Arachidonic acid and other free fatty acid changes during abortion induced by prostaglandin Fza PAUL

L.

OGBURN,

WILLIAM R. C.

E. BRENNER, REITZ,

FREDERICK

PH.D.**

G. STAUROVSKY,

JAMES

R. DINGFELDER, Hill,

M.D.

P. ZUSPAN,

LINDA

Chapel

M.D.*

JR.,

North

Carolina,

M.D. C.N.M. M.D.

and

Columbus,

Ohio

Serum free fatty acids (FFA’s) were measured after intra-amniotic injection of prostaglandin F,, (PGF*,) for the induction of abortion in eight heathy women in the midtrimester of pregnancy. The total FFA levels increased in all cases during the period between PGF,, administration and abortion. This lipolysis most likely is secondary to the effect of the catecholamines as indicated by the temporal relationship between increased plasma and urine catecholamine and serum FFA levels. The percentage of arachidonic acid in the total amount of FFA’s decreased after administration of PGF,,. This proportional decrease in arachidonic acid (p < 0.05) may be due to selective utilization for the production of endogenous prostaglandins. (AM. J. OBSTET. GYNECOL. 130: 188, 1978.)

Serum nonesterified fatty acids (or FFA’s) normally increase in human pregnancy, then quickly decrease post parturn.* FFA levels increase during labor and reach a maximum in the second stage.5 Oxytocin catecholamines will increase FFA levels6, 7 Because of this indirect evidence for an important role of FFA’s in parturition, serum FFA levels, with special attention to arachidonic acid and catecholamines, were evaluated during second-trimester abortions induced with intraamniotic PGF,,.

EXOGENOUS PROSTAGLANDIN F,, (PGF*,) will cause uterine contractions with resultant abortion or labor when administered via one of several routes. Endogenous PGF,, has been implicated as the initiator of spontaneous labor.’ Because arachidonic acid in its nonesterified form is the precursor of PGF,, and prostaglandin I$, some* have hypothesized that it plays an important part in the initiation of parturition. Arachidonic acid and other free fatty acids (FFA’s) will stimulate smooth muscle contractions in vitro.3

Material and methods Eight physically healthy gravid women from 18 to 2 1 years of age and at 18 to 2 1 menstrual weeks’ gestation were studied during abortion induced with intraamniotically administered PGFz, (40 mg. as the Tham salt). The characteristics and course of each patient are reported elsewhere.8 All subjects were monitored fat intra-amniotic pressure by the open-end catheter technique.g The abortion was declared complete when both the fetus and placenta were completely expelled from the uterus. Abortion was considered incompiete if any part of the placenta was retained in the uterus; abortion was completed with vacuum aspiration. Heart and respiration rates, blood pressure, and fetal heart rates (FHR’s) were observed at least each hour, with fetal distress defined as a change of 20 or more beats per minute (b.p.m.) in FHR. Patients received nothing

From the Departments of Obstetrics and Gynecology, University of North Carolina School of Medicine and Ohio State University. Supported in part by grants from the International Fertilitv Research Proeram. Research Triangle Park. North bwolina (AIDTcsd 2979), and the F&d Founaiztion (No. 690-0108). Received for pu&lication Revised Accepted

October

May

27, 1977.

5, 1977

November

17, 1977.

Reprint requests: Dr. William E. Brenner, Department of Obstetrics and Gynecology, University of North Carolina, Old Clinic Bldg., 226 H, Chapel Hill, North Carolina 27514. *Present Obstetrics

address: University and Gynecology,

**Present address: University Biochemistry, Reno, Nevada.

of Minnesota, Department Minneapolis, Minnesota. of Nevada,

Department

of of

188

Serum

Table

I. Serum

.Wean ilulividual FFA Olgic acid (18:l) (nmole/ml.) Palmitic acid (16:O) (nmolei ml.) Linoleic acid (18:2) (nmolei ml.) Stearic acid ( 18 : 0) (nmolei ml.) Arachidonic acid (20:4) (nmole/ ml.) Total FFA (nmole/ml.)

FFA after 40 mg. of PGF,, injected Preinjection base line

Time of fetal distress

abortioninducedby

PGF,,

189

intra-amniotically

4 hours

6 hours

8 hours

16 huurs

24 hours

111.5

? 48.8

245.8

t 92.4*

212.2

? 98.8

246.1

t 62.6

277.2

c 96.0

238.0

-c 64.6

98.0 -e 66. I

88.9

k 32.0

168.0

2 44.9

147.8

-r- 53.3

172.7

? 40.6

190.6

+- 53.2

165.7

+ 35.6

77.1 it 36.4

57.6 it 22.2

105.9

? 32.3t

92.9 + 37.8

103.5

-r- 22.7

114.6

2 29.9

100.6

-t 37.3

44.7

67.7

ck 21.4

65.2

77.4 " 21.7

80.9

rt 20.8

64.4

-t 13.3$

33.2 _' 17.3

19.3 * 4.3

17.3 r 5.8

42.0

ir 13.1

17.6 2 7.0

323.4

r

121.6

18.3 f 5.0

621.4

-r- 275.6

Values are means + standard deviations. *Significant compared to base-line value t Significant compared to base-line value $ Significant compared to base-line value

_' 23.8

18.7 +- 7.9

550.9

in 215.6

Science Labs., 900, Perkin-Elmer

634.9

+ 149.5

695.1

t

197.2

19.4 -f 4.8

604.9

I 144.7

iz 24.7

14.1 -r 5.2

272.1

+ 149.9

at p = 0.01. at p = 0.002. at p = 0.00 1.

by mouth for nine to 10.5 hours before induction of abortion. After administration of prostaglandin, the patients ingested regular diets with the exception that they received no caffeine. Blood, urine, and amniotic fluid samples were collected, stabilized in preservative, frozen, and analyzed to determine epinephrine (E) and norepinephrine (NE) levels by the method of Zuspan and Cooley.lo Blood for FFA analysis was drawn before administration and at four, six, eight, 16, and 24 hours after administration of PGFz:,,. The serum for FFA analysis was obtained from blood which was allowed to clot at room temperature for not more than two hours; the serum samples were then frozen at -4” C. until the time of analysis. Catecholamines and FFA’s are stable under these conditions of storage. All analyses on these samples were done at one time. The serum FFA’s were then extracted by the method of Hagenfeldt. *I Methyl esters of these FFA’s were prepared by a previously reported method.‘* The methyl esters were dissolved in one to four volumes of carbon disulfide and injected onto a 12 foot by ‘/8 inch glass column packed with 12 per cent EGSS-Y on lOO/ 120 mesh Gas Chrom Q* in a gas-liquid chromatography machine.? The column temperature was 195” C., and the carrier gas was nitrogen; a hydrogen flame detector was used to measure the methyl esters as they emerged from the column. An internal standard of heptadecanoic acid was used for column recovery cal*Applied tMode1

FFAchangesdurirg

Inc., State College, Corp., Norwalk,

Pennsylvania. Connecticut.

culations. An integrator measured the relative areas under each peak; by iomparing the areas of the major peaks with the major peak of the heptadecanoic acid internal standard, the amount of each major FFA in each sample was calculated. The total FFA’s were computed as the sums of the respective major individual FFA in each sample. RSSUltS

Seven patients had complete abortions and one underwent vacuum aspiration for a retained placenta. The times for abortion ranged from nine hours and 50 minutes to 32 hours and 35 minutes after PGF2, administration. All patients had increases in total FFA’s during the period of time between PGFzc, dosage and abortion. Mean values for the major individual FFA’s with levels of significance, are presented in Table I. Mean total FFA’s increased significantly (p < O.OOl), from 323.4,-e 121.6 to levels of 634.9 * 149.5, 695.1 +: 197.2, and 604.9 + 144.7 nmole per milliliter at 6, 8 and 16 hours, respectively, after PGFz,. Serum levels increased to amounts as high as 1,065.l nmole per milliliter froma base line of 496.9 nmole per milliliter in one patient. The major individual FFA’s which make up total FFA’s (palmitic acid, 16 : 0; stearic acid, 18 : 0; oleic acid, 18: 1; linoleic acid, 18: 2) followed the pattern of the total FFA’s in each patient by increasing and decreasing roughly as a proportion of the total EFA’s (Fig. I). Arachidonic acid (20: 4), unlike the other FFA’s, did not change significantly after injection of PGF,,; as a

190

Ogburn et al.

A. TOTAL

January Am. J. Obstet.

AND INDIVIDUAL

FFA

soor

800

A. TOTAL AND INDIVIDUAL

15, 1978

Gynecol.

FFA

c TOTAL FFA

600 t

.

r

l

ii t- B. PROPORTION

n

ARACHIDONIC

ACID

ARACHl~olU&A~~~ZO:4)

6

8

IO

12

TIME

14

16

18

x ,oo

20

22

B. PROPORTION

0 2

T

%

0’-10

24

(HRS)

total FFA and the standard deviations in nanomoles per milliliter, the mean levels of palmitic acid (16: 0), stearic acid (18: 0), oleic acid (18: l), linoleic acid (18: 2), and arachidonic acid (20: 4) in nanomoles per milliliter, and the mean proportion of total FFA’s consisting of arachidonic acid in per cent from eight women in midtrimester pregnancy after the intra-amniotic administration of 40 mg. of PGF,, are shown (see text). The mean serum levels obtained at the time of fetal distress (F.D.) are plotted at the mean time that fetal distress occurred. percentage

arachidonic

acid

5.90

to 2.63

k 3.39

PGF,, 6.70

+ 3.94

per

mean over to the

increase

the

from cent

then

the

course line

of the

and

per

FFA

made the

up

crease

in

base

at eight

proportion

of

(Fig.

line

hours

increased

after

to a level

of

FFA

serum

of the

abortion

abortion

made

course

FFA’s

also

and (Fig.

in-

the

then

correlated

up of arachidonic

plasma well

,

,

,

,

temporally

with

(Fig.

3). The

correlated made

up

plasma

negatively by

E and with

the

arachidonic

acid

activity

Montevideo the

increased units

proportion acid

after

of

(20:4)

the

decreased

to levels PGF,,

between

300

administration

total

FFA

(Fig.

that

was

5).

Comment

acid

FFA’s

are

the

blood.

adipose state

urinary the

FFA

FFA

uterine

400

in

E and

total

re-

of abortion.

mean

,

4).

arachidonic

2). Again,

plasma catecholamines were measured the procedures as described elsewhere.* in

and

mean of

Mean while

major

after

I,,

catecholamines

by

at 24 hours. of

base

over

catecholamines

cent

the

the proportion Urine throughout

a drop

? 1.30

levels

turned decreased

serum

(p < 0.05),

creased

The

showed

injection

The

of

I

IO 20 30 40 50 60 70 80 90 loo Post PROPORTION OF ABORTION TIME (%)

total FFA and the standard deviations in nanomoles per milliliter, the mean levels of palmitic acid (16:0), stearic acid (18:0), oleic acid (18: l), linoleic acid (18: 2), and arachidonic acid (20: 4) in nanomoles per milliliter, and the mean proportion of total FFA consisting of arachidonic acid in per cent from eight women in midtrimester pregnancy over the course of abortion after the intraamniotic administration of 40 mg. of PGF,, are shown (see text).

urinary the

I

ACID

Fig. 2. The mean major

Fig. 1. The mean major

result,

I,

0

ARACHIDONIC

in-

tissue bound

the

most They and

rather

metabolically are

are

released

transported

loosely

as anions

E and NE stimulate lipolysis vitro studies have demonstrated

in vitro that

active by

lipids

found

lipolysis

from

in the

unesterified

to albumin.13 and in vivo.14 prostaglandin

Both In E1

Volume Number

Serum FFA changes during abortion induced by PGF2,

130 ?

A FATTY ACID

(TOTAL

FFAI

191

I6 [ A. PLASMA

700650600550

-

-6 1 500 z

-

0450-

E g400-

0 = 350300250-

,eB,UAINE ASMA

CATECHOLAMINES

17 -EPlNEPHRlNE -NOREPINEPHRINE

c--d

EPINEPHRINE

-

NOREPINEPHRINE

-

16 OE~NEPHRINE 15 14

DNOREPINEPHRINE

-

12 I I , 2 C URINARY I I

p

CATECHOLAMINES

r

$9

q EPINEPHRINE ¤N~REPINEF~-~RINE

IO 9 8i

IO

5s 7

r

6 5 4 3 2 I

&F2~~~0RT~0~

w moRnoN

(w

16

.IME

(I-IRS

)

Cn PlWORTI(lN

ARACHIIXMIC

Fig. 3. The mean serum total major FFA in nanomoles per milliliter, the plasma E and NE levels in micrograms per liter, and the mean urinary output of E and N in micrograms per four hours among eight women in midtrimester pregnancy after intra-amniotic administration of 40 mg. of PGF,, at time 0 are shown (see text). The values at the time of fetal distress (F.D.) are shown at the mean time of the distress. inhibits

the

infusing

lipolytic

subjects

with

levek7*

I4 This

indirect

effect

stimulated which

effects

pregnant

and

PGEi

of E and

results

lipolytic in that

stimulate

In the

present

the

increase

study

there

of plasma nervous

of plasma

FFA.i4

is a temporal

correlation

30

40

50

60

70

so

90

loo

x 1oo

Post

FFA

system

catecholamines

20

(20:4)

PROPORTION OF ABORTION (k)

to be an

to release

ARACHIDONIC ACID TOTAL FFA

IO

human

is postulated

sympathetic

by prostaglandins

In contrast,

normal

in elevation effect

the

NE.

nonpregnant

ACID

is

Fig. 4. The mean plasma E and NE in micrograms per liter, the mean urinary E and NE in micrograms per four hours, and the mean proportion of the total major FFA consisting of arachidonic acid in per cent over the course of abortion in eight women in midtrimester pregnancy who received 40 mg. of PGF,, as detailed in the text.

192

Ogburn et al.

A. 2

January 15, 1978 Am. J. Obstet. Gpnecol.

PROPORTION

ARACHIDONIC

ACID

8,

0’ -0

’ 0

’ ’ ’ ’ ’ ’ ’ ’ 1020504050.60m8090100Pos1 PROPORTION

B. UTERINE

OF ABORTION

TIME

’

’

’

(%)

ACTIVITY

700 r c

600

3-

500

t

‘“t40 0

IO 20 30 40 50 60 m a0 90 too post PROPORTION

OF ABORTION

TIME

(%)

Fig. 5. The mean the total major

proportion in Per cent of arachidonic acid in FFA and the mean uterine activity in Montevideo units that equal the number of contractions in 10 minutes times the mean intensity of contractions in 10 minutes, in eight women in midtrimester pregnancy after the intra-amniotic administration of 40 mg. of PGF*,. between mean serum FFA levels, plasma E, and urinary E and NE. The time of greatest urinary excretion of NE and E occurred in the eight-hour period following the peak FFA level and the highest mean plasma E value occurred at the same time as the FFA peak. Thus, in these patients it may be postulated that (1) the administration of PGF,, resulted in increased NE and E either directly or indirectly (as stress or pain secondary to uterine contractions), and (2) the increased NE and E then elevated the FFA levels through lipolysis. FFA levels increase in normal human pregnancy in the period between 20 and 40 weeks of gestation with a return to prepregnancy values within five days post partum. Levels of 200 to 300 per cent of prepregnancy values were observed.4 FFA’s increase progressively during the course of normal labor and decrease almost immediately after delivery.r5* r6 The peak FFA level occurs at the end of the second stage5 and it was postulated to be secondaryto the endogenous release of epinephrine.r6 Other causes of increased FFA’s in labor

REFERENCES

1. Karim, S. M. M.: Prostaglandins and reproduction, In MacDonald, R. R., editor: Scientific Basis of Obstetrics

have also been postulated. Although oxytocin administration resulted in increased blood FFA levels in dogsI and in puerperal women,rs FFA levels were not significantly different in women with oxytocininduced or augmented labor as compared to women in spontaneous labor.r5 Both prostaglandins and catecholamines may have caused the elevation of FFA. Certain lipids may play an important part in the initiation of labor since administration of a fat emulsion at term has resulted in labor.rs~ 2o Arachidonic acid (20 : 4), the obligate precursor for PGF,,, is stored in the fetal membranes in an esterified form before labor and is elevated in amniotic fluid during labor as an FFA. Intra-amniotic administration of arachidonic acid has resulted in uterine. contractions and abortion.2 It has been proposed that arachidonic acid is released from the chorion laeve by phospholipase AZ and diffuses to the uterine decidua where it is converted into PGF,, (and prostaglandin I$) which, in turn, causes the uterine contractions of labor.2s 21

Summary 1. Intra-amniotic administration of PGF,, resulted in increased uterine activity, increased plasma E, increased urine NE and E excretion, increased serum FFA levels, and a decrease in the portion of the total FFA made up by arachidonic acid (20: 4) in eight women at 18 to 21 weeks’ gestation during abortion. 2. The peak levels of catecholamines corresponded temporally with the peak levels of FFA. 3. After the abortions were completed, plasma E, urinary NE and E, total FFA’s, and the portion of FFA’s made up by arachidonic acid all returned to base-line levels. It is hypothesized that the following sequence of events occurred: (1) Intrauterine administration of PGF,, resulted in uterine contractions; (2) either the stress of uterine contractions or PGF,, triggered the release of amines (E and NE); (3) elevation of NE and E caused lipolysis which resulted in the increase in serum FFA; (4) arachidonic acid (20 : 4), a precusor of PGq,, may have been selectively removed from the circulating bloodstream by uterine and placental tissues and subsequently converted to PGF,, which resulted in uterine contractions; (5) there may be a positive feedback mechanism which is interrupted only when the products of conception, which selectively utilized arachidonic acid to produce PGF,,, were expelled.

and Gynecology, London, pp. 215-338. 2. MacDonald, P. C., Schultz,

1971, J. & A. Churchill, M., Duenhoelter,

Ltd.,

J. H., Gant,

Volume

Serum FFA changes during abortion induced by PGF,,

130

193

Number2

3. 4.

5.

6. 7.

8.

9.

10.

11.

N. F., Jimenez, J. M.. Pritchard, J. A., Porter, J. C., and Johnston, J. M.: Initiation of human parturition. I. Mechanism of action of arachidonic acid. Obstet. Gynecol. 44: 629, 1974. Jager. F. C.: Effect of fatty acids on the contraction of guinea pig ileum in vitro, Experientia 26: 731, 1970. Burr. R. L.: Plasma nonesterified fatty acids in normal pregnancy and the puerperium, Obstet. Gynecol. 15: 460. 1960. Nieschlag, E., Wombacher, H.. Kremer, G. J., and Martin. K.: Plasma corticosteroids, free fatty acids, insulin and glucose in maternal blood during delivery, Klin. Wochenschr. 48: 1066, 1970. Gusdon, J. P., and Burt, R. L.: Effects of oxytocin on nonesterified fatty acids, Obstet. Gynecol. 38: 444, 197 1. Zuspan. F. P.. Whaley. W. H., Nelson, G. H., and Ahlquist, R. P.: Placental transfer of epinephrine. I. Maternal-fetal metabolic alterations of glucose and nonesterified fatty acids. AM. J. OBSTET. GYNECOL. 95: 284, 1966. Brenner. W. E., Ogburn, P. L., Jr.. Dingfelder, J. R., Staurovsky, L. G., and Zuspan, F. P.: Catecholamines during therapeutic abortion induced with intraamniotic prostaglandin F,,, AM. J. OBSTET. GYNECOL. 130: 178,1978. Alvarez. H., and Caldeyro, R. Contractility of the human uterus recorded by new methods, Surg. Gynecol. Obstet. 91: I. 1950. Zuspan. F. P.. and Cooley, M. A. Semiautomated fiuorometric trihydroxyindole method for determining epinephrine (E) and norepinephrine (NE), in Proceedings of the Technicon International Congress on Advane es in Autoanalysis, New York, 1969. 1970, vol. 1. p. 351. Hagenfeldt, L.: A gas chromatographic method for the determination of individual free fatty acids in plasma, Clin. Chim. Acta 13: 266, 1966.

12. Metcalfe, L. D., Schmitz, A. A., and Pelka. J. R.: Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis, Anal. Chem. 38: 514. 1966. 13. Olson, R. E., and Vester, J. W.: Nutrition-endocrine interrelationships in the control of fat transport in man, Physiol. Rev. 40: 677. 1960. 14. Horton, E. W.: Adipose tissue. ltl Prostaglandins, Monographs on Endocrinology. New York, 1972, Springer-Verlag, chap. 8, pp. 1OY- 1 16. 15. Fairweather, D. V. I.: Changes in serum non-esterified fatty acid levels in spontaneous and in oxytocin induced labour, Br. J. Obstet. Gynaecol. 72: 408. 1965. 16. Whaley, M‘. H., Zuspan, F. P.. Nelson, G. H., and Ahlquist, R. P.: Alterations of plasma free fatty acids and glucose during labor, AM. J. OBSTET. GYWCXIL.. 97: 875, 1967. 17. Burt, R. L.. Leake, N. H., and Dannenburg, W. N.: Effect of synthetic oxytocin on plasma nonesteritied fatty acids, triglycerides, and blood glucose, Obster. Gynecol. 21: 708, 1963. 18. Jaisle, F.: Lipide Wahrend Schwangerschaft und Wehentatigkeit, Med. Klin. 64: 139. 1969. 19. Luukkainen, T.. Jarvinen, P. A., and Pvor%. .r.: Induction of labour with intravenous fat emulsion at term, Br. J. Obstet. Gynaecol. 71: 45, 1964. 20. Schultz. F. M.. Schwarz, B. E., MacDonald, P. C., and Johnston, J. M.: Initiation of human parturition. II. Identification of phospholipase A, in fetal chorioamnion and uterine decidua. AM. J. OBSTET. GYNECOL. 123: 650, 1975. 21. Schwarz. B. E., Schultz, M., MacDonald, P. C., and Johnston, J. M.: Initiation of human parturition: III. Fetal membrane content of PGE, and PGF,, precursor, Obstet. Gynecol. 46: 564, 3975.

lnformrlion for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: “The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published.” Authors will be consulted, when possible, regarding republication of their material.