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Placental transfer and distribution of nicotine in the pregnant rhesus monkey
Placental transfer and distribution
of nicotine
in the pregnant rhesus monkey KOTARO
SUZUKI,
TERLJSADA
PH.D.
HORIGUCHI,
ARSENIO
C.
M.D.
COMAS-URRUTIA,
EBERHARD
M.D.
MUELLER-HEUBACH,
HISAYO
0.
KARLIS
ADAMSONS,
Boston,
M.D.,
M.D.
MORISHIMA,
Massachusetts,
M.D., M.D.,
and New
York,
PH.D.
PH.D. New
York
Placental transfer and distribution of nicotine in fetal tissues were determined in IO pregnant monkeys in the third trimester. Nicotine was exchanged rapidly across the placenta between mother and fetus when it ~4s administered into the maternal circulation in a dose of 1 mg. per kilogram of body weight. The concentration of nicotine in the fetal circulation surpassed the maternal level rapidly, reaching maximum in 16 minutes and remaining at 4 higher concentration for osver two hours following the injection. Disappearance of nicotine from the fetal circulation was slower than that from the maternal circulation. The concentration of nicotine in adrenal glands, heart, kidneys, stomach wall, and spleen of fetuses was high; that in placenta and skeletal muscles was low. The amount of nicotine contained in a fetus, however, was only a small fraction of the total dose administered to the mother.
disturbances in both mother and fetus and lead to fetal asphyxia.l These pharmacologic effects were thought to be due mainly to a disturbance in the uterine perfusion rather than to the direct effect of transmitted nicotine upon the fetus. The present report concerns itself with the transmission of nicotine from the mother to the fetus and with the distribution of the transmitted nicotine in the fetal tissues of the rhesus monkey.
W E H A v E P R E v I 0 u s L Y reported that nicotine administered to the pregnant rhesus monkey can cause marked cardiovascular From the Deaartment of Obstetrics and Gynecology, karvard dedical School, Beth Israel Hosbital, Boston, Massachusetts; ihe Department of Obstetrics and Gynecology and the Department of Anesthesiology, College of Physicians and Surgeons, Columbia University, and the Department of Obstetrics and Gynecology and the Department of Pharmacology, The Mount Sinai School of Medicine of the City University of New York, New York.
Material and method Nine pregnant rhesus monkeys (Macaca mulatta) and one pregnant Javanese monkey of gestational ages ranging from 107 to 150 days (term, 168 days) were used in this study. They were bred in our colony and were free of disease. The gestational age was determined from menstrual history and mating date. The preparation was identical with that previously described in detai1.l The mother received pentobarbital, 14 to 25 mg. per kilogram intramuscularly, prior
Supported by grants-in-aid from the American Medical Association Education and Research Foundation, Committee for Research on Tobacco and Health, and United States Public Health Service Grants HD-118 and GM-0906. Received for publication August 13, 1973. Revised Accepted
October October
25, 1973. 30,
1973.
Reprint requests: Dr. Kotaro Suzuki, Department of Obstetrics and Gynecology, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215.
253
254
Suzuki et al.
to induction of anesthesia rvith N,O and halothane. The femoral artery was catheterized with a polyethylene tube to the level of the abdominal aorta for pressure recording and blood samp1in.g. In six monkeys the uterus was exposed through a midline abdominal incision and the fetal head and neck were delivered through a lo\\zr uterine segment incision without exteriorizing the uterus. The neck of the fetus was incised and the left carotid artery and right jugular vein were esposed. A polyethylene catheter was inserted into the left carotid artery to the level of the aortic arch in five cases, and into the right jugular vein to the level above the right atrium in two cases, for pressure recording and blood sampling. After the fetal skin \vas closed the head was replaced back into the amniotic cavity. An additional polyethylene catheter was inserted into the amniotic cavity for pressure recording and for replacement and sampling of the fluid. The uterine and abdominal lvalls were then closed. The original amniotic fluid volume was restored with warm saline. The animal \vas allowed to recolrer from anesthesia in the lrft recumbent position, and maintained restrained under light sedation with pentobarbital. The animal’s deep colonic temperature Ivas maintained at 37 to 38O C. with a heating pad and covers. Blood pressure and heart rate of both mother and fetus, as \cell as amniotic fluid pressure, were recorded continuously throughout the cxperimental period on a multichannel recorder. In three monkeys only maternal vessels were catheterized for injection of nicotine, sampling of maternal blood. and maternal blood pressure and heart rate monitoring. This preparation was used for thv study of distribution of nicotine in various fetal tissurs in a rrlativcly normal undisturbed state. In nine animals “H-labeled nicotine (1 mg. per kilogram) was administered into the maternal inferior vena cava over a period of five seconds to five minutes. In the remaining animal the same dose was infused over 20 minutes.
May 15, 1974 Am. J. Obstet. Gynecol.
The nicotine solution for injection was prepared from pure nicotine (Eastman Organic Chemicals, Kochester, New York) \\,hich was dissolved in normal saline to give 3 concentration of 1 to 3 mg. per milliliter. A lcno\vn amount of “H-nicotine (specific 324 to 390 mCi. per millimole. activity, Amcrsham/Searle, Des Plaines, Illinois) was added to the unlabeled nicotine solution of normal saline in a tracer amount so that 1 mg. per kilogram of body lveight (B. W.) of nicotine containing radioactivity of 10,000 c.p.m. per gram of B. W. could be given into the maternal circulation. A series of blood samples from the maternal aorta (5 ml. each) and the fetal aorta or superior vena cava (1.2 ml. each) were collected over 60 to 120 minutes. Preinjection control samples were obtained and then serial collections made at first every few minutes and then at longer intervals follolving the start of injection. The number of samples never exceeded six. An aliquot of each blood sample was analyed for acid-base status and oxygenation to \,crify the results reported earlier.’ Then the plasma was separated from the aample and used for determination of nicotine concentration. Follolving the administration of ‘H-nirotinc, hysterotomy was performed at 0.5. 1 .O, or 1.5 hours and various frtal organs were dissected to determine the tissue level of nirotinr. In those cases where no fetal cathrtcr \\‘as placed, a terminal cardiac blood sample was also collected from the fetus for acid-base analysis. All tissue and plasma samples lcere lcept frozen until analysts for nicotine \serkl done. Isolation and quantitation of nicotine. One milliliter of maternal plasma or 0.5 ml. of fetal plasma (whole blood in one case) \vas mixed with 0.5 ml. of distilled \T.ater. 0.5 ml. of ION NaOH. and 15 ml. of dichloromethanc. The mixture was shaken for two hours and then centrifuged for 10 minutes at 1.400 ,g in a conical centrifuge tuhe. ‘l‘\\o layers were formed and a measurcsd amount of the lower layer (dichloromethane extract) ranKing from 8 to 12.5 ml. was takrn into a fresh tube. A tinv amount
Volume Number
119 2
Placental
Injection
of
transfer
and
distribution
of
nicotine
255
Nicotine
1%+out Fig. 1. Two-compartment model for the present study. M, maternal plasma; F, fetal plasma; V,,, maternal plasma volume; VI, fetal plasma volume; Qm.l, quantity of nicotine in maternal plasma at time t; Q,.t, quantity of nicotine in fetal plasma at time t; Km+,, transfer rate (rate constant) of nicotine from maternal plasma to fetal plasma; K I+&, transfer rate of nicotine from fetal plasma to maternal plasma; K m+out, excretion of nicotine from maternal plasma to outside.
of anhydrous Na,SO, was added by microspatula to the tube, which was then shaken gently for about five minutes. The anhydrous Na$O, was allowed to settle to the bottom of the tube. The dichloromethane extract was then decanted into a new centrifuge tube and evaporated to dryness. Methyl alcohol (0.1 to 0.5 ml.) was added to the residue left in the tube and warmed slightly to aid in dissolving. The entire volume of this dissolved residue was spotted on a thin-layer chromatography plate coated with aluminum oxide G. The plate was developed for 30 minutes in a tank containing 100 ml. of chloroform and 0.7 ml. of methyl alcohol. Simultane1: 1,000 nicotine : methanol soluously, a tion was run on the other part of the same plate as a standard and developed. The plate
lowed
was
removed
from
the
tank
to dry. The portion for the sample was covered and the portion standard nicotine was sprayed with dorfT’s reagent to visualize the spot
and
al-
plasma for the Dragenof nico-
tine. The spot in the sample portion on the plate corresponding to Rf value of the standard nicotine solution was then scraped off the plate completely with the aid of suction. This sample was mixed with 15 ml. of scintillation solution consisting of 1,000 ml. of toluene, 7 Gm. of 2,5-diphenyloxazole (PPO) , 0.36 Gm. of 1,4-bis[2- (5-phenyloxazolyl)] benzene (POPOP), and 200 ml. of Beckman BBS-3,” and counted for its radioactivity. For analysis of tissues, each fetal organ was weighed and homogenized. Then the exact amount of tissue homogenate (10 to 500 mg.) was mixed with 1 ml. of distilled water, 1 ml. of 10N NaOH, and 30 ml. of dichloromethane. The rest of the procedure was carried out in the same way as for plasma or whole blood, except that 23 to 25 ml. of the dichloromethane extract was used. The recovery rate for nicotine in plas-
‘Solubilizer which ostensibly counting (Beckman Instruments
enhances Co., Palo
liquid Alto,
scintillation California).
256
Suzuki
May 15, 1974 Am. J. Ohstrt. Gyn~col.
et al.
1000~ 1 -
z a ;:
500-
5
200
-i g c 0) z
100 _ r -0 50
E a ,” z z
NICOTINE CONCENTRATION AND AMNIOTIC FLUID
IN
--Q----4
PLASMA
OF MOTHER
----_
---_
AND
-D----4
0
FETUS
20 t I
MOTHER
10 “:
5
FETUS,
AMNIOTIC FLUID
5
z i
2-
z I 0
MONKEY NO. GESTATIONAL I I5
I 30
446 AGE
121 I 45
DAYS I 60
I 75
1 90
I 105
I I20
I 135
150
MINUTES
Fig. 2. Nicotine concentration in the plasma of the mother and fetus and the amniotic fluid of the rhesus monkey following the single injection of “H-nicotine (1 mg. per kilogram of body weight) into the maternal circulation. Concentration unit for the amniotic fluid is the same as for the plasma.
ma and whole blood by this method was rather constant, yielding 52 _+ 2.8 (S.E.M.) per cent and 60 + 2.0 (S.E.M.) per cent, respectively. Recovery rate in tissues was assumed to be about the same as in whole blood. The concentration of nicotine in maternal and fetal plasma and fetal tissues was then calculated from the specific activity of ,,H-nicotine of the solution injected, taking two correction factors into consideration, namely, the ratio of the volume of the dichloromethane extract actually used for analysis against that of the initial dichloromethane. and the recovery rate for nicotine by the extraction procedure. Transfer rate of nicotine. Transfer rates of nicotine between maternal and fetal compartments (k,, + f, Kf _ m) and elimination rate of nicotine from the maternal plasin six monma (K, -+ <,“t) were estimated keys in whirh a single injection of “H-nicotine ( 1 mg. per kilogram of B.W.) was given to the maternal circulation. A two-compartment model was utilized for this purpose (Fiq. 1 1. It was assumed that the volume
of the maternal and fetal plasmas remained constant and that the injected “H-nicotine was instantaneously distributed throughout the entire maternal plasma compartment. The exponential curve of nicotine concentration in maternal plasma as a function of time on a semilogarithmic scale was broken down into two straight lines by the “peeling” technique. Each line depicted an exponential term. An equation for the maternal plasma concentration curve of nicotine was determined by deriving exponential constants and coefficients from the values of the slope (LY,. -) and y-intercept (A,, z) of the two straight lines. From this equation the relevant transfer and excretion rates of nicotine were calculated (see Appendix) Maternal and fetal conditions during the preinjection control period were deemed to be normal on the basis of cardiovascular and acid-base monitorin,g. All the mothers and fetuses were alive at the end of the experiment. In three preparations in which no fetal catheter had been placed, the terminal cardiac blood sample obtained from the fetuses showed pH values of 7.215 to 7.335.
Volume Number
119 2
Placental
Table I. Plasma concentration ( 1 mg. per kilogram
of nicotine of body weight)
Gestational age [days j
Mode of administration
446
121
Single injection
443 494 447 458 444
147 145 118 134 127
Monkey NO.
“F refers to tDatum
fetal
on whole
Single injection Single injection Single injection Single injection Constant infusion over 20 minutes value; M, maternal value; blood
following
its administration
6
F>M F>M F>M F M
15 10
distribution
Slope of decline*
F
23
FhM
257
in fetus Maximum concentration (pg per 100 ml.)
Time to maximum concentration (min.)
92
32
got 70 58
25 85 45 16 60
F
20
of nicotine
to the mother
of nicotine
Time to F = M’ (min.)
F>M
amniotic
and
Concentration __-.__
~~~ Relation to maternal concentmtion at end point*
AF,
transfer
94 670
fluid.
concentration.
Results
Placental transfer of nicotine. The
typical changes in concentration of nicotine in the maternal and fetal plasmas are shown in Fig. 2 and descriptive data for the nicotine concentration curve in each experiment are presented in Table I. Nicotine crossed the placenta rapidly from the mother to the fetus. In five of six monkeys in which the nicotine concentrations were determined in both the maternal and fetal plasma, fetal levels exceeded maternal. It took only six to 20 minutes for the fetal plasma or blood levels to reach the maternal in four cases after a single injection of nicotine (1 mg. per kilogram of B.W.) to the maternal circulation. In another case where a constant infusion of nicotine was given with same dose to the mother over 20 minutes, 23 minutes elapsed before maternal levels were reached in the fetus. It took 16 to 85 minutes for the fetal level to reach its maximum value ranging from 58 to 94 pg per 100 ml. of plasma or blood after a single injection and 670 pg per 100 ml. following constant infusion. In only one case the fetal concentration did not reach the maternal level. In every experiment the fetal plasma or blood concentration of nicotine declined either more slowly than the maternal concentration or, at most, at a similar rate. In four of five cases the fetal plasma or blood
Table II. Fractional transfer rates (rate constants) of nicotine between maternal fetal plasma compartments (minute -I) Monkey No.
K
446 443 494 447 458 491
Mean S.E.M. *For
m + f*
0.437 0.202 0.106 0.052 0.020 0.273 0.182 0.064 explanation
see legend
Kr
-+ m*
-a out+
0.046 0.098 0.272 0.056 0.046 0.034 0.092 0.037 ______
0.225 0.021 0.026 0.063 0.025 0.263 0.104 0.045 to Fig.
Km
and
1.
still retained 72 to 89 per cent of the maximal concentration of nicotine at 120 minutes following a single injection of nicotine to the mother. In one case (No. 458) only 16 per cent remained. In one case with constant infusion of nicotine to the mother, 48 per cent of the maximal concentration still remained in the fetal circulation at 120 minutes following the infusion. Fractional transfer rates of nicotine between maternal and fetal plasma compartments in six animals where the “H-nicotine was administered to the mother by single injection are shown in Table II. Differences among the mean transfer rates between these two compartments and the excretion rate from the mother are not statistically significant because of the large individual variability. An average of 18 per cent of the
258
Suzuki et al.
Am.
J.
May 15, 1974 Obstet. Gynecol.
Table III. Nicotine distribution in fetal organs of the rhesus monkey (milligram of nicotine per kilogram of tissue and percentage of the total dose administered to the mother in each organ) Time
after
Monkey
injection
30
No.
Gestation Fetal
(min.)
465
(days)
weight
(grams)
0.46t (0.423)X 0.68 (0.177) 0.82 (0.121)
wall
Adrenals
0.47 (0.038)
Spleen
0.69 (0.055) 1.12 (0.017)
wall
Liver Skeletal
I
--.-~ <
410
Lungs
Stomach
~-..
I -J”-
146
Brain
Intestinal
~---?!L
muscle
Kidneys Heart Placenta
150
I40
410
0.15 (0.095) 0.14 (0.010) 2.05 10.169) 5.74 (0.056) 1.35 (0.011)
l’hymus
491 . /.
0.39 (0.242) 1.N (0.112) 0.58 (0.063) 1.68 (0.045)
)
0.28 (0.037 1 0.79 (0.864 ) 1.34 (0.113 ) 2.34 (0.061) 0.46 (1.121) 0.50 (0.001)
per
cent
4.167
2Javanese monkey. tconcentration in milligrams :Percentage of the total dose calculated
as
(nicotine
of nicotine nicotine
of concentration
s
2.559 per kilogram
0.07 (o.0003) 0.82 (0.004)
1.34 (0.017) 1.18 (cr.013) (1.85 (0.1 15 ) (1.92 (1.274 ) 0.67 (0.036 ) 1.79 (0.048 0.54 (0.876 0.44 (0.012
0.62 (0.045 0.02 (0.012 1.06 (0.049 0.08 (0.001) 0.19 (0.252)
2.853
--,-L& 0.28 (0.182 ) 0.41 (0.025) 0.28 (0.027) 2.33 (0.005) cl.75 f CJ.003) 1.10 10.012) 0.43 (0.054) 0.29 (0.357) 0.67 (0.032
j
0.63 (0.151)
0.55 (0.010 j 0.25 (0.350) 1.47 (0.008) 0.70 (0.345)
2.999
1.410
of tisaw.
administered to the mother organ weight/total nicotine
nicotine in the maternal plasma could be transferred to the fetal plasma per minute and an average of 10 per cent of the nicotine in the fetal plasma could be transmitted back to the maternal plasma per minute. Excretion rates of nicotine from the mother via the urine averaged nine per cent of the amount in her plasma per minute. Distribution of nicotine in fetal tissues. The distribution of nicotine in various fetal tissues of five monkeys is presented in Table III. Higher concentrations (> 1.0 mg. per kilogram) were found in adrenals, heart, kidneys, stomach, and spleen in both mature and immature fetuses; this pertained equally
107 /j" 168 7.02 (2.425) 0.65 (0.026) 0.76 (0.034)
Blood Total
90 ~. ~~~ 494
,p_---
467
120
1.75 (0.021
1.22 (0.244) 0.63 (1.30) 1.73 (0.242) 1.65 (0.099) 0.53 (1.452)
60
(1 mu. ‘< body dose administrledi
weight x
in kilograms) 100.
in an entire
organ,
to samples obtained 30 minutes or 60 minutes following the injection of nicotine to the mother. Brain and intestines had high concfmtrations of nicotine in tissue samples obtained at these intervals in fetuses of less than 120 days’ gestational age, whereas these organs showed consistently lower concentrations in more mature fetuses. The converse was true for lungs and liver. The concentration of nicotine in the adrenals of less mature fetuses was as high as 5.74 mg. per kilogram of tissue at 30 minutes after injection but fell to 0.07 mg. per kilogram at 60 minutes. Adrenals in more mature fetuses. however, showed progressive increase in levels from 0.47 mg. per kilogram
Volume Number
119 2
at 30 minutes to 1.68 mg. per kilogram at 60 minutes and 2.33 mg. per kilogram at 90 minutes. The brain tissue of a fetus of 107 days of gestation (No. 491) showed the highest concentration of nicotine among all the tissues, 7.02 mg. per kilogram at 60 minutes. The thymus of a mature fetus (No. 494) contained 1.47 mg. per kilogram of nicotine at 90 minutes, whereas that of less mature fetuses had only 0.50 mg. per kilogram at 30 minutes and 0.44 mg. per kilogram at 60 minutes. Because of their relatively large mass, skeletal muscle and placenta took up larger quantities of nicotine than other organs, even though the concentrations of the alkaloid in these organs were low. When the total percentage of nicotine contained in a fetus and placenta was estimated by adding the amount in each organ as a fraction of the total dose of nicotine administered, it was found to range from four per cent at 30 minutes after injection to one per cent at 90 minutes. Comment The dosage of nicotine used in this study, 1.0 mg. per kilogram, is much larger than the amount the pregnant woman would be expected to absorb in the course of cigarette smoking. The pharmacologically equivalent effect is attainable in the human subject, however, due to the difference in sensitivity to nicotine between man and m0nkey.l The monkey is at least six times as tolerant to nicotine as the human being, as evidenced by data on the lethal dose.2 Thus, a nicotine dose of 1.0 mg. per kilogram in the monkey should correspond to no more than 0.17 mg. per kilogram in the human being. Two to 3 mg. of nicotine may be absorbed during cigarette smoking on average, yielding a concentration of nicotine in a smoking mother similar to that used in this study. Rapid transfer of nicotine from the mother to the fetus has been proved and the nicotine concentrations in maternal and fetal blood and in the fetal tissues have been determined in the pregnant primate for the first time. The methods we developed for isolation and quantitation of nicotine in blood and tissues were sensitive enough to measure
Placental
transfer
and
distribution
of nicotine
259
as low as 3 pg per 100 ml. of nicotine in plasma and reliable enough to have reliably constant recovery rates. Unexpectedly, the majority of both mature and immature fetuses showed persistently higher plasma or blood concentrations of nicotine than the mothers after six to 23 minutes following administration of nicotine to the mother. Disappearance of nicotine from the fetal blood compartment was slower than from the maternal blood. Thus, cardiovascular disturbances, acidosis, and hypoxia which have been observed in the mature fetus following administration of 1 mg. per kilogram of nicotine to the mother1 must be considered possibly the result of the nicotine transmitted to the fetus. Our previous finding that cardiovascular responses of the fetus differed according to whether comparable dosages of nicotine was administered to the mother or to the fetus directly’ may now be partly explained by our current observation that the fetal plasma or blood level of nicotine administered to the mother (of 1 mg. per kilogram of maternal body weight) is much lower than the level produced by direct injection (of 1 mg. per kilogram of fetal body weight) into the fetal circulation. The effect of maternally administered nicotine and its metabolites on uterine perfusion might also contribute to the disparate fetal cardiovascular responses. Similarly, our data helped to provide an explanation for the previous finding that mature fetuses, but not immature ones, sustained rather prolonged acidosis and hypoxia following nicotine administration to the mother. This is believed to be based on differences in the developmental stage of autonomic nervous system in the fetus in view of our finding that both mature and lessmature fetuses have similar plasma or blood concentrations of nicotine. Another factor to explain the apparently greater vulnerability of mature fetuses to nicotine could be the more prolonged tissue concentrations of nicotine found in mature fetuses as compared with those that are less mature. Although concentrations in many organs of the younger fetus (such as heart, adrenals, kidneys, spleen, intestines, and stomach) were
260
Suzuki
May
et al.
Am.
higher than or similar to those in the older fetus at 30 minutes after injection of nicotine, they fell with time so that some tissues of the mature fetus (especially heart, adrenals, and spleen) had higher values at 60 minutes after injection. Lungs, stomach, skeletal muscle, and placenta also had similar late peak values in the older fetus. Thus, one could postulate that the transmitted nicotine might disturb the umbilical circulation or adversely affect cellular metabolism in the mature primate fetus. However, the total amount of nicotine present in a fetus from 30 to 90 minutes after the nicotine injection to the mother is small, accounting for approximately one to four per cent of the total dose of nicotine injected (Table III). Admittedly these percentages are only approximations because some tissues, such as subcutaneous fat, skin, and pancreas. have not been included. Since distribution in these organs in the dog is reported to be either insignificant or very small,” these figures can be considered to be reasonably accurate. The time for complete excretion of nicotine via urine is about 48 hours in the dog3 following the administration of nicotine, 1 mg. per kilogram, intravenously. It is reasonable to assume that most of the nicotine administered to the pregnant monkey is still present during the 90 minutes of observation after the injection here. The fetus and placenta constitute approximately seven to 10 per cent of the total body weight of the pregnant monkey. Thus one to four per cent of the total dose of nicotine present in the fetus is indeed a smaller proportion than that which can be expected with uniform distribution of nicotine in the gravid organism. Uneven distribution to the fetus may be the result of diminished perfusion due to uterine vasoconstric-
J. Obstet.
15, 1971 Gynecol.
tion. Working synergistically with the transmitted nicotine in the fetus, this vasoconstriction in thr uterine circulation and its resultant diminished intervillous space perfusion could be another factor causing adverse fetal effects as previously contended.’ High nicotine concentrations in the kidneys are expected because the main excretory pathway is via the urine and the kidney is partly involved in metabolism of nicotine.4 The main excretion of nicotine from the fetus, however, must be via the placenta since the fetal kidney is known to be far less functionally effective as an excretory organ than the placenta. The liver shows fairly high concentrations in the mature fetus at 30 minutes, but these decline progressively to very low levels at 60 and 90 minutes even though the blood levels of nicotine are still near their peak. In less mature fetuses the concentrations in the liver are persistentIy low. This indicates that the fetal liver does not serve as the major site for detoxication of nicotine, as does the adult liver; this is likely due to its immature enzyme systems. Most of the nicotine in the fetus is probably excreted by way of the placenta without being metabolized. High concentrations in the heart and the stomach wall of the fetus are in agreement with the findings made by Fishman:’ in an adult dog. Although the number of monkeys used in the current experiment is limited, it is clear that nicotine is rapidly transferred across the placenta and that nicotine transmitted to the fetus is excreted more slowly than that in the mother. We gratefully acknowledge Miss Lydia O’Brien’s valuable technical assistance for chemical assay of nicotine, and Dr. Emanuel A. Friedman’s review of our manuscript.
REFERENCES
1. Suzuki, K., Horiguchi, A. C., Mueller-Heubach, and Adamsons, K.: AM. 2. 3.
T., Comas-Urrutia, E., Morishima, H. O., J. OBSTET.
GYNECOL.
111: 1092, 1971. Feurt, S. D., Jenkins, J. H., Hayes, F. A., and Crockford, H. A.: Science 127: 1054, 1958. Fishman, S. S.: Arch. Int. Pharmacodyn. 145: 123, 1963.
4. Goodman, L. S., and Gilman, A.: The Pharmacological Basis of Therapeutics, ed. 3, New York, 1965, The Macmillan Company, p. 582. 5.
Riggs, D. Physiological
Williams
S.: The Mathematical Problems, Baltimore,
Approach 1963,
& Wilkins Company, pp. 204-206.
to The
Volume Number
119 2
Placental
transfer
Appendix
and
exp
The explicit two-compartment lows5 :
[-%
equation for the generalized system (Fig. 3) is as fol-
=
KS” - Kt
(
(K,
of
+ Kf
1 _ K,,
nicotine
+ Z)
- Ki
t]
+ Z
22 exp
F,.t
distribution
[-%(K,,
+ Kr
-
Z)
t])
+ Z
22
Injection
of Nicotine I
F
"f
Qf-t
1
Kf+out
Km = 5lb.f + KIF-0,t Fig.
3. A general
Kf = Kf pm + Kf-+out
two-compartment
71000 z Y) 500 I! L
AND
model.
Symbols
denote
the same as in Fig.
OF MOTHER
AMNIOTIC
AND
1.
FETUS,
FLUID
: Ill
\\THER
FLUID
;
2-
z I
MONKEY NO GESTATIONAL
0
1 IS
1 30
AA6 AGE
121
I A5
DAYS I 60
I 75
I 90
I 105
I 120
I 135
1
0
MINUTES
Fig. 4. Determination as a function of time.
of the
equation
for
the maternal
plasma
concentration
curve
of nicotine
261
262
Where
Suzuki
et al.
F,,,.t =
Am. J.
the fraction of the total dose of nicotine in the compartment M at time t.
z = \/(K,,, - Kr)” + 4K,,-+r KY+,,, Boundary conditions: at t = 0. F,, n = 1, FL,, z (1 In the present study the equation for the nicotine concentration curve of the maternal plasma is expressed as: c,,, t x A,f.-,‘I’ + .4,e-““L Where
(2) (Fig. 4)
C,,,.t =
the concentration of nicotine in the maternal plasma in micrograms per 100 c.c. t = the time in minutes At time zero, Eq. (2) gives: C 111 0 = ,4, + .4,
Since the dose of nicotine administered, was 1 mg. x body weight, the initial tot, Q volume of distribution in the compartment M (the volume of the compartment M)
v,,,
z
May 15, 1974 Obstet. Gynecol.
$y ID 0
Multiplying both sides of VJQ,,,, to convert it to F ,,,.t F 1,1t I~: A’@1
Eq.
+ i\‘re-“g’
(2)
by (3)
Eq. (3 ) is a particular example of Eq. (1). We can, therefore, equate the numerical values in Eq. (3) to the corresponding algebraic values of Eq. (1) : s (Ku, tKr+Z)
=uI
(4)
% (Ku, +Kt-Z)
=a:
(5)
K,,, - Kt +-- Z -- A’, ?Z
(6)
Solving Eqs. (4), (5), and (6) with the knowledge of Kf - INlt = 0 (Therefore Kf = ~ ,j , WC can obtain the values for K, + f. Kr Kf -+ ,“, and
K,, + Ollt.
of nicotine
in the pregnant rhesus monkey KOTARO
SUZUKI,
TERLJSADA
PH.D.
HORIGUCHI,
ARSENIO
C.
M.D.
COMAS-URRUTIA,
EBERHARD
M.D.
MUELLER-HEUBACH,
HISAYO
0.
KARLIS
ADAMSONS,
Boston,
M.D.,
M.D.
MORISHIMA,
Massachusetts,
M.D., M.D.,
and New
York,
PH.D.
PH.D. New
York
Placental transfer and distribution of nicotine in fetal tissues were determined in IO pregnant monkeys in the third trimester. Nicotine was exchanged rapidly across the placenta between mother and fetus when it ~4s administered into the maternal circulation in a dose of 1 mg. per kilogram of body weight. The concentration of nicotine in the fetal circulation surpassed the maternal level rapidly, reaching maximum in 16 minutes and remaining at 4 higher concentration for osver two hours following the injection. Disappearance of nicotine from the fetal circulation was slower than that from the maternal circulation. The concentration of nicotine in adrenal glands, heart, kidneys, stomach wall, and spleen of fetuses was high; that in placenta and skeletal muscles was low. The amount of nicotine contained in a fetus, however, was only a small fraction of the total dose administered to the mother.
disturbances in both mother and fetus and lead to fetal asphyxia.l These pharmacologic effects were thought to be due mainly to a disturbance in the uterine perfusion rather than to the direct effect of transmitted nicotine upon the fetus. The present report concerns itself with the transmission of nicotine from the mother to the fetus and with the distribution of the transmitted nicotine in the fetal tissues of the rhesus monkey.
W E H A v E P R E v I 0 u s L Y reported that nicotine administered to the pregnant rhesus monkey can cause marked cardiovascular From the Deaartment of Obstetrics and Gynecology, karvard dedical School, Beth Israel Hosbital, Boston, Massachusetts; ihe Department of Obstetrics and Gynecology and the Department of Anesthesiology, College of Physicians and Surgeons, Columbia University, and the Department of Obstetrics and Gynecology and the Department of Pharmacology, The Mount Sinai School of Medicine of the City University of New York, New York.
Material and method Nine pregnant rhesus monkeys (Macaca mulatta) and one pregnant Javanese monkey of gestational ages ranging from 107 to 150 days (term, 168 days) were used in this study. They were bred in our colony and were free of disease. The gestational age was determined from menstrual history and mating date. The preparation was identical with that previously described in detai1.l The mother received pentobarbital, 14 to 25 mg. per kilogram intramuscularly, prior
Supported by grants-in-aid from the American Medical Association Education and Research Foundation, Committee for Research on Tobacco and Health, and United States Public Health Service Grants HD-118 and GM-0906. Received for publication August 13, 1973. Revised Accepted
October October
25, 1973. 30,
1973.
Reprint requests: Dr. Kotaro Suzuki, Department of Obstetrics and Gynecology, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215.
253
254
Suzuki et al.
to induction of anesthesia rvith N,O and halothane. The femoral artery was catheterized with a polyethylene tube to the level of the abdominal aorta for pressure recording and blood samp1in.g. In six monkeys the uterus was exposed through a midline abdominal incision and the fetal head and neck were delivered through a lo\\zr uterine segment incision without exteriorizing the uterus. The neck of the fetus was incised and the left carotid artery and right jugular vein were esposed. A polyethylene catheter was inserted into the left carotid artery to the level of the aortic arch in five cases, and into the right jugular vein to the level above the right atrium in two cases, for pressure recording and blood sampling. After the fetal skin \vas closed the head was replaced back into the amniotic cavity. An additional polyethylene catheter was inserted into the amniotic cavity for pressure recording and for replacement and sampling of the fluid. The uterine and abdominal lvalls were then closed. The original amniotic fluid volume was restored with warm saline. The animal \vas allowed to recolrer from anesthesia in the lrft recumbent position, and maintained restrained under light sedation with pentobarbital. The animal’s deep colonic temperature Ivas maintained at 37 to 38O C. with a heating pad and covers. Blood pressure and heart rate of both mother and fetus, as \cell as amniotic fluid pressure, were recorded continuously throughout the cxperimental period on a multichannel recorder. In three monkeys only maternal vessels were catheterized for injection of nicotine, sampling of maternal blood. and maternal blood pressure and heart rate monitoring. This preparation was used for thv study of distribution of nicotine in various fetal tissurs in a rrlativcly normal undisturbed state. In nine animals “H-labeled nicotine (1 mg. per kilogram) was administered into the maternal inferior vena cava over a period of five seconds to five minutes. In the remaining animal the same dose was infused over 20 minutes.
May 15, 1974 Am. J. Obstet. Gynecol.
The nicotine solution for injection was prepared from pure nicotine (Eastman Organic Chemicals, Kochester, New York) \\,hich was dissolved in normal saline to give 3 concentration of 1 to 3 mg. per milliliter. A lcno\vn amount of “H-nicotine (specific 324 to 390 mCi. per millimole. activity, Amcrsham/Searle, Des Plaines, Illinois) was added to the unlabeled nicotine solution of normal saline in a tracer amount so that 1 mg. per kilogram of body lveight (B. W.) of nicotine containing radioactivity of 10,000 c.p.m. per gram of B. W. could be given into the maternal circulation. A series of blood samples from the maternal aorta (5 ml. each) and the fetal aorta or superior vena cava (1.2 ml. each) were collected over 60 to 120 minutes. Preinjection control samples were obtained and then serial collections made at first every few minutes and then at longer intervals follolving the start of injection. The number of samples never exceeded six. An aliquot of each blood sample was analyed for acid-base status and oxygenation to \,crify the results reported earlier.’ Then the plasma was separated from the aample and used for determination of nicotine concentration. Follolving the administration of ‘H-nirotinc, hysterotomy was performed at 0.5. 1 .O, or 1.5 hours and various frtal organs were dissected to determine the tissue level of nirotinr. In those cases where no fetal cathrtcr \\‘as placed, a terminal cardiac blood sample was also collected from the fetus for acid-base analysis. All tissue and plasma samples lcere lcept frozen until analysts for nicotine \serkl done. Isolation and quantitation of nicotine. One milliliter of maternal plasma or 0.5 ml. of fetal plasma (whole blood in one case) \vas mixed with 0.5 ml. of distilled \T.ater. 0.5 ml. of ION NaOH. and 15 ml. of dichloromethanc. The mixture was shaken for two hours and then centrifuged for 10 minutes at 1.400 ,g in a conical centrifuge tuhe. ‘l‘\\o layers were formed and a measurcsd amount of the lower layer (dichloromethane extract) ranKing from 8 to 12.5 ml. was takrn into a fresh tube. A tinv amount
Volume Number
119 2
Placental
Injection
of
transfer
and
distribution
of
nicotine
255
Nicotine
1%+out Fig. 1. Two-compartment model for the present study. M, maternal plasma; F, fetal plasma; V,,, maternal plasma volume; VI, fetal plasma volume; Qm.l, quantity of nicotine in maternal plasma at time t; Q,.t, quantity of nicotine in fetal plasma at time t; Km+,, transfer rate (rate constant) of nicotine from maternal plasma to fetal plasma; K I+&, transfer rate of nicotine from fetal plasma to maternal plasma; K m+out, excretion of nicotine from maternal plasma to outside.
of anhydrous Na,SO, was added by microspatula to the tube, which was then shaken gently for about five minutes. The anhydrous Na$O, was allowed to settle to the bottom of the tube. The dichloromethane extract was then decanted into a new centrifuge tube and evaporated to dryness. Methyl alcohol (0.1 to 0.5 ml.) was added to the residue left in the tube and warmed slightly to aid in dissolving. The entire volume of this dissolved residue was spotted on a thin-layer chromatography plate coated with aluminum oxide G. The plate was developed for 30 minutes in a tank containing 100 ml. of chloroform and 0.7 ml. of methyl alcohol. Simultane1: 1,000 nicotine : methanol soluously, a tion was run on the other part of the same plate as a standard and developed. The plate
lowed
was
removed
from
the
tank
to dry. The portion for the sample was covered and the portion standard nicotine was sprayed with dorfT’s reagent to visualize the spot
and
al-
plasma for the Dragenof nico-
tine. The spot in the sample portion on the plate corresponding to Rf value of the standard nicotine solution was then scraped off the plate completely with the aid of suction. This sample was mixed with 15 ml. of scintillation solution consisting of 1,000 ml. of toluene, 7 Gm. of 2,5-diphenyloxazole (PPO) , 0.36 Gm. of 1,4-bis[2- (5-phenyloxazolyl)] benzene (POPOP), and 200 ml. of Beckman BBS-3,” and counted for its radioactivity. For analysis of tissues, each fetal organ was weighed and homogenized. Then the exact amount of tissue homogenate (10 to 500 mg.) was mixed with 1 ml. of distilled water, 1 ml. of 10N NaOH, and 30 ml. of dichloromethane. The rest of the procedure was carried out in the same way as for plasma or whole blood, except that 23 to 25 ml. of the dichloromethane extract was used. The recovery rate for nicotine in plas-
‘Solubilizer which ostensibly counting (Beckman Instruments
enhances Co., Palo
liquid Alto,
scintillation California).
256
Suzuki
May 15, 1974 Am. J. Ohstrt. Gyn~col.
et al.
1000~ 1 -
z a ;:
500-
5
200
-i g c 0) z
100 _ r -0 50
E a ,” z z
NICOTINE CONCENTRATION AND AMNIOTIC FLUID
IN
--Q----4
PLASMA
OF MOTHER
----_
---_
AND
-D----4
0
FETUS
20 t I
MOTHER
10 “:
5
FETUS,
AMNIOTIC FLUID
5
z i
2-
z I 0
MONKEY NO. GESTATIONAL I I5
I 30
446 AGE
121 I 45
DAYS I 60
I 75
1 90
I 105
I I20
I 135
150
MINUTES
Fig. 2. Nicotine concentration in the plasma of the mother and fetus and the amniotic fluid of the rhesus monkey following the single injection of “H-nicotine (1 mg. per kilogram of body weight) into the maternal circulation. Concentration unit for the amniotic fluid is the same as for the plasma.
ma and whole blood by this method was rather constant, yielding 52 _+ 2.8 (S.E.M.) per cent and 60 + 2.0 (S.E.M.) per cent, respectively. Recovery rate in tissues was assumed to be about the same as in whole blood. The concentration of nicotine in maternal and fetal plasma and fetal tissues was then calculated from the specific activity of ,,H-nicotine of the solution injected, taking two correction factors into consideration, namely, the ratio of the volume of the dichloromethane extract actually used for analysis against that of the initial dichloromethane. and the recovery rate for nicotine by the extraction procedure. Transfer rate of nicotine. Transfer rates of nicotine between maternal and fetal compartments (k,, + f, Kf _ m) and elimination rate of nicotine from the maternal plasin six monma (K, -+ <,“t) were estimated keys in whirh a single injection of “H-nicotine ( 1 mg. per kilogram of B.W.) was given to the maternal circulation. A two-compartment model was utilized for this purpose (Fiq. 1 1. It was assumed that the volume
of the maternal and fetal plasmas remained constant and that the injected “H-nicotine was instantaneously distributed throughout the entire maternal plasma compartment. The exponential curve of nicotine concentration in maternal plasma as a function of time on a semilogarithmic scale was broken down into two straight lines by the “peeling” technique. Each line depicted an exponential term. An equation for the maternal plasma concentration curve of nicotine was determined by deriving exponential constants and coefficients from the values of the slope (LY,. -) and y-intercept (A,, z) of the two straight lines. From this equation the relevant transfer and excretion rates of nicotine were calculated (see Appendix) Maternal and fetal conditions during the preinjection control period were deemed to be normal on the basis of cardiovascular and acid-base monitorin,g. All the mothers and fetuses were alive at the end of the experiment. In three preparations in which no fetal catheter had been placed, the terminal cardiac blood sample obtained from the fetuses showed pH values of 7.215 to 7.335.
Volume Number
119 2
Placental
Table I. Plasma concentration ( 1 mg. per kilogram
of nicotine of body weight)
Gestational age [days j
Mode of administration
446
121
Single injection
443 494 447 458 444
147 145 118 134 127
Monkey NO.
“F refers to tDatum
fetal
on whole
Single injection Single injection Single injection Single injection Constant infusion over 20 minutes value; M, maternal value; blood
following
its administration
6
F>M F>M F>M F
15 10
distribution
Slope of decline*
F
23
FhM
257
in fetus Maximum concentration (pg per 100 ml.)
Time to maximum concentration (min.)
92
32
got 70 58
25 85 45 16 60
F
20
of nicotine
to the mother
of nicotine
Time to F = M’ (min.)
F>M
amniotic
and
Concentration __-.__
~~~ Relation to maternal concentmtion at end point*
AF,
transfer
94 670
fluid.
concentration.
Results
Placental transfer of nicotine. The
typical changes in concentration of nicotine in the maternal and fetal plasmas are shown in Fig. 2 and descriptive data for the nicotine concentration curve in each experiment are presented in Table I. Nicotine crossed the placenta rapidly from the mother to the fetus. In five of six monkeys in which the nicotine concentrations were determined in both the maternal and fetal plasma, fetal levels exceeded maternal. It took only six to 20 minutes for the fetal plasma or blood levels to reach the maternal in four cases after a single injection of nicotine (1 mg. per kilogram of B.W.) to the maternal circulation. In another case where a constant infusion of nicotine was given with same dose to the mother over 20 minutes, 23 minutes elapsed before maternal levels were reached in the fetus. It took 16 to 85 minutes for the fetal level to reach its maximum value ranging from 58 to 94 pg per 100 ml. of plasma or blood after a single injection and 670 pg per 100 ml. following constant infusion. In only one case the fetal concentration did not reach the maternal level. In every experiment the fetal plasma or blood concentration of nicotine declined either more slowly than the maternal concentration or, at most, at a similar rate. In four of five cases the fetal plasma or blood
Table II. Fractional transfer rates (rate constants) of nicotine between maternal fetal plasma compartments (minute -I) Monkey No.
K
446 443 494 447 458 491
Mean S.E.M. *For
m + f*
0.437 0.202 0.106 0.052 0.020 0.273 0.182 0.064 explanation
see legend
Kr
-+ m*
-a out+
0.046 0.098 0.272 0.056 0.046 0.034 0.092 0.037 ______
0.225 0.021 0.026 0.063 0.025 0.263 0.104 0.045 to Fig.
Km
and
1.
still retained 72 to 89 per cent of the maximal concentration of nicotine at 120 minutes following a single injection of nicotine to the mother. In one case (No. 458) only 16 per cent remained. In one case with constant infusion of nicotine to the mother, 48 per cent of the maximal concentration still remained in the fetal circulation at 120 minutes following the infusion. Fractional transfer rates of nicotine between maternal and fetal plasma compartments in six animals where the “H-nicotine was administered to the mother by single injection are shown in Table II. Differences among the mean transfer rates between these two compartments and the excretion rate from the mother are not statistically significant because of the large individual variability. An average of 18 per cent of the
258
Suzuki et al.
Am.
J.
May 15, 1974 Obstet. Gynecol.
Table III. Nicotine distribution in fetal organs of the rhesus monkey (milligram of nicotine per kilogram of tissue and percentage of the total dose administered to the mother in each organ) Time
after
Monkey
injection
30
No.
Gestation Fetal
(min.)
465
(days)
weight
(grams)
0.46t (0.423)X 0.68 (0.177) 0.82 (0.121)
wall
Adrenals
0.47 (0.038)
Spleen
0.69 (0.055) 1.12 (0.017)
wall
Liver Skeletal
I
--.-~ <
410
Lungs
Stomach
~-..
I -J”-
146
Brain
Intestinal
~---?!L
muscle
Kidneys Heart Placenta
150
I40
410
0.15 (0.095) 0.14 (0.010) 2.05 10.169) 5.74 (0.056) 1.35 (0.011)
l’hymus
491 . /.
0.39 (0.242) 1.N (0.112) 0.58 (0.063) 1.68 (0.045)
)
0.28 (0.037 1 0.79 (0.864 ) 1.34 (0.113 ) 2.34 (0.061) 0.46 (1.121) 0.50 (0.001)
per
cent
4.167
2Javanese monkey. tconcentration in milligrams :Percentage of the total dose calculated
as
(nicotine
of nicotine nicotine
of concentration
s
2.559 per kilogram
0.07 (o.0003) 0.82 (0.004)
1.34 (0.017) 1.18 (cr.013) (1.85 (0.1 15 ) (1.92 (1.274 ) 0.67 (0.036 ) 1.79 (0.048 0.54 (0.876 0.44 (0.012
0.62 (0.045 0.02 (0.012 1.06 (0.049 0.08 (0.001) 0.19 (0.252)
2.853
--,-L& 0.28 (0.182 ) 0.41 (0.025) 0.28 (0.027) 2.33 (0.005) cl.75 f CJ.003) 1.10 10.012) 0.43 (0.054) 0.29 (0.357) 0.67 (0.032
j
0.63 (0.151)
0.55 (0.010 j 0.25 (0.350) 1.47 (0.008) 0.70 (0.345)
2.999
1.410
of tisaw.
administered to the mother organ weight/total nicotine
nicotine in the maternal plasma could be transferred to the fetal plasma per minute and an average of 10 per cent of the nicotine in the fetal plasma could be transmitted back to the maternal plasma per minute. Excretion rates of nicotine from the mother via the urine averaged nine per cent of the amount in her plasma per minute. Distribution of nicotine in fetal tissues. The distribution of nicotine in various fetal tissues of five monkeys is presented in Table III. Higher concentrations (> 1.0 mg. per kilogram) were found in adrenals, heart, kidneys, stomach, and spleen in both mature and immature fetuses; this pertained equally
107 /j" 168 7.02 (2.425) 0.65 (0.026) 0.76 (0.034)
Blood Total
90 ~. ~~~ 494
,p_---
467
120
1.75 (0.021
1.22 (0.244) 0.63 (1.30) 1.73 (0.242) 1.65 (0.099) 0.53 (1.452)
60
(1 mu. ‘< body dose administrledi
weight x
in kilograms) 100.
in an entire
organ,
to samples obtained 30 minutes or 60 minutes following the injection of nicotine to the mother. Brain and intestines had high concfmtrations of nicotine in tissue samples obtained at these intervals in fetuses of less than 120 days’ gestational age, whereas these organs showed consistently lower concentrations in more mature fetuses. The converse was true for lungs and liver. The concentration of nicotine in the adrenals of less mature fetuses was as high as 5.74 mg. per kilogram of tissue at 30 minutes after injection but fell to 0.07 mg. per kilogram at 60 minutes. Adrenals in more mature fetuses. however, showed progressive increase in levels from 0.47 mg. per kilogram
Volume Number
119 2
at 30 minutes to 1.68 mg. per kilogram at 60 minutes and 2.33 mg. per kilogram at 90 minutes. The brain tissue of a fetus of 107 days of gestation (No. 491) showed the highest concentration of nicotine among all the tissues, 7.02 mg. per kilogram at 60 minutes. The thymus of a mature fetus (No. 494) contained 1.47 mg. per kilogram of nicotine at 90 minutes, whereas that of less mature fetuses had only 0.50 mg. per kilogram at 30 minutes and 0.44 mg. per kilogram at 60 minutes. Because of their relatively large mass, skeletal muscle and placenta took up larger quantities of nicotine than other organs, even though the concentrations of the alkaloid in these organs were low. When the total percentage of nicotine contained in a fetus and placenta was estimated by adding the amount in each organ as a fraction of the total dose of nicotine administered, it was found to range from four per cent at 30 minutes after injection to one per cent at 90 minutes. Comment The dosage of nicotine used in this study, 1.0 mg. per kilogram, is much larger than the amount the pregnant woman would be expected to absorb in the course of cigarette smoking. The pharmacologically equivalent effect is attainable in the human subject, however, due to the difference in sensitivity to nicotine between man and m0nkey.l The monkey is at least six times as tolerant to nicotine as the human being, as evidenced by data on the lethal dose.2 Thus, a nicotine dose of 1.0 mg. per kilogram in the monkey should correspond to no more than 0.17 mg. per kilogram in the human being. Two to 3 mg. of nicotine may be absorbed during cigarette smoking on average, yielding a concentration of nicotine in a smoking mother similar to that used in this study. Rapid transfer of nicotine from the mother to the fetus has been proved and the nicotine concentrations in maternal and fetal blood and in the fetal tissues have been determined in the pregnant primate for the first time. The methods we developed for isolation and quantitation of nicotine in blood and tissues were sensitive enough to measure
Placental
transfer
and
distribution
of nicotine
259
as low as 3 pg per 100 ml. of nicotine in plasma and reliable enough to have reliably constant recovery rates. Unexpectedly, the majority of both mature and immature fetuses showed persistently higher plasma or blood concentrations of nicotine than the mothers after six to 23 minutes following administration of nicotine to the mother. Disappearance of nicotine from the fetal blood compartment was slower than from the maternal blood. Thus, cardiovascular disturbances, acidosis, and hypoxia which have been observed in the mature fetus following administration of 1 mg. per kilogram of nicotine to the mother1 must be considered possibly the result of the nicotine transmitted to the fetus. Our previous finding that cardiovascular responses of the fetus differed according to whether comparable dosages of nicotine was administered to the mother or to the fetus directly’ may now be partly explained by our current observation that the fetal plasma or blood level of nicotine administered to the mother (of 1 mg. per kilogram of maternal body weight) is much lower than the level produced by direct injection (of 1 mg. per kilogram of fetal body weight) into the fetal circulation. The effect of maternally administered nicotine and its metabolites on uterine perfusion might also contribute to the disparate fetal cardiovascular responses. Similarly, our data helped to provide an explanation for the previous finding that mature fetuses, but not immature ones, sustained rather prolonged acidosis and hypoxia following nicotine administration to the mother. This is believed to be based on differences in the developmental stage of autonomic nervous system in the fetus in view of our finding that both mature and lessmature fetuses have similar plasma or blood concentrations of nicotine. Another factor to explain the apparently greater vulnerability of mature fetuses to nicotine could be the more prolonged tissue concentrations of nicotine found in mature fetuses as compared with those that are less mature. Although concentrations in many organs of the younger fetus (such as heart, adrenals, kidneys, spleen, intestines, and stomach) were
260
Suzuki
May
et al.
Am.
higher than or similar to those in the older fetus at 30 minutes after injection of nicotine, they fell with time so that some tissues of the mature fetus (especially heart, adrenals, and spleen) had higher values at 60 minutes after injection. Lungs, stomach, skeletal muscle, and placenta also had similar late peak values in the older fetus. Thus, one could postulate that the transmitted nicotine might disturb the umbilical circulation or adversely affect cellular metabolism in the mature primate fetus. However, the total amount of nicotine present in a fetus from 30 to 90 minutes after the nicotine injection to the mother is small, accounting for approximately one to four per cent of the total dose of nicotine injected (Table III). Admittedly these percentages are only approximations because some tissues, such as subcutaneous fat, skin, and pancreas. have not been included. Since distribution in these organs in the dog is reported to be either insignificant or very small,” these figures can be considered to be reasonably accurate. The time for complete excretion of nicotine via urine is about 48 hours in the dog3 following the administration of nicotine, 1 mg. per kilogram, intravenously. It is reasonable to assume that most of the nicotine administered to the pregnant monkey is still present during the 90 minutes of observation after the injection here. The fetus and placenta constitute approximately seven to 10 per cent of the total body weight of the pregnant monkey. Thus one to four per cent of the total dose of nicotine present in the fetus is indeed a smaller proportion than that which can be expected with uniform distribution of nicotine in the gravid organism. Uneven distribution to the fetus may be the result of diminished perfusion due to uterine vasoconstric-
J. Obstet.
15, 1971 Gynecol.
tion. Working synergistically with the transmitted nicotine in the fetus, this vasoconstriction in thr uterine circulation and its resultant diminished intervillous space perfusion could be another factor causing adverse fetal effects as previously contended.’ High nicotine concentrations in the kidneys are expected because the main excretory pathway is via the urine and the kidney is partly involved in metabolism of nicotine.4 The main excretion of nicotine from the fetus, however, must be via the placenta since the fetal kidney is known to be far less functionally effective as an excretory organ than the placenta. The liver shows fairly high concentrations in the mature fetus at 30 minutes, but these decline progressively to very low levels at 60 and 90 minutes even though the blood levels of nicotine are still near their peak. In less mature fetuses the concentrations in the liver are persistentIy low. This indicates that the fetal liver does not serve as the major site for detoxication of nicotine, as does the adult liver; this is likely due to its immature enzyme systems. Most of the nicotine in the fetus is probably excreted by way of the placenta without being metabolized. High concentrations in the heart and the stomach wall of the fetus are in agreement with the findings made by Fishman:’ in an adult dog. Although the number of monkeys used in the current experiment is limited, it is clear that nicotine is rapidly transferred across the placenta and that nicotine transmitted to the fetus is excreted more slowly than that in the mother. We gratefully acknowledge Miss Lydia O’Brien’s valuable technical assistance for chemical assay of nicotine, and Dr. Emanuel A. Friedman’s review of our manuscript.
REFERENCES
1. Suzuki, K., Horiguchi, A. C., Mueller-Heubach, and Adamsons, K.: AM. 2. 3.
T., Comas-Urrutia, E., Morishima, H. O., J. OBSTET.
GYNECOL.
111: 1092, 1971. Feurt, S. D., Jenkins, J. H., Hayes, F. A., and Crockford, H. A.: Science 127: 1054, 1958. Fishman, S. S.: Arch. Int. Pharmacodyn. 145: 123, 1963.
4. Goodman, L. S., and Gilman, A.: The Pharmacological Basis of Therapeutics, ed. 3, New York, 1965, The Macmillan Company, p. 582. 5.
Riggs, D. Physiological
Williams
S.: The Mathematical Problems, Baltimore,
Approach 1963,
& Wilkins Company, pp. 204-206.
to The
Volume Number
119 2
Placental
transfer
Appendix
and
exp
The explicit two-compartment lows5 :
[-%
equation for the generalized system (Fig. 3) is as fol-
=
KS” - Kt
(
(K,
of
+ Kf
1 _ K,,
nicotine
+ Z)
- Ki
t]
+ Z
22 exp
F,.t
distribution
[-%(K,,
+ Kr
-
Z)
t])
+ Z
22
Injection
of Nicotine I
F
"f
Qf-t
1
Kf+out
Km = 5lb.f + KIF-0,t Fig.
3. A general
Kf = Kf pm + Kf-+out
two-compartment
71000 z Y) 500 I! L
AND
model.
Symbols
denote
the same as in Fig.
OF MOTHER
AMNIOTIC
AND
1.
FETUS,
FLUID
: Ill
\\THER
FLUID
;
2-
z I
MONKEY NO GESTATIONAL
0
1 IS
1 30
AA6 AGE
121
I A5
DAYS I 60
I 75
I 90
I 105
I 120
I 135
1
0
MINUTES
Fig. 4. Determination as a function of time.
of the
equation
for
the maternal
plasma
concentration
curve
of nicotine
261
262
Where
Suzuki
et al.
F,,,.t =
Am. J.
the fraction of the total dose of nicotine in the compartment M at time t.
z = \/(K,,, - Kr)” + 4K,,-+r KY+,,, Boundary conditions: at t = 0. F,, n = 1, FL,, z (1 In the present study the equation for the nicotine concentration curve of the maternal plasma is expressed as: c,,, t x A,f.-,‘I’ + .4,e-““L Where
(2) (Fig. 4)
C,,,.t =
the concentration of nicotine in the maternal plasma in micrograms per 100 c.c. t = the time in minutes At time zero, Eq. (2) gives: C 111 0 = ,4, + .4,
Since the dose of nicotine administered, was 1 mg. x body weight, the initial tot, Q volume of distribution in the compartment M (the volume of the compartment M)
v,,,
z
May 15, 1974 Obstet. Gynecol.
$y ID 0
Multiplying both sides of VJQ,,,, to convert it to F ,,,.t F 1,1t I~: A’@1
Eq.
+ i\‘re-“g’
(2)
by (3)
Eq. (3 ) is a particular example of Eq. (1). We can, therefore, equate the numerical values in Eq. (3) to the corresponding algebraic values of Eq. (1) : s (Ku, tKr+Z)
=uI
(4)
% (Ku, +Kt-Z)
=a:
(5)
K,,, - Kt +-- Z -- A’, ?Z
(6)
Solving Eqs. (4), (5), and (6) with the knowledge of Kf - INlt = 0 (Therefore Kf = ~ ,j , WC can obtain the values for K, + f. Kr Kf -+ ,“, and
K,, + Ollt.