. Sixteen amniotic fluid amino acids were significantly lower at 36 to 40 weeks than 7 to 18 weeks, whereas no significant differences were found for most of the plasma amino acids of the 2 groups.
COMPARATIVE STUDY of amino acids in amniotic fluid and maternal plasma has been inadequately studied even though it may well reflect the biochemical environment of the developing fetus. Few reports have appeared in the literature within recent months dealing with the quantitative lefvels of amino acids in human amniotic fluid. The first1 was a study of amniotic fluid and maternal and fetal plasma amino acid levels at term only, and amino acid levels were generally found to be lower in amniotic fluid than in maternal plasma, the exception being taurine. The second report2 was of amniotic fluid amino acid levels at various stages of gestation. Here, there was a general decrease of amniotic fluid amino acids from the first trimester to term. It was also noted
that in early pregnancy the amniotic fluid amino acids were generally higher than adult plasma, whereas at term the reverse was true. However, no distinction was made between samples from normal and abnormal (Rh incompatible) pregnancies, and maternal plasma levels were not reported for each gestational stage. Rhesus monkeys have been extensively studied,3 the results differing in some respects from those reported on human beings, indicating a possible species difference in the mechanisms involved between maternal and fetal plasma and amniotic fluid. More recently, Cockburn and associates4 reported preliminary findings on amino acid levels in maternal plasma and amniotic fluid and fetal urine from 8 normal pregnancies of 15 to 20 weeks’ gestation. The concentrations in amniotic fluid were greater than in maternal venous plasma and fetal urine. Little correlation was found between amino acid levels in maternal plasma and amniotic fluid, whereas some significant
THE
From the DeQartment of Obstetrics Gynaecology and the Department Clinical Pathology, The University Alberta HosQital. Supported by the Medical Council of Canada, Grant
and of of
Research MRC3619.
251
252
Reid,
Campbell,
and
Yakymyshyn
positive correlation was found to exist between early fetal urine and amniotic fluid levels. The importance of amino acid levels in amniotic fluid, especially during the first half of pregnancy, may manifest itself in the detection of early and possibly abnormal changes in the developing fetus, first appearing as changes in the free amino acid levels in amniotic fluid. One recent report5 indicates from a close correlation found between fetal weight and amniotic fluid volume during the first half of pregnancy that the amniotic fluid may even be an extension of fetal extracellular fluid and space during early pregnancy. Recently, Saifer and colleague? have emphasized the value of determining amniotic fluid amino acid levels in early pregnancy and have reported 3 amino acids not found in maternal serum. The increasing availability of amniotic fluid at various gestational times, mainly the first and third trimesters, has made such analyses much more meaningful in defining fetal maturity, isoimmunization processes, and genetic disorders. Fetal growth depends upon the utilization of free amino acids in the synthesis of structural and functional proteins. The levels of certain amino acids higher in amniotic fluid than maternal plasma, during the first half of pregnancy, imply the selectivity of the placenta.’ This is a preliminary study of 21 amino acids in amniotic fluid and maternal plasma of 27 normal pregnant women at 2 periods of gestation. Plasma levels of the same amino acids were determined for a group of 16 normal nonpregnant adults. This study is an attempt to establish normal levels of amino acids at 2 gestation periods, 7 to 18 weeks and 36 to 40 weeks, in the amniotic fluid and plasmas of normal, healthy pregnant women. Materials
and
methods
The subjects were normal, healthy pregnant women with no history of metabolic disorders, toxemia, or Rh incompatibility. Samples of amniotic fluid were collected by amniocentesis from 16 term pregnancies (36
to 40 weeksi just prior to delivery and from 11 early pregnancies (7 to 18 weeks’ being therapeutically aborted. Fasting samples of maternal blood were also obtained from an antecubital vein and collected in EDTA.” Both blood and amniotic fluid were centrifuged at approximately 2,000 r.p.m. for 10 minutes within 30 minutes of sampling. The separated plasma and clear amniotic fluid supernatant were deproteinized with 30 mg. of crystalline sulfosalicylic acid per milliliter of plasma and 20 mg. per milliliter of amniotic fluid and again centrifuged. The clear protein-free supernatants of both fluids were then frozen at -20° C. pending analysis for no longer than 2 weeks. The quantitative amino acid analysis by ion-exchange column chromatography was carried out on a sample volume containing 0.5 ml. of plasma or 1.0 ml. of amniotic fluid and 0.25 pmoles of norleucine standard prepared in 12.5 per cent sucrose. A Technicon amino acid analyzer (Model NCl) -f was used, with the use of Type “B” resin and a sodium citrate buffer gradient of pH 2.875 to 5.00. A flow rate of 30 ml. per hour from a single long column (140 cm.) at 60’ C. was maintained over a 21 hour period. The separated amino acids were simultaneously determined calorimetrically with ninhydrin in the presence of the reducing agent sodium cyanide. The incorporation of sodium cyanide directly into the manifold at the time of reaction greatly reduces stability problems and is a recent development in this laboratory from the method recommended by Technicon.* The peaks were automatically recorded at 2 wavelengths (570 and 440 nm.) to include the prolines, and a Technicon integrator/calculator was subsequently used to calculate the results in micromoles per liter. Results Mean values, ranges, and standard deviations for the concentrations of 21 free amino acids (in micromoles per liter) for each of *EDTA tTcchnicon
=
ethylenediaminetetra-acetic Corp.,
Tarrytown,
New
acid anticoagulant. Ymk.
Volume Number
111 2
Amino
acids in amniotic
fluid and maternal
plasma
253
254
Reid,
Campbell,
and
Yakymyshyn
Table II. Comparison between amino acid concentrations in maternal plasma and amniotic fluid at 7 to 18 weeks’ and 36 to 40 weeks’ gestation and nonpregnancy plasma ~---. -~_---.___--.~~~ ... ..__.__......._ ___.__~~. .~ .~~ Maternal Amino
acid
Taurine Aspartic Threonine Serine Glutamic Proline Citrulline Glycine Alanine o-Amino butyric Valine Half-cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine
7-18 wk. us. nonpreanancy 0* 0 - Trend - Trend 0 -
36-40 wk. nonpregnancy -t 0
Trend$
11 Trend +§ -
0 0 -
- Trend -
- Trend 0 0 - Trend
- Trend - Trend 0 0 -
0 -. 0 0 0
- Trend
l 0 = no significant difference. t- = that it is significantly lower. STrend = significant at a probability of 0.10. O+ = a significantly greater amino acid concentration
plasma
in the first-named
the groups studied are given in Table I. The plasma amino acid values established on 16 normal nonpregnant controls were used as a basis for determining any changes of plasma amino acids during normal pregnancy. A t test for unpaired data was used to calculate probability values between the various groups, the differences being considered as statistically significant where chances of occurrence were 5 per cent probability or less. These results are shown in Tables II and III. At 7 to 18 weeks’ gestation, 10 plasma amino acids were significantly lower (p < 0.05) than corresponding nonpregnancy levels (Table II). Six other amino acidsthreonine, serine, alpha-amino butyric acid, methionine, isoleucine, and arginine-also showed a tendency to be lower (p < 0.10). Taurine, aspartic acid, glutamic acid, leutine, and phenylalanine showed no significant change. Between 36 and 40 weeks’ gestation, the plasma amino acids were either
dmniotic ---~--‘_----us. 7-18 wk. us. nonpregnancy
fluid 36-40 wk. nonpregnancy
0 0 0 -
0 0 .._ 0 -.
fi _. --. Trend 0 0 0 0 0 0 0 0 +
- Trend _ -
; 0
group
than in the second
_--. _. us.
(p < 0.05).
decreased or did not change from those of the nonpregnant control group. The only exceptions were glutamic acid which showed a significant increase and taurine and leucine which showed a trend to drop from early to late pregnancy. When the amniotic fluid from 7 to 18 weeks’ gestation was compared to nonpregnancy plasma, no significant differences were noted for 14 amino acids (Table II). As shown in Table III, most of the amino acids were significantly increased in the amniotic fluid as compared to the maternal plasma. At term (36 to 40 weeks), the reverse was found. Fifteen amino acids were lower in amniotic fluid as compared to maternal plasma, and 6 others showed no difference. On comparing amniotic fluid at term with nonpregnancy plasma, all of the amino acids were significantly lower in the amniotic fluid with the exceptions of taurine, aspartic, and glutamic acid, showing no change. Sixteen amniotic fluid amino acids decreased during
Volume Number
111 2
Amino
acids in amniotic
fluid
and maternal
plasma
255
Table III. Comparison between amino acid concentrations in maternal plasma and amniotic fluid at 7 to 18 weeks’ and 36 to 40 weeks’ gestation
Amino
Plasma, 36-40 wk. vs. 7-18 wk.
acid
Taurine Aspartic Threonine Serine
0* 0 +t 0
Glutamic Proline Citrulline Glycine Alanine o-Amino butyric Valine Half-cystine Methionine Isoieucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine
0 + Trends 0 0 + Trend 0 0 t + Trend 0 0 0 0 0
‘0 = no cgniicant
t+ = a significantly f = it is lower. BTrertd = significant
Amniotic fluid, 36-40 wk. vs. 7-18 wk. 0 f:
t
- Trend - Trend -
,-
t
t
Trend
Amniotic fluid us. Amniotic maternal plasma, maternal 7-18 wk. 36-40
fluid vs. plasma, wk.
0 0 t -
0 0 -
+ + 0 0 t 0 + Trend + + 0 0 t t 0 t + + Trend
0 0 0
0
difference.
greater
amino
at a probability
acid
concentration
in the first-named
group
than in the second
(p < 0.05).
of 0.10.
pregnancy, being significantly lower at 36 to 40 weeks’ than at 7 to 18 weeks’ gestation. Two others showed a trend to be lower while taurine, aspartic acid, and serine showed no significant change. No significant change for 13 plasma amino acids was found between early and late pregnancy. Threonine, half-cystine, lysine, and histidine were lower in early pregnancy, while proline, alanine, methionine, and arginine showed a trend to be lower. Figs. 1 and 2 show that amniotic fluid valine values are lower in late pregnancy versus early pregnancy. It is obvious also that the maternal plasma is similar at both periods of gestation. During the present study, beta-alanine, carnosine, and hydroxyproline were frequently detected in amniotic fluid but considered to be in quantities too insignificant to accurately calculate. Ethanolamine was detected in both maternal plasma and amniotic fluid so infrequently that it was not considered in the determination of a normal set of values.
Comments
As can be seen in Table II, there is a significant drop in the level of plasma amino acids during early pregnancy. However, as seen in Table III, there is little change in the plasma amino acid levels after 18 weeks. There are only two reports in the literaturegl lo comparing maternal plasma amino acids at term with nonpregnancy plasma levels. These workers singled out threonine and histidine as being increased in maternal plasma. As seen in Table II, we only found a significant increase in plasma glutamic acid in late pregnancy compared to the nonpregnant state. This may be due to the conversion of glutamine to glutamic acid which normally occurs on sample storage. Thus this finding may not have physiologic significance. Our work has shown that there is a significant drop in plasma amino acids during the first 18 weeks of pregnancy. The cause of plasma amino acid changes during pregnancy is a subject of considerable
256
Reid, Campbell,
and Yakymyshyn
Fig. 1. Probability plot for normalized population distribution of saline from the amniotic: fluid and maternal plasma of 11 normal pregnant subjects at 7 to 18 xweks gestation. controversy. There is some evidence that hormonal control is involved. Progesterone is known to be increased in plasma during pregnancy. I1 Landau and Lugibihll* recently described a selective plasma amino acid decrease in subjects following intramuscular injections of progesterone. Phenylalanine alone remained unchanged, while 12 other plasma amino acids were lowered. In early and late pregnancy versus nonpregnancy, these results showed similar differences to those found by Landau and Lugibihl in their experiments. Closer agreement was noted with early than with late pregnancy. For both groups, phenylalanine was among those amino acids showing no significant change. Landau and Lugibihll* suggest that progesterone has a relatively selective influence on plasma amino acid concentrations, several of which may be critical in regulating peripheral protein catabolism. Other+ have also reported a drop of tyrosine levels in women taking estrogen-progestogen-type oral contraceptives. Whether a steroid effect can be used to explain the drop in the amniotic
fluid amino acids we have reportrd requires further work. Regarding amino acids in amniotic fluid, these are maintained at nonpregnancy levels during early pregnancy, dropping to levels below maternal plasma levels at term. The amniotic fluid amino acids at the 7 to 18 week gestational period with the exception of serine were either unchanged or significantly higher than corresponding maternal plasma levels. Serine was significantly lower. Cockburn and colleagues* reported similar findings for a group studied at 15 to 20 weeks’ gestation. In the present study, 3 amino acids, glutamic acid, phenylalanine, and lysine, were even higher in amniotic fluid than in nonpregnancy plasma during early pregnancy (Table II). Where no significant differences were noted for most plasma amino acids from early to late pregnancy, amniotic fluid levels all dropped with the exception of taurine, aspartic acid, and glycine (showing no change), below corresponding maternal plasma levels. High levels of amniotic fluid
Volume Number
Amino
111 2
Fig. and
REFERENCES
Levy, 3:
113,
fluid
and maternal
2. Probability plot for normalized population distribution of valine from the amniotic maternal plasma of 16 normal pregnant subjects at 36 to 40 weeks’ gestation.
taurim as opposed to maternal serum have been reported.lp 2 However, we were unable to show any significant difference for this particular amino acid on applying the t test to our data. The exact source of amniotic fluid throughout pregnancy is still not known.14 Plently5 demonstrated, through the use of isotope tracers, a bidirectional transfer in the 3 compartments of fetus, mother, and amniotic fluid. Some feel that the amniotic fluid during early pregnancy4 and at term” is a reflection of the fetal urine amino acid content. Others5 have suggested that the amniotic fluid during the first half of pregnancy may be an extension of the fetal extracellular space. This raises the possibility that amniol:ic fluid amino acids during early pregnancy may reflect corresponding fetal blood levels. Cockburn and colleagues4 suggest that the presence of a considerable amount of
1.
acids in amniotic
H.
L., ancl 1969.
P. P.: Pediatr.
Res.
257
fluid
Wharton’s jelly between chorion and amnion during early pregnancy may contribute amino acids to amniotic fluid. The presence of maternal proteins in amniotic fluidI and genetic evidence17 support the theory that the amniotic fluid proteins are at least partly maternal in origin. Normal values and ranges for 21 ammo acids in early and late pregnancy have been established in maternal venous plasma and amniotic fluid from 27 normal, healthy pregnant women. These results should provide a basis for further study of abnormal pregnancies or pregnancies where mothers are possible carriers of inborn errors of metabolism. The knowledge of amniotic fluid levels of amino acids, especially during early pregnancy, may very well provide the physician with forewarning of pending fetal problems or abnormal fetal growth and development.
2. Montag,
plasma
Emery, A. E. H., and Schrimgeour, 1970.
Burt, D., Nelson, J. B.: Laneet
M. 1:
D., 1307,
258
3. 4. 5. 6. 7.
8.
9. 10.
Reid,
Campbell,
and
Yakymyshyn
Kerr, G. R., and Kennan, A. L.: AM, J. OBSTET. GYNECOL. 105: 363, 1969. Cockburn, F., Robins, S. P., and Forfar, J. 0.: Br. Med. J. 3: 747, 1970. Lind, T., and Hytten, F. E.: Lancet 1: 1147, 1970. Saifer, A., A-zary, E., Valenti, C., and Schneck, L.: Clin. Chem. 16: 891, 1970. Litonjua, A. D., Canlas, M., Soliman, J., and Paulino, D. Q.: AM. J. OBSTET. GYNECOL. 99: 242, 1967. Yakymyshyn, L. Y., Reid, D. W., and Campbell, D. J.: 14th Annual Meeting, Can. Sot. Clin. Chem., Quebec City, Canada, July, 1970, Abstract. Lorincz, A. B., and Kuttner, R. E.: AM. J. OBSTET. GYNECOL. 105: 925. 1969. Armstrong, M. D., and Yates, K. N.: AM. J. OBSTET. GYNECOL. 88: 381. 1964.
11.
12. 13. 14.
15. 16. 17.
Van Der Molen, H. J .? and Aakvaag, .A.: Xn Gray, C. H., and Bacharack, A. L., editors: Hormones in Blood, ed. 2. New York, 1967, Academic Press, Inc., p. 221. Landau, R. L., and Lugibihl, K.: Metabolism 16: 1114, 1967. Rose, D. P., and Cramp, D. G.: Clin. Chim. Acta 29: 49, 1970. Schulman, H.: In Mann, L. I., and Frederick, M. A., editors: Clinical Obstetrics and Gynecology, New York, 1970, vol. 13, Harper Sr Row, Publishers, p, 542. Plentl, A. ‘4.: Ann. N. Y. Acad. Sci. 75: 746, 1959. Usategui-Gomez, and Morgan, D. F.: Nature 212: 1600, 1966. Seppala, M., Ruoslahti, E., and Tallberg, T. H.: Ann Med. Exp. Biol. Fenn. 44: 6, 1966.
DAVID
report
W.
J.
REID,
M.D.
DONALD
J.
CAMPBELL,
LUELLA
Y.
YAKYMYSHYN,
Edmonton,
Alberta,
PH.D. B.Sc.
Canada
Twenty-one amino acids were quantitatively determined in amniotic fluid and maternal plasma of 27 normal women at 2 periods of normal gestation. The plasma levels of the same 21 amino acids were also predetermined for a grouQ of normal nonpregnant adults. Significant differences were found for levels of plasma amino acids in both the 7 to 18 week and 36 to 40 week gestational groups (u compared to the nonpregnant group. The free amino acid levels of amniotic @uid did not reflect maternal plasma levels. At 7 to 18 weeks, IO amino acids were significantly higher in amniotic fluid than maternal plasma, while at term the reverse was found, 15 amino acids being lower in amniotic fluid than maternal plasma. Sixteen amniotic fluid amino acids were significantly lower at 36 to 40 weeks than 7 to 18 weeks, whereas no significant differences were found for most of the plasma amino acids of the 2 groups.
COMPARATIVE STUDY of amino acids in amniotic fluid and maternal plasma has been inadequately studied even though it may well reflect the biochemical environment of the developing fetus. Few reports have appeared in the literature within recent months dealing with the quantitative lefvels of amino acids in human amniotic fluid. The first1 was a study of amniotic fluid and maternal and fetal plasma amino acid levels at term only, and amino acid levels were generally found to be lower in amniotic fluid than in maternal plasma, the exception being taurine. The second report2 was of amniotic fluid amino acid levels at various stages of gestation. Here, there was a general decrease of amniotic fluid amino acids from the first trimester to term. It was also noted
that in early pregnancy the amniotic fluid amino acids were generally higher than adult plasma, whereas at term the reverse was true. However, no distinction was made between samples from normal and abnormal (Rh incompatible) pregnancies, and maternal plasma levels were not reported for each gestational stage. Rhesus monkeys have been extensively studied,3 the results differing in some respects from those reported on human beings, indicating a possible species difference in the mechanisms involved between maternal and fetal plasma and amniotic fluid. More recently, Cockburn and associates4 reported preliminary findings on amino acid levels in maternal plasma and amniotic fluid and fetal urine from 8 normal pregnancies of 15 to 20 weeks’ gestation. The concentrations in amniotic fluid were greater than in maternal venous plasma and fetal urine. Little correlation was found between amino acid levels in maternal plasma and amniotic fluid, whereas some significant
THE
From the DeQartment of Obstetrics Gynaecology and the Department Clinical Pathology, The University Alberta HosQital. Supported by the Medical Council of Canada, Grant
and of of
Research MRC3619.
251
252
Reid,
Campbell,
and
Yakymyshyn
positive correlation was found to exist between early fetal urine and amniotic fluid levels. The importance of amino acid levels in amniotic fluid, especially during the first half of pregnancy, may manifest itself in the detection of early and possibly abnormal changes in the developing fetus, first appearing as changes in the free amino acid levels in amniotic fluid. One recent report5 indicates from a close correlation found between fetal weight and amniotic fluid volume during the first half of pregnancy that the amniotic fluid may even be an extension of fetal extracellular fluid and space during early pregnancy. Recently, Saifer and colleague? have emphasized the value of determining amniotic fluid amino acid levels in early pregnancy and have reported 3 amino acids not found in maternal serum. The increasing availability of amniotic fluid at various gestational times, mainly the first and third trimesters, has made such analyses much more meaningful in defining fetal maturity, isoimmunization processes, and genetic disorders. Fetal growth depends upon the utilization of free amino acids in the synthesis of structural and functional proteins. The levels of certain amino acids higher in amniotic fluid than maternal plasma, during the first half of pregnancy, imply the selectivity of the placenta.’ This is a preliminary study of 21 amino acids in amniotic fluid and maternal plasma of 27 normal pregnant women at 2 periods of gestation. Plasma levels of the same amino acids were determined for a group of 16 normal nonpregnant adults. This study is an attempt to establish normal levels of amino acids at 2 gestation periods, 7 to 18 weeks and 36 to 40 weeks, in the amniotic fluid and plasmas of normal, healthy pregnant women. Materials
and
methods
The subjects were normal, healthy pregnant women with no history of metabolic disorders, toxemia, or Rh incompatibility. Samples of amniotic fluid were collected by amniocentesis from 16 term pregnancies (36
to 40 weeksi just prior to delivery and from 11 early pregnancies (7 to 18 weeks’ being therapeutically aborted. Fasting samples of maternal blood were also obtained from an antecubital vein and collected in EDTA.” Both blood and amniotic fluid were centrifuged at approximately 2,000 r.p.m. for 10 minutes within 30 minutes of sampling. The separated plasma and clear amniotic fluid supernatant were deproteinized with 30 mg. of crystalline sulfosalicylic acid per milliliter of plasma and 20 mg. per milliliter of amniotic fluid and again centrifuged. The clear protein-free supernatants of both fluids were then frozen at -20° C. pending analysis for no longer than 2 weeks. The quantitative amino acid analysis by ion-exchange column chromatography was carried out on a sample volume containing 0.5 ml. of plasma or 1.0 ml. of amniotic fluid and 0.25 pmoles of norleucine standard prepared in 12.5 per cent sucrose. A Technicon amino acid analyzer (Model NCl) -f was used, with the use of Type “B” resin and a sodium citrate buffer gradient of pH 2.875 to 5.00. A flow rate of 30 ml. per hour from a single long column (140 cm.) at 60’ C. was maintained over a 21 hour period. The separated amino acids were simultaneously determined calorimetrically with ninhydrin in the presence of the reducing agent sodium cyanide. The incorporation of sodium cyanide directly into the manifold at the time of reaction greatly reduces stability problems and is a recent development in this laboratory from the method recommended by Technicon.* The peaks were automatically recorded at 2 wavelengths (570 and 440 nm.) to include the prolines, and a Technicon integrator/calculator was subsequently used to calculate the results in micromoles per liter. Results Mean values, ranges, and standard deviations for the concentrations of 21 free amino acids (in micromoles per liter) for each of *EDTA tTcchnicon
=
ethylenediaminetetra-acetic Corp.,
Tarrytown,
New
acid anticoagulant. Ymk.
Volume Number
111 2
Amino
acids in amniotic
fluid and maternal
plasma
253
254
Reid,
Campbell,
and
Yakymyshyn
Table II. Comparison between amino acid concentrations in maternal plasma and amniotic fluid at 7 to 18 weeks’ and 36 to 40 weeks’ gestation and nonpregnancy plasma ~---. -~_---.___--.~~~ ... ..__.__......._ ___.__~~. .~ .~~ Maternal Amino
acid
Taurine Aspartic Threonine Serine Glutamic Proline Citrulline Glycine Alanine o-Amino butyric Valine Half-cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine
7-18 wk. us. nonpreanancy 0* 0 - Trend - Trend 0 -
36-40 wk. nonpregnancy -t 0
Trend$
11 Trend +§ -
0 0 -
- Trend -
- Trend 0 0 - Trend
- Trend - Trend 0 0 -
0 -. 0 0 0
- Trend
l 0 = no significant difference. t- = that it is significantly lower. STrend = significant at a probability of 0.10. O+ = a significantly greater amino acid concentration
plasma
in the first-named
the groups studied are given in Table I. The plasma amino acid values established on 16 normal nonpregnant controls were used as a basis for determining any changes of plasma amino acids during normal pregnancy. A t test for unpaired data was used to calculate probability values between the various groups, the differences being considered as statistically significant where chances of occurrence were 5 per cent probability or less. These results are shown in Tables II and III. At 7 to 18 weeks’ gestation, 10 plasma amino acids were significantly lower (p < 0.05) than corresponding nonpregnancy levels (Table II). Six other amino acidsthreonine, serine, alpha-amino butyric acid, methionine, isoleucine, and arginine-also showed a tendency to be lower (p < 0.10). Taurine, aspartic acid, glutamic acid, leutine, and phenylalanine showed no significant change. Between 36 and 40 weeks’ gestation, the plasma amino acids were either
dmniotic ---~--‘_----us. 7-18 wk. us. nonpregnancy
fluid 36-40 wk. nonpregnancy
0 0 0 -
0 0 .._ 0 -.
fi _. --. Trend 0 0 0 0 0 0 0 0 +
- Trend _ -
; 0
group
than in the second
_--. _. us.
(p < 0.05).
decreased or did not change from those of the nonpregnant control group. The only exceptions were glutamic acid which showed a significant increase and taurine and leucine which showed a trend to drop from early to late pregnancy. When the amniotic fluid from 7 to 18 weeks’ gestation was compared to nonpregnancy plasma, no significant differences were noted for 14 amino acids (Table II). As shown in Table III, most of the amino acids were significantly increased in the amniotic fluid as compared to the maternal plasma. At term (36 to 40 weeks), the reverse was found. Fifteen amino acids were lower in amniotic fluid as compared to maternal plasma, and 6 others showed no difference. On comparing amniotic fluid at term with nonpregnancy plasma, all of the amino acids were significantly lower in the amniotic fluid with the exceptions of taurine, aspartic, and glutamic acid, showing no change. Sixteen amniotic fluid amino acids decreased during
Volume Number
111 2
Amino
acids in amniotic
fluid
and maternal
plasma
255
Table III. Comparison between amino acid concentrations in maternal plasma and amniotic fluid at 7 to 18 weeks’ and 36 to 40 weeks’ gestation
Amino
Plasma, 36-40 wk. vs. 7-18 wk.
acid
Taurine Aspartic Threonine Serine
0* 0 +t 0
Glutamic Proline Citrulline Glycine Alanine o-Amino butyric Valine Half-cystine Methionine Isoieucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine
0 + Trends 0 0 + Trend 0 0 t + Trend 0 0 0 0 0
‘0 = no cgniicant
t+ = a significantly f = it is lower. BTrertd = significant
Amniotic fluid, 36-40 wk. vs. 7-18 wk. 0 f:
t
- Trend - Trend -
,-
t
t
Trend
Amniotic fluid us. Amniotic maternal plasma, maternal 7-18 wk. 36-40
fluid vs. plasma, wk.
0 0 t -
0 0 -
+ + 0 0 t 0 + Trend + + 0 0 t t 0 t + + Trend
0 0 0
0
difference.
greater
amino
at a probability
acid
concentration
in the first-named
group
than in the second
(p < 0.05).
of 0.10.
pregnancy, being significantly lower at 36 to 40 weeks’ than at 7 to 18 weeks’ gestation. Two others showed a trend to be lower while taurine, aspartic acid, and serine showed no significant change. No significant change for 13 plasma amino acids was found between early and late pregnancy. Threonine, half-cystine, lysine, and histidine were lower in early pregnancy, while proline, alanine, methionine, and arginine showed a trend to be lower. Figs. 1 and 2 show that amniotic fluid valine values are lower in late pregnancy versus early pregnancy. It is obvious also that the maternal plasma is similar at both periods of gestation. During the present study, beta-alanine, carnosine, and hydroxyproline were frequently detected in amniotic fluid but considered to be in quantities too insignificant to accurately calculate. Ethanolamine was detected in both maternal plasma and amniotic fluid so infrequently that it was not considered in the determination of a normal set of values.
Comments
As can be seen in Table II, there is a significant drop in the level of plasma amino acids during early pregnancy. However, as seen in Table III, there is little change in the plasma amino acid levels after 18 weeks. There are only two reports in the literaturegl lo comparing maternal plasma amino acids at term with nonpregnancy plasma levels. These workers singled out threonine and histidine as being increased in maternal plasma. As seen in Table II, we only found a significant increase in plasma glutamic acid in late pregnancy compared to the nonpregnant state. This may be due to the conversion of glutamine to glutamic acid which normally occurs on sample storage. Thus this finding may not have physiologic significance. Our work has shown that there is a significant drop in plasma amino acids during the first 18 weeks of pregnancy. The cause of plasma amino acid changes during pregnancy is a subject of considerable
256
Reid, Campbell,
and Yakymyshyn
Fig. 1. Probability plot for normalized population distribution of saline from the amniotic: fluid and maternal plasma of 11 normal pregnant subjects at 7 to 18 xweks gestation. controversy. There is some evidence that hormonal control is involved. Progesterone is known to be increased in plasma during pregnancy. I1 Landau and Lugibihll* recently described a selective plasma amino acid decrease in subjects following intramuscular injections of progesterone. Phenylalanine alone remained unchanged, while 12 other plasma amino acids were lowered. In early and late pregnancy versus nonpregnancy, these results showed similar differences to those found by Landau and Lugibihl in their experiments. Closer agreement was noted with early than with late pregnancy. For both groups, phenylalanine was among those amino acids showing no significant change. Landau and Lugibihll* suggest that progesterone has a relatively selective influence on plasma amino acid concentrations, several of which may be critical in regulating peripheral protein catabolism. Other+ have also reported a drop of tyrosine levels in women taking estrogen-progestogen-type oral contraceptives. Whether a steroid effect can be used to explain the drop in the amniotic
fluid amino acids we have reportrd requires further work. Regarding amino acids in amniotic fluid, these are maintained at nonpregnancy levels during early pregnancy, dropping to levels below maternal plasma levels at term. The amniotic fluid amino acids at the 7 to 18 week gestational period with the exception of serine were either unchanged or significantly higher than corresponding maternal plasma levels. Serine was significantly lower. Cockburn and colleagues* reported similar findings for a group studied at 15 to 20 weeks’ gestation. In the present study, 3 amino acids, glutamic acid, phenylalanine, and lysine, were even higher in amniotic fluid than in nonpregnancy plasma during early pregnancy (Table II). Where no significant differences were noted for most plasma amino acids from early to late pregnancy, amniotic fluid levels all dropped with the exception of taurine, aspartic acid, and glycine (showing no change), below corresponding maternal plasma levels. High levels of amniotic fluid
Volume Number
Amino
111 2
Fig. and
REFERENCES
Levy, 3:
113,
fluid
and maternal
2. Probability plot for normalized population distribution of valine from the amniotic maternal plasma of 16 normal pregnant subjects at 36 to 40 weeks’ gestation.
taurim as opposed to maternal serum have been reported.lp 2 However, we were unable to show any significant difference for this particular amino acid on applying the t test to our data. The exact source of amniotic fluid throughout pregnancy is still not known.14 Plently5 demonstrated, through the use of isotope tracers, a bidirectional transfer in the 3 compartments of fetus, mother, and amniotic fluid. Some feel that the amniotic fluid during early pregnancy4 and at term” is a reflection of the fetal urine amino acid content. Others5 have suggested that the amniotic fluid during the first half of pregnancy may be an extension of the fetal extracellular space. This raises the possibility that amniol:ic fluid amino acids during early pregnancy may reflect corresponding fetal blood levels. Cockburn and colleagues4 suggest that the presence of a considerable amount of
1.
acids in amniotic
H.
L., ancl 1969.
P. P.: Pediatr.
Res.
257
fluid
Wharton’s jelly between chorion and amnion during early pregnancy may contribute amino acids to amniotic fluid. The presence of maternal proteins in amniotic fluidI and genetic evidence17 support the theory that the amniotic fluid proteins are at least partly maternal in origin. Normal values and ranges for 21 ammo acids in early and late pregnancy have been established in maternal venous plasma and amniotic fluid from 27 normal, healthy pregnant women. These results should provide a basis for further study of abnormal pregnancies or pregnancies where mothers are possible carriers of inborn errors of metabolism. The knowledge of amniotic fluid levels of amino acids, especially during early pregnancy, may very well provide the physician with forewarning of pending fetal problems or abnormal fetal growth and development.
2. Montag,
plasma
Emery, A. E. H., and Schrimgeour, 1970.
Burt, D., Nelson, J. B.: Laneet
M. 1:
D., 1307,
258
3. 4. 5. 6. 7.
8.
9. 10.
Reid,
Campbell,
and
Yakymyshyn
Kerr, G. R., and Kennan, A. L.: AM, J. OBSTET. GYNECOL. 105: 363, 1969. Cockburn, F., Robins, S. P., and Forfar, J. 0.: Br. Med. J. 3: 747, 1970. Lind, T., and Hytten, F. E.: Lancet 1: 1147, 1970. Saifer, A., A-zary, E., Valenti, C., and Schneck, L.: Clin. Chem. 16: 891, 1970. Litonjua, A. D., Canlas, M., Soliman, J., and Paulino, D. Q.: AM. J. OBSTET. GYNECOL. 99: 242, 1967. Yakymyshyn, L. Y., Reid, D. W., and Campbell, D. J.: 14th Annual Meeting, Can. Sot. Clin. Chem., Quebec City, Canada, July, 1970, Abstract. Lorincz, A. B., and Kuttner, R. E.: AM. J. OBSTET. GYNECOL. 105: 925. 1969. Armstrong, M. D., and Yates, K. N.: AM. J. OBSTET. GYNECOL. 88: 381. 1964.
11.
12. 13. 14.
15. 16. 17.
Van Der Molen, H. J .? and Aakvaag, .A.: Xn Gray, C. H., and Bacharack, A. L., editors: Hormones in Blood, ed. 2. New York, 1967, Academic Press, Inc., p. 221. Landau, R. L., and Lugibihl, K.: Metabolism 16: 1114, 1967. Rose, D. P., and Cramp, D. G.: Clin. Chim. Acta 29: 49, 1970. Schulman, H.: In Mann, L. I., and Frederick, M. A., editors: Clinical Obstetrics and Gynecology, New York, 1970, vol. 13, Harper Sr Row, Publishers, p, 542. Plentl, A. ‘4.: Ann. N. Y. Acad. Sci. 75: 746, 1959. Usategui-Gomez, and Morgan, D. F.: Nature 212: 1600, 1966. Seppala, M., Ruoslahti, E., and Tallberg, T. H.: Ann Med. Exp. Biol. Fenn. 44: 6, 1966.