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Caffeine and its dimethylxanthines and fetal cerebral development in rat
ORIGINAL AR TICLES
Caffeine and Its Dimethylxanthines and Fetal Cerebral Development in Rat Harumi Tanaka, MD, Kazuharu Nakazawa, Masataka Arima, MD and Setsuo Iwasaki, PhD The relationship between the distribution and pharmacokinetic behavior of caffeine and its dimethylxanthines in pregnant rats and fetuses and fetal cerebral development was compared in four groups with different modes of oral caffeine ingestion by the mothers. During the premating period and pregnancy, female Wistar rats were divided into 0.04% caffeine (C) and water (W) groups, respectively. When the groups are expressed as W or C before mating- W or C during pregnancy, the fetal body weight was low in the three caffeine-treated groups (W-C, C-W and C-C) and the fetal cerebral weight was the lowest in the W-C group. The mean concentration of caffeine or metabolites in maternal plasma, maternal liver, placenta and fetal cerebrum on gestational day (g d) 21 was increased in the W-C group compared to in the C-C group. The concentration of caffeine in fetal cerebrum was increased but that of metabolites was not, compared to the concentration of caffeine or metabolites in the placenta. Radioactivity in fetal cerebrum after intraperitoneal injection of 14C-caffeine was higher in the W-C group than in the other three groups. After intravenous injection of caffeine the apparent volume of distribution of caffeine in maternal plasma was markedly decreased in the W-C group, and the plasma molar concentration ratio of theophylline to caffeine was significantly increased in both the W-C and C-C groups. The adverse effect of maternal caffeine ingestion on the fetal cerebrum may be associated with the decreased apparent volume of distribution of caffeine in maternal plasma and the high caffeine content of fetal cerebrum. Tanaka H, Nakazawa K, Arima M, Iwasaki S. Caffeine and its dimethylxanthines and fetal cerebral development in rat. Brain Dev 1984;6:355-61
The possible harmful effect of caffeine in human pregnancy can not be neglected because of the pharmacological and embryo-fetopathic action of caffeine or its dimethylxanthines under experimental conditions [1, 2]. In a
From the Division of Mental Retardation and Birth Defect Research, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for pUblication: April 20, 1984. Accepted for pUblication: June 10, 1984.
Key words: Caffeine, dimethylxanthine, pregnancy, fetal rat, cerebrum, placenta, volume of distribution. Correspondence address: Dr. Harumi Tanaka, Division of Mental Retardation and Birth Defect Research, National Center for Nervous, Mental and Muscular Disorders, Ogawa-higashi-machi, Kodaira, Tokyo 187, Japan.
previous study we demonstrated the adverse effect of maternal caffeine ingestion on the fetal cerebrum in rat and suggested that this effect on the fetus depends on the mode of maternal caffeine ingestion during the premating and/or gestational periods [3]. The object of the present study was to elucidate the relationship between the distribution and metabolism of caffeine in pregnant rats and fetuses and the fetal growth with different modes of maternal caffeine ingestion during the premating period and pregnancy.
Materials and Methods Experimental procedures for preparation of fetal models were essentially as described previously [3]. Virgin female albino rats of the
Wistat strain were divided into caffeine and control groups, receiving 0 .04% caffeine (C) via drinking water and drinking water (W), respectively. The groups are expressed as W or C before mating - W or C during pregnancy. According to the experimental step, two experiments were performed.
1. Distribution of Caffeine and Metabolites in Mothers and Fetuses (Exp 1). Female rats at 8 weeks of age were pretreated for 134 days with C or W. During mating with male rats at 15 weeks of age, all rats were given water only. Both groups received C or W throughout pregnancy . The day on which a sperm-positive vaginal smear was obtained following mating was considered as gestational day (g d) O. Pregnancies were terminated on g d 21 at 8: 30 am by cesarean sectioning. Pregnant rats were examined for body weight, liver weight and consumed weights of solid diet , water and 0.04% caffeine. Fetuses were examined for litter size, fetal viability 1-1.5 hours after cesarean sectioning, placental weight , body weight and cerebral weight. Radioactivity in fetal cerebrum at 2 hours after intraperitoneal injection of 50 J..Ll [1-methyl14C]-caffeine (9.8 J..Lg = 2.5 J..LCi/fetus, diluted with isotonic saline, New England Nuclear, Boston, Mass, USA) into lively fetuses 1 hour after cesarean sectioning was counted with a scintillation counter. After weighing and freezing, analysis of caffeine and its three dimethylxanthines was performed by high-performance liquid chromatography (HPLC) in maternal plasma, maternal liver and placenta at cesarean sectioning and in fetal cerebrum 1 hour after cesarean sectioning. 2. Parameters for Caffeine Metabolism in Maternal Plasma (Exp 2). Female rats at 8-13 weeks of age were given C or W during the premating period for about 38 days and the gestational period for 17 days. In the morning on g d 17 , caffeine treatment was discontinued and drinking water was given in both the W-C and C-C groups. On g d 18 at least 24 hours after initiation of water treatment, pregnant rats of the four groups were injected intravenously with 10 mg/kg body weight of caffeine. Caffeine and its three dimethylxanthines in plasma were analyzed at 2 to 12 hours after injection. A report on
356 Brain & Development, Vol 6, No 4, 1984
precise pharmacokinetic analysis is in preparation (Nakazawa K and Tanaka H) .
3. Analysis of Caffeine and Three Dimethylxanthines Concentration of caffeine, theophylline , paraxanthine and theobromine in maternal plasma, maternal liver, placenta and fetal cerebrum were determined essentially by the methods of Bonati et al [4] and Tse et al [5] , with minor modifications. To 0 .2 ml of plasma and 0 .5 ml of 10% whole homogenate with isotonic saline of three kinds of tissues, 7-[2-hydroxyethyl]theophylline for plasma, placenta and ce rebrum or 7-[~-hydroxypropyl ]theophylline for liver as an internal standard was added. After extraction with chloroform: isopropyl alcohol (75: 25, v/v), the organic phase was evaporated and . the residue dissolved in 0.005 M sodium acetate pH 5 buffer. A 20 J..Ll aliquot of the supernatant was injected into the HPLC apparatus . Analysis at 280 nm was performed with a 30 em x 3.9 mm ID 10-J..Lm J..LBondapak CIS column (Waters Assoc , Milford , Mass, USA) and a mobile phase of 0.005 M sodium acetate pH 5 buffer: methyl alcohol: acetonitrile: tetrahydrofuran (92.5:3:2.8:1.7, v/v). The assay was performed in duplicate or triplicate. Chemicals: Analytical grade caffeine, theophilline and theobromine, ultraviolet grade methyl alcohol, chloroform, isopropyl alcohol, tetrahydrofuran and acetonitrile (Wako Pure Chemical Industries , Osaka, Japan); paraxanthine and 7-[~-hydroxypropyl]theophylline (Sigma Chemical Co, St Louis, Mo , USA), 7[2-hydroxyethyl]theophylline (Tokyo Kasei Kogyo Co , Tokyo , Japan). 4. Statistical Analysis Mean significant difference was calculated according to the Student's t test. Results 1. Maternal and Fetal Factors The effect of four modes of maternal oral ad libitum ingestion on maternal and fetal factors in Exp 1 was almost the same as that described previously [3]. Mean maternal solid food intake during pregnancy for 21 days did not show a significant difference between the four groups; 459 g in WoW, 497 g in W-C, 467 g in CoW and 498 g in CoCo Mean maternal caffeine
intake with 0.04% caffeine solution in tap water also showed almost the same weight in two groups; 438 mg of caffeine in W-C and 457 mg of caffeine in C-C during pregnancy for 21 days. There was no significant difference between the four groups in maternal body weights on g d 0 and 21; 350 gin WoW, 409 g in W-C, 371 g in CoW and 367 g in C-C on g d 21. Maternal liver weight on g d 21 and litter size also did not show any significant difference between the four groups. Mean placental weight was 0.63 g in WoW and 0.82 g in CoW, but 0.49 g in W-C and 0.59 g in C-C, in which a significant difference was only observed between W-C and C-C (p < 0.05). Mean percent survival was 94% in WoW, 98% in CoW, 86% in W-C and only 30% in C-C, in which prolonged maternal administration of caffeine shows an adverse effect on the fetal survival rate. Fetal body weight after cesarean sectioning and fetal cerebral weights with two kinds of treatment after cesarean sectioning are shown in Table 1. In the present study but not in the previous study a significant difference was found in body weight, reduced weight being demonstrated in the three caffeine-treated groups; W-C, CoW and CoCo Mean fetal cerebral weight was reduced in caffeine-treated groups, especially in the W-C group, compared to the control WOW group.
2. Caffeine and Metabolite Levels in Mothers and Fetuses There was a good positive correlation between the concentration of caffeine or metabolites in
maternal plasma, maternal liver and placenta in three dams at cesarean sectioning. The mean concentrations or contents of caffeine, paraxanthine or theophylline in maternal liver, maternal plasma, placenta and fetal cerebrum with two modes of maternal oral ad libitum ingestion of caffeine during pregnancy in Exp 1 are shown in Table 2. As no essential or conclusive difference in distribution between the three dimethyxanthines was seen, the values for theobromine are omitted from Table 2. The concentration of caffeine or metabolites was almost the same in maternal liver, maternal plasma and placenta, however, a high mean concentration of the three materials was found in maternal liver followed by maternal plasma and placenta, in that order. The mean concentration of caffeine or metabolites in maternal liver or maternal plasma or placenta was always higher in W-C than in CoCo The mean concentration of caffeine in fetal cerebrum was increased but that of metabolites was not compared to the concentration of caffeine or metabolites in the placenta in both the W-C and C-C groups; the fetal cerebral value as a percentage of placental value was 113% and 119% for caffeine, 91 % and 92% for paraxanthine and 92% and 89% for theophylline in the W-C and C-C groups, respectively. The concentration or content of caffeine or metabolites in fetal cerebrum was all Significantly higher in the W-C group than in the C-C group. In Fig 1 the relationship between the concentration of caffeine or theophylline in fetal cerebrum and cerebral weight on g d 21 is
Table 1 Fetal body and cerebral weights on g d 21
WOW Body weight (g)
5.57
±
0.23 (13)
5.12
±
c-c
COW
W-C 0.33 (22)
4.85
±
0.58 (23)
5.07
II L - ****~*~**** ________________ I
Cerebral weight (mg) intact
146
± I
3
( 4)
ip injection
146
±
6
( 9)
I
0.70 (15)
I
-----.J
L ._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
±
~.
136 ± 9 ** ----.J
( 9)
132
±
15
(12)
137
±
12
( 6)
138
( 8)
144
±
8
(10)
140
±
13
( 8)
±
* ----.J
12
W: water, C: 0.04% caffeine, W or CoW or C: W or C before mating period-W or C during pregnancy. Data expressed as means ± SD (no of fetuses). Significant difference between groups; ****: p < 0.01, ***: p < 0.02, **: p < 0.05, *: p < 0.10.
Tanaka et al: Caffeine in fetal cerebrum 357
Table 2
Maternal and fetal caffeine or metabolite levels on g d 21 Maternal liver (lJ,g/g wet wt)
Maternal plasma (lJ,g/ml)
Caffeine W-C C-C
9.9 ± 2.9 [3] 4.4 ± 1.6 [3] **
9.5 ± 3.6 [3] 4.0 ± 1.7 [3] *
8.1 ± 3.4 [3] 3.2 ± 1.3 [3] *
8.23 ± 3.41 (6) 3.55 ± 1.05 (6)****
1.07 ± 0.41 (6) 0.47 ± 0.17(6)****
Paraxanthine W-C C-C
4.1 ± 0.9 [3] 2.2 ± 0.2 [3] **
3.6 ± 0.7 [3] 3.0 ± 0.6 [3]
3.0 ± 0.4[3] 2.1 ± 0.2 [3] **
2.66 ± 0.39 (6) 1.91 ± 0.23 (6)****
0.35 ± 0.04 (6) 0.25 ± 0.03 (6)****
3.6 ± 0.4 [3] 3.4 ± 0.9 [3]
2.7 ± 0.1 [3] 2.1 ± 0.2 [3] ***
2.44 ± 0.16 (6) 1.79 ± 0.15 (6)****
0.32 ± 0.02 (6) 0.24 ± 0.02(6)****
Theophylline W-C 3.9 ± 0.7 [3] C-C 3.4 ± 1.5 [3]
Placenta (lJ,g/g wet wt)
Fetal cerebrnm (lJ,g/g wet wt) (lJ,g/cerebrnm)
Data expressed as means ± SD [no of dams] or (no of fetuses). . Significant difference between W-C and C-C; ****: p < 0.01, ***: p < 0.02, **: p < 0.05, *: p < 0.10. For the fetal cerebrum assay, one to three fetuses were obtained from one dam.
12.0
• • •
10.0
'-' 3:
'-' Q)
3: 01
"'3' 5.0
•
Q)
0
c: Q) ~ ~
c
u
•
0
•
0 0
Q)
= c:
3.0
'TI~O '- - - - - - - '1~6:' 1-~~b~___ ;~ ._O~l~ O 00
o
:_O____O_O___
Cerebral weight (mg)
demonstrated in both the W-C and C-C groups. There was no clear correlation between the two factors in these two groups.
3. Radioactivity in Fetal Cerebrum The mean percent radioactivity in fetal cerebrum of the four groups 2 hours after intraperitoneal injection of [1-methyl- 14 C ]-caffeine is presented in Table 3. The recovery of radioactivity in the cerebral homogenate, in which caffeine and its metabolites were not identified, accounted for about only 1.7% of the intraperitonealy administered radioactivity of 14C_
358 Brain & Development, Vol 6, No 4, 1984
Fig 1 Relationship between caffeine or theophylline concentration in fetal cerebrnm and cerebral weight on g d 21. e: W-C group, 0: C-C group.
caffeine. The highest values of the radioactive concentration in the whole homogenate and acid-soluble fraction of fetal cerebrum were observed in the W-C group.
4. Caffeine Metabolism in Mothers A few parameters for maternal caffeine metabolism in plasma are presented in Table 4. As to the pharmacokinetic parameters of caffeine, the apparent volume of distribution was significantly decreased and elimination of caffeine increased in the pregnant rats given caffeine only during pregnancy (W-C). On the other
Discussion
Table j Radioactivity in fetal cerebrum on g d 21 after intraperitoneal injection of [[1-methyl-14 CJj-caffeine
WOw w-c cow C-C (6)
(7)
(9)
(8)
Dpm in whole homogenate 100% 118% 1l0% 103% /g wet wt Dpm in 0_3N PCA soluble fraction /g wet wt 100% 117 % 1l0% 102% Data expressed as mean percent of WoW value (no of fetuses) _
Table 4 Parameters for caffeine metabolism in maternal plasma on g d 18 after intravenous injection of caffeine
WOw w-c COw C-C [7}
Volume of distribution (ml/kg) Elimination rate constant
lQO%
[6}
[7}
[7}
~i~% llf%
100% 141%
83% 117%
Molar concentration ratio; theophylline to caffeine at 6 hrs 100% 175 %
83% 188%
(h
O ,)
I L..--J
at 8 Ius
L - - J L-.J
I
100% 223% 68% 195%
Il--l
L--.J L-.J
I
Data expressed as mean percent of WoW value [no of dams] _ Statistical difference between groups shown at p < 0_01 with bars_
hand, plasma molar concentration ratios of theophylline to caffeine at 6 or 8 hours after injection were significantly increased in the pregnant rats of both groups given caffeine during pregnancy (W-C and C-C)_ The molar concentration ratios of paraxanthine or theobromine to caffeine showed similar tendencies_ These results show that the apparent volume of distribution of caffeine in plasma was influenced by caffeine administered during not only the gestational period but also the premating period, however, the molar concentration ratio of dimethylxanthine to caffeine was essentially influenced only by caffeine administered during the gestational period_
It remains to be determined that how fetal and neonatal growth is influenced by the maternal oral ingestion mode for caffeine with regard to the consumed quantity and duration during the premating and gestational periods in rat or man_ The teratogenic potential of coffee or caffeine in rats fed via the oral route before mating and throughout gestation could only be demonstrated after administration of quantities of about 20 mg/kg/day by Palm et al [6]- They also demonstrated that the F 1 fetus from dams treated with coffee, 38 mg/kg/day, was similar in body weight but the mean fetal brain weight was significantly lower as a percentage of body weight compared to the controL On the other hand, the reduced growth potential of caffeine in rat offspring was shown by the administration of a low level of 10 mg/kg/day throughout sequential pregnancies and lactations by Dunlop et al [7] , who also demonstrated a progressive reduction in birth weight and brain weight of offspring of sequential pregnancy 4 without accompanying teratogenic effects_ In our previous study, a reduction in mean fetal cerebral weight without reduction in body weight was induced by maternal caffeine ad libitum ingestion during the premating period for 47125 days and throughout pregnancy [3]- However, in our present study, maternal caffeine ingestion during a further prolonged premating period of 134 days and throughout pregnancy produced reduction in both body and cerebral weight. Our pregnant rats received a daily dose of about 57 mg/kg of caffeine when calculated without consideration of loss at drinking _ Based on these results, it may be suggested that relatively high daily oral doses throughout pregnancy are required to induce the teratogenic effect or one part of the inhibitory effect on fetal cerebral growth, and prolonged, relatively low daily oral doses with or without administration during pregnancy have an inhibitory effect on fetal body growth_ In other words, there may be some cumulative effect in caf, feine-treated mothers on the reduction in fetal body growth, but a relatively high acute effect during pregnancy on the reduction in fetal cerebral growth_ In human studies, a positive association between the daily consumption of eight or more cups of coffee and congenital anomalies [8] or that of five or more cups of
Tanaka et al: Caffeine in fetal cerebrum 359
coffee and small-for-date babies [9] coincides with the results of our studies on rat. The possible nature of the reduction in fetal body weight by caffeine may be on either placental growth or on fetal growth directly . In our previous and present studies, caffeine-exposure throughout pregnancy, especially in the W-C group , has been found to affect the placental growth to some extent. However , there was no clear correlation between the reduction in fetal body weight demonstrated on prolonged administration and the reduction in placental weight demonstrated on acute administration during pregnancy in the present study. The inhibitory effect of maternal caffeine on fetal growth may be accounted for in part by the inhibition of growth hormone and thyrotropin secretion [10] after further study. There have only been a few studies on association between fetal abnormalities and caffeine or metabolite levels with different maternal administration modes of caffeine. As N-demethylation is the main pathway of in vitro caffeine metabolism in the rat liver at all ages [11] , maternal and fetal caffeine or metabolite levels were determined to elucidate the factors for fetal cerebral development. The concentration of caffeine or metabolites showed similar values in mothers and fetuses in our study. This good placental transfer in rat coincides with the finding of the passage of caffeine into human gonadal and fetal tissue [12] or the passage of caffeine and many metabolites into the mouse embryo [13]. Although the caffeine dose ingested ad libitum by mothers was not different between W-C and C-C during pregnancy , the mean value of caffeine and metabolites of mothers and fetuses was always higher in W-C than C-C in our study, which stresses the importance of the caffeine ingestion mode of mothers. As to caffeine and the fetal cerebrum, our study showed some evidence that the level of caffeine was higher in fetuses than in mothers but the metabolites were not , and that the fetal cerebral concentration might be highest in the W-C group compared to the other three groups was shown by an isotopic study . It is important to clarify for the fetal development, why the fetal cerebral caffeine level was increased in the W-C group compared to the C-C group. The developmental pattern of caffeine metabolism demonstrates that the liver
360 Brain & Development, Vol 6, No 4, 1984
of the newborn has an extremely limited capacity to metabolize caffeine [11, 14] , and that the adult metabolite pattern was attained by weaning in rat [II] and by 7 to 9 months in man [14]. It is also known that the metabolism of theophylline to caffeine occurs in the human fetus and newborn infant [15]. Based on the above mentioned results on fetal caffeine metabolism , the caffeine levels may increase slightly but significantly in the fetus by accumulation. This caffeine accumulation may potentially produce a toxic effect if the mother has toxic caffeine plasma concentrations. However , this metabolical specificity in the fetus could not explain the difference in fetal cerebrum between W-C and C-C. How may the different concentration of fetal cerebral caffeine between W-C and C-C be explained by the maternal caffeine metabolism or kinetics? In our study , the molar concentration ratio of theophylline to caffeine at 6 and 8 hours was increased in both W-C and C-C to similar levels. This result may be explained by the stimulatory effect of caffeine administered twice daily for only 3 days on drug metabolizing activities which lasted for only 24 hours after the last dose in male mature rat [16, 17]. According to the dog study by Axelrod et al [18], caffeine is distributed in tissues in approximate proportion to their water content. From this view point , the much decreased apparent volume of distribution of caffeine in the W-C group compared to the other three groups is reasonable. The low value of the volume of distribution in maternal plasma may result in the high concentration of caffeine in mother and fetus , although explanation of this phenomenon must await further experiments. Recently there have been several studies on the effect of pregnancy on pharmacokinetics of caffeine for estimating the caffeine effect on the fetus in man and animals [l , 19-21]. Delayed elimination of caffeine in pregnant women [1, 19] or a greatly increased half-life of elimination of caffeine [20] could produce the side effects of coffee in pregnancy at a lower rate of consumption than in the nonpregnant state. These findings may prompt further study to assess the pharmacokinetic changes of caffeine in pregnant women and fetus or neonate and the possibility of its adverse effect.
Acknowledgments This study was partly supported by Grant No 83-05 from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan.
10.
11. References 1. Brazier JL, Ritter J, Berland M, Khenfer D, Faucon G. Pharmacokinetics of caffeine during and after pregnancy. Dev Pharmacol Ther 1983; 6:315-22. 2. Fujii T, Nishimura H. Adverse effects of prolonged administration of caffeine on rat fetus. Toxicol Appl Pharmacol 1972;22:449-57. 3. Tanaka H, Nakazawa K, Arima M. Adverse effect of maternal caffeine ingestion on fetal cerebrum in rat. Brain Dev (Tokyo) 1983;5:397-406. 4. Bonati M, Castelli D, Latini R, Garattini S. Comparison of gas-liquid chromatography with nitrogen-phosphorus selective detection and high-performance liquid chromatography methods for caffeine determination in plasma and tissues. J Chromatogr 1979; 164:109-13. 5. Tse FLS, Szeto DW. Reversed-phase high-performance liquid chromatographic determination of caffeine and its N-demethylated metabolites in dog plasma. J Chromatogr 1981;226:231-6. 6. Palm PE, Arnold EP, Rachwall PC, Leyczek JC, Teague KW, Kensler CJ. Evaluation of the teratogenic potential of fresh-brewed coffee and caffeine in the rat. Toxicol Appl Pharmacol 1978;44:1-16. 7. Dunlop M, Court JM. Effect of maternal caffeine ingestion on neonatal growth in rat. Bioi Neonate 1981;39:178-84. 8. Jacobson MF, Goldman AS, Syme RH. Coffee and birth defects. Lancet 1981;1:1415-6. 9. Hoshiai H, Uehara S, Ryuh Y, et al. Caffeine and pregnancy. In: The study report on the prevention of fetal disorders which can be realized by improving the care of pregnant women (in Japanese). Research subgroup for handicapped children of the Ministry of Health and Welfare
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13. 14. 15.
16. 17.
18.
19. 20. 21.
studying the care of pregnant women, 1981: 34-6. Spindel E, Arnold M, Cusack B, Wurtman RJ. Effects of caffeine on anterior pituitary and thyroid function in the rat. J Pharmacol Exp Ther 1980;214:58-62. Warszawski D, Ben-Zvi Z, Gorodischer R. Caffeine metabolism in liver slices during postnatal development in the rat. Biochem Pharmacol 1981; 30:3145-50. Goldstein A, Warren R. Passage of caffeine into human gonadal and fetal tissue. Biochem PharmacoI1962;11:166-8. Scott WJ. Caffeine-induced limb malformations: Description of malformations and quantitation of placental transfer. Teratology 1983;28:427-35. Aldridge A, Aranda JV, Neims AH. Caffeine metabolism in the newborn. Gin Pharmacol Ther 1979;25:447-53. Aranda JV, Louridas AT, Vitullo BB, Thorn P, Aldridge A, Habner R. Metabolism of theophylline to caffeine in human fetal liver. Science 1979;206: 1319-21. Mitoma C, Sorich TJ, Neubauer SE. The effect of caffeine on drug metabolism. Life Sci 1968; 7:145-51. Mitoma C, Lombrozo L, LeValley SE, Dehn F. Nature of the effect of caffeine on the drugmetabolizing enzymes. Arch Biochem Biophys 1969; 134:434-41. Axelrod J, Reichenthal J. The fate of caffeine in man and a method for its estimation in biological material. J Pharmacol Exp Ther 1953;107:51923. Parsons WD, Pelletier JG. Delayed elimination of caffeine by women in the last 2 weeks of pregnancy. Can Med Assoc J 1982;127:377-80. Knitti R, Rothweiler H, Schlatter Ch. Effect of pregnancy on the pharmacokinetics of caffeine. EurJ Pharmacol 1981;21:121-6. Christensen HD, Manion CV, Kling OR. Caffeine kinetics during late pregnancy. In: Soyka LF, Redmond GP, eds. Drug metabolism in the immature human. New York: Raven Press, 1981: 163-81.
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361
Caffeine and Its Dimethylxanthines and Fetal Cerebral Development in Rat Harumi Tanaka, MD, Kazuharu Nakazawa, Masataka Arima, MD and Setsuo Iwasaki, PhD The relationship between the distribution and pharmacokinetic behavior of caffeine and its dimethylxanthines in pregnant rats and fetuses and fetal cerebral development was compared in four groups with different modes of oral caffeine ingestion by the mothers. During the premating period and pregnancy, female Wistar rats were divided into 0.04% caffeine (C) and water (W) groups, respectively. When the groups are expressed as W or C before mating- W or C during pregnancy, the fetal body weight was low in the three caffeine-treated groups (W-C, C-W and C-C) and the fetal cerebral weight was the lowest in the W-C group. The mean concentration of caffeine or metabolites in maternal plasma, maternal liver, placenta and fetal cerebrum on gestational day (g d) 21 was increased in the W-C group compared to in the C-C group. The concentration of caffeine in fetal cerebrum was increased but that of metabolites was not, compared to the concentration of caffeine or metabolites in the placenta. Radioactivity in fetal cerebrum after intraperitoneal injection of 14C-caffeine was higher in the W-C group than in the other three groups. After intravenous injection of caffeine the apparent volume of distribution of caffeine in maternal plasma was markedly decreased in the W-C group, and the plasma molar concentration ratio of theophylline to caffeine was significantly increased in both the W-C and C-C groups. The adverse effect of maternal caffeine ingestion on the fetal cerebrum may be associated with the decreased apparent volume of distribution of caffeine in maternal plasma and the high caffeine content of fetal cerebrum. Tanaka H, Nakazawa K, Arima M, Iwasaki S. Caffeine and its dimethylxanthines and fetal cerebral development in rat. Brain Dev 1984;6:355-61
The possible harmful effect of caffeine in human pregnancy can not be neglected because of the pharmacological and embryo-fetopathic action of caffeine or its dimethylxanthines under experimental conditions [1, 2]. In a
From the Division of Mental Retardation and Birth Defect Research, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for pUblication: April 20, 1984. Accepted for pUblication: June 10, 1984.
Key words: Caffeine, dimethylxanthine, pregnancy, fetal rat, cerebrum, placenta, volume of distribution. Correspondence address: Dr. Harumi Tanaka, Division of Mental Retardation and Birth Defect Research, National Center for Nervous, Mental and Muscular Disorders, Ogawa-higashi-machi, Kodaira, Tokyo 187, Japan.
previous study we demonstrated the adverse effect of maternal caffeine ingestion on the fetal cerebrum in rat and suggested that this effect on the fetus depends on the mode of maternal caffeine ingestion during the premating and/or gestational periods [3]. The object of the present study was to elucidate the relationship between the distribution and metabolism of caffeine in pregnant rats and fetuses and the fetal growth with different modes of maternal caffeine ingestion during the premating period and pregnancy.
Materials and Methods Experimental procedures for preparation of fetal models were essentially as described previously [3]. Virgin female albino rats of the
Wistat strain were divided into caffeine and control groups, receiving 0 .04% caffeine (C) via drinking water and drinking water (W), respectively. The groups are expressed as W or C before mating - W or C during pregnancy. According to the experimental step, two experiments were performed.
1. Distribution of Caffeine and Metabolites in Mothers and Fetuses (Exp 1). Female rats at 8 weeks of age were pretreated for 134 days with C or W. During mating with male rats at 15 weeks of age, all rats were given water only. Both groups received C or W throughout pregnancy . The day on which a sperm-positive vaginal smear was obtained following mating was considered as gestational day (g d) O. Pregnancies were terminated on g d 21 at 8: 30 am by cesarean sectioning. Pregnant rats were examined for body weight, liver weight and consumed weights of solid diet , water and 0.04% caffeine. Fetuses were examined for litter size, fetal viability 1-1.5 hours after cesarean sectioning, placental weight , body weight and cerebral weight. Radioactivity in fetal cerebrum at 2 hours after intraperitoneal injection of 50 J..Ll [1-methyl14C]-caffeine (9.8 J..Lg = 2.5 J..LCi/fetus, diluted with isotonic saline, New England Nuclear, Boston, Mass, USA) into lively fetuses 1 hour after cesarean sectioning was counted with a scintillation counter. After weighing and freezing, analysis of caffeine and its three dimethylxanthines was performed by high-performance liquid chromatography (HPLC) in maternal plasma, maternal liver and placenta at cesarean sectioning and in fetal cerebrum 1 hour after cesarean sectioning. 2. Parameters for Caffeine Metabolism in Maternal Plasma (Exp 2). Female rats at 8-13 weeks of age were given C or W during the premating period for about 38 days and the gestational period for 17 days. In the morning on g d 17 , caffeine treatment was discontinued and drinking water was given in both the W-C and C-C groups. On g d 18 at least 24 hours after initiation of water treatment, pregnant rats of the four groups were injected intravenously with 10 mg/kg body weight of caffeine. Caffeine and its three dimethylxanthines in plasma were analyzed at 2 to 12 hours after injection. A report on
356 Brain & Development, Vol 6, No 4, 1984
precise pharmacokinetic analysis is in preparation (Nakazawa K and Tanaka H) .
3. Analysis of Caffeine and Three Dimethylxanthines Concentration of caffeine, theophylline , paraxanthine and theobromine in maternal plasma, maternal liver, placenta and fetal cerebrum were determined essentially by the methods of Bonati et al [4] and Tse et al [5] , with minor modifications. To 0 .2 ml of plasma and 0 .5 ml of 10% whole homogenate with isotonic saline of three kinds of tissues, 7-[2-hydroxyethyl]theophylline for plasma, placenta and ce rebrum or 7-[~-hydroxypropyl ]theophylline for liver as an internal standard was added. After extraction with chloroform: isopropyl alcohol (75: 25, v/v), the organic phase was evaporated and . the residue dissolved in 0.005 M sodium acetate pH 5 buffer. A 20 J..Ll aliquot of the supernatant was injected into the HPLC apparatus . Analysis at 280 nm was performed with a 30 em x 3.9 mm ID 10-J..Lm J..LBondapak CIS column (Waters Assoc , Milford , Mass, USA) and a mobile phase of 0.005 M sodium acetate pH 5 buffer: methyl alcohol: acetonitrile: tetrahydrofuran (92.5:3:2.8:1.7, v/v). The assay was performed in duplicate or triplicate. Chemicals: Analytical grade caffeine, theophilline and theobromine, ultraviolet grade methyl alcohol, chloroform, isopropyl alcohol, tetrahydrofuran and acetonitrile (Wako Pure Chemical Industries , Osaka, Japan); paraxanthine and 7-[~-hydroxypropyl]theophylline (Sigma Chemical Co, St Louis, Mo , USA), 7[2-hydroxyethyl]theophylline (Tokyo Kasei Kogyo Co , Tokyo , Japan). 4. Statistical Analysis Mean significant difference was calculated according to the Student's t test. Results 1. Maternal and Fetal Factors The effect of four modes of maternal oral ad libitum ingestion on maternal and fetal factors in Exp 1 was almost the same as that described previously [3]. Mean maternal solid food intake during pregnancy for 21 days did not show a significant difference between the four groups; 459 g in WoW, 497 g in W-C, 467 g in CoW and 498 g in CoCo Mean maternal caffeine
intake with 0.04% caffeine solution in tap water also showed almost the same weight in two groups; 438 mg of caffeine in W-C and 457 mg of caffeine in C-C during pregnancy for 21 days. There was no significant difference between the four groups in maternal body weights on g d 0 and 21; 350 gin WoW, 409 g in W-C, 371 g in CoW and 367 g in C-C on g d 21. Maternal liver weight on g d 21 and litter size also did not show any significant difference between the four groups. Mean placental weight was 0.63 g in WoW and 0.82 g in CoW, but 0.49 g in W-C and 0.59 g in C-C, in which a significant difference was only observed between W-C and C-C (p < 0.05). Mean percent survival was 94% in WoW, 98% in CoW, 86% in W-C and only 30% in C-C, in which prolonged maternal administration of caffeine shows an adverse effect on the fetal survival rate. Fetal body weight after cesarean sectioning and fetal cerebral weights with two kinds of treatment after cesarean sectioning are shown in Table 1. In the present study but not in the previous study a significant difference was found in body weight, reduced weight being demonstrated in the three caffeine-treated groups; W-C, CoW and CoCo Mean fetal cerebral weight was reduced in caffeine-treated groups, especially in the W-C group, compared to the control WOW group.
2. Caffeine and Metabolite Levels in Mothers and Fetuses There was a good positive correlation between the concentration of caffeine or metabolites in
maternal plasma, maternal liver and placenta in three dams at cesarean sectioning. The mean concentrations or contents of caffeine, paraxanthine or theophylline in maternal liver, maternal plasma, placenta and fetal cerebrum with two modes of maternal oral ad libitum ingestion of caffeine during pregnancy in Exp 1 are shown in Table 2. As no essential or conclusive difference in distribution between the three dimethyxanthines was seen, the values for theobromine are omitted from Table 2. The concentration of caffeine or metabolites was almost the same in maternal liver, maternal plasma and placenta, however, a high mean concentration of the three materials was found in maternal liver followed by maternal plasma and placenta, in that order. The mean concentration of caffeine or metabolites in maternal liver or maternal plasma or placenta was always higher in W-C than in CoCo The mean concentration of caffeine in fetal cerebrum was increased but that of metabolites was not compared to the concentration of caffeine or metabolites in the placenta in both the W-C and C-C groups; the fetal cerebral value as a percentage of placental value was 113% and 119% for caffeine, 91 % and 92% for paraxanthine and 92% and 89% for theophylline in the W-C and C-C groups, respectively. The concentration or content of caffeine or metabolites in fetal cerebrum was all Significantly higher in the W-C group than in the C-C group. In Fig 1 the relationship between the concentration of caffeine or theophylline in fetal cerebrum and cerebral weight on g d 21 is
Table 1 Fetal body and cerebral weights on g d 21
WOW Body weight (g)
5.57
±
0.23 (13)
5.12
±
c-c
COW
W-C 0.33 (22)
4.85
±
0.58 (23)
5.07
II L - ****~*~**** ________________ I
Cerebral weight (mg) intact
146
± I
3
( 4)
ip injection
146
±
6
( 9)
I
0.70 (15)
I
-----.J
L ._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
±
~.
136 ± 9 ** ----.J
( 9)
132
±
15
(12)
137
±
12
( 6)
138
( 8)
144
±
8
(10)
140
±
13
( 8)
±
* ----.J
12
W: water, C: 0.04% caffeine, W or CoW or C: W or C before mating period-W or C during pregnancy. Data expressed as means ± SD (no of fetuses). Significant difference between groups; ****: p < 0.01, ***: p < 0.02, **: p < 0.05, *: p < 0.10.
Tanaka et al: Caffeine in fetal cerebrum 357
Table 2
Maternal and fetal caffeine or metabolite levels on g d 21 Maternal liver (lJ,g/g wet wt)
Maternal plasma (lJ,g/ml)
Caffeine W-C C-C
9.9 ± 2.9 [3] 4.4 ± 1.6 [3] **
9.5 ± 3.6 [3] 4.0 ± 1.7 [3] *
8.1 ± 3.4 [3] 3.2 ± 1.3 [3] *
8.23 ± 3.41 (6) 3.55 ± 1.05 (6)****
1.07 ± 0.41 (6) 0.47 ± 0.17(6)****
Paraxanthine W-C C-C
4.1 ± 0.9 [3] 2.2 ± 0.2 [3] **
3.6 ± 0.7 [3] 3.0 ± 0.6 [3]
3.0 ± 0.4[3] 2.1 ± 0.2 [3] **
2.66 ± 0.39 (6) 1.91 ± 0.23 (6)****
0.35 ± 0.04 (6) 0.25 ± 0.03 (6)****
3.6 ± 0.4 [3] 3.4 ± 0.9 [3]
2.7 ± 0.1 [3] 2.1 ± 0.2 [3] ***
2.44 ± 0.16 (6) 1.79 ± 0.15 (6)****
0.32 ± 0.02 (6) 0.24 ± 0.02(6)****
Theophylline W-C 3.9 ± 0.7 [3] C-C 3.4 ± 1.5 [3]
Placenta (lJ,g/g wet wt)
Fetal cerebrnm (lJ,g/g wet wt) (lJ,g/cerebrnm)
Data expressed as means ± SD [no of dams] or (no of fetuses). . Significant difference between W-C and C-C; ****: p < 0.01, ***: p < 0.02, **: p < 0.05, *: p < 0.10. For the fetal cerebrum assay, one to three fetuses were obtained from one dam.
12.0
• • •
10.0
'-' 3:
'-' Q)
3: 01
"'3' 5.0
•
Q)
0
c: Q) ~ ~
c
u
•
0
•
0 0
Q)
= c:
3.0
'TI~O '- - - - - - - '1~6:' 1-~~b~___ ;~ ._O~l~ O 00
o
:_O____O_O___
Cerebral weight (mg)
demonstrated in both the W-C and C-C groups. There was no clear correlation between the two factors in these two groups.
3. Radioactivity in Fetal Cerebrum The mean percent radioactivity in fetal cerebrum of the four groups 2 hours after intraperitoneal injection of [1-methyl- 14 C ]-caffeine is presented in Table 3. The recovery of radioactivity in the cerebral homogenate, in which caffeine and its metabolites were not identified, accounted for about only 1.7% of the intraperitonealy administered radioactivity of 14C_
358 Brain & Development, Vol 6, No 4, 1984
Fig 1 Relationship between caffeine or theophylline concentration in fetal cerebrnm and cerebral weight on g d 21. e: W-C group, 0: C-C group.
caffeine. The highest values of the radioactive concentration in the whole homogenate and acid-soluble fraction of fetal cerebrum were observed in the W-C group.
4. Caffeine Metabolism in Mothers A few parameters for maternal caffeine metabolism in plasma are presented in Table 4. As to the pharmacokinetic parameters of caffeine, the apparent volume of distribution was significantly decreased and elimination of caffeine increased in the pregnant rats given caffeine only during pregnancy (W-C). On the other
Discussion
Table j Radioactivity in fetal cerebrum on g d 21 after intraperitoneal injection of [[1-methyl-14 CJj-caffeine
WOw w-c cow C-C (6)
(7)
(9)
(8)
Dpm in whole homogenate 100% 118% 1l0% 103% /g wet wt Dpm in 0_3N PCA soluble fraction /g wet wt 100% 117 % 1l0% 102% Data expressed as mean percent of WoW value (no of fetuses) _
Table 4 Parameters for caffeine metabolism in maternal plasma on g d 18 after intravenous injection of caffeine
WOw w-c COw C-C [7}
Volume of distribution (ml/kg) Elimination rate constant
lQO%
[6}
[7}
[7}
~i~% llf%
100% 141%
83% 117%
Molar concentration ratio; theophylline to caffeine at 6 hrs 100% 175 %
83% 188%
(h
O ,)
I L..--J
at 8 Ius
L - - J L-.J
I
100% 223% 68% 195%
Il--l
L--.J L-.J
I
Data expressed as mean percent of WoW value [no of dams] _ Statistical difference between groups shown at p < 0_01 with bars_
hand, plasma molar concentration ratios of theophylline to caffeine at 6 or 8 hours after injection were significantly increased in the pregnant rats of both groups given caffeine during pregnancy (W-C and C-C)_ The molar concentration ratios of paraxanthine or theobromine to caffeine showed similar tendencies_ These results show that the apparent volume of distribution of caffeine in plasma was influenced by caffeine administered during not only the gestational period but also the premating period, however, the molar concentration ratio of dimethylxanthine to caffeine was essentially influenced only by caffeine administered during the gestational period_
It remains to be determined that how fetal and neonatal growth is influenced by the maternal oral ingestion mode for caffeine with regard to the consumed quantity and duration during the premating and gestational periods in rat or man_ The teratogenic potential of coffee or caffeine in rats fed via the oral route before mating and throughout gestation could only be demonstrated after administration of quantities of about 20 mg/kg/day by Palm et al [6]- They also demonstrated that the F 1 fetus from dams treated with coffee, 38 mg/kg/day, was similar in body weight but the mean fetal brain weight was significantly lower as a percentage of body weight compared to the controL On the other hand, the reduced growth potential of caffeine in rat offspring was shown by the administration of a low level of 10 mg/kg/day throughout sequential pregnancies and lactations by Dunlop et al [7] , who also demonstrated a progressive reduction in birth weight and brain weight of offspring of sequential pregnancy 4 without accompanying teratogenic effects_ In our previous study, a reduction in mean fetal cerebral weight without reduction in body weight was induced by maternal caffeine ad libitum ingestion during the premating period for 47125 days and throughout pregnancy [3]- However, in our present study, maternal caffeine ingestion during a further prolonged premating period of 134 days and throughout pregnancy produced reduction in both body and cerebral weight. Our pregnant rats received a daily dose of about 57 mg/kg of caffeine when calculated without consideration of loss at drinking _ Based on these results, it may be suggested that relatively high daily oral doses throughout pregnancy are required to induce the teratogenic effect or one part of the inhibitory effect on fetal cerebral growth, and prolonged, relatively low daily oral doses with or without administration during pregnancy have an inhibitory effect on fetal body growth_ In other words, there may be some cumulative effect in caf, feine-treated mothers on the reduction in fetal body growth, but a relatively high acute effect during pregnancy on the reduction in fetal cerebral growth_ In human studies, a positive association between the daily consumption of eight or more cups of coffee and congenital anomalies [8] or that of five or more cups of
Tanaka et al: Caffeine in fetal cerebrum 359
coffee and small-for-date babies [9] coincides with the results of our studies on rat. The possible nature of the reduction in fetal body weight by caffeine may be on either placental growth or on fetal growth directly . In our previous and present studies, caffeine-exposure throughout pregnancy, especially in the W-C group , has been found to affect the placental growth to some extent. However , there was no clear correlation between the reduction in fetal body weight demonstrated on prolonged administration and the reduction in placental weight demonstrated on acute administration during pregnancy in the present study. The inhibitory effect of maternal caffeine on fetal growth may be accounted for in part by the inhibition of growth hormone and thyrotropin secretion [10] after further study. There have only been a few studies on association between fetal abnormalities and caffeine or metabolite levels with different maternal administration modes of caffeine. As N-demethylation is the main pathway of in vitro caffeine metabolism in the rat liver at all ages [11] , maternal and fetal caffeine or metabolite levels were determined to elucidate the factors for fetal cerebral development. The concentration of caffeine or metabolites showed similar values in mothers and fetuses in our study. This good placental transfer in rat coincides with the finding of the passage of caffeine into human gonadal and fetal tissue [12] or the passage of caffeine and many metabolites into the mouse embryo [13]. Although the caffeine dose ingested ad libitum by mothers was not different between W-C and C-C during pregnancy , the mean value of caffeine and metabolites of mothers and fetuses was always higher in W-C than C-C in our study, which stresses the importance of the caffeine ingestion mode of mothers. As to caffeine and the fetal cerebrum, our study showed some evidence that the level of caffeine was higher in fetuses than in mothers but the metabolites were not , and that the fetal cerebral concentration might be highest in the W-C group compared to the other three groups was shown by an isotopic study . It is important to clarify for the fetal development, why the fetal cerebral caffeine level was increased in the W-C group compared to the C-C group. The developmental pattern of caffeine metabolism demonstrates that the liver
360 Brain & Development, Vol 6, No 4, 1984
of the newborn has an extremely limited capacity to metabolize caffeine [11, 14] , and that the adult metabolite pattern was attained by weaning in rat [II] and by 7 to 9 months in man [14]. It is also known that the metabolism of theophylline to caffeine occurs in the human fetus and newborn infant [15]. Based on the above mentioned results on fetal caffeine metabolism , the caffeine levels may increase slightly but significantly in the fetus by accumulation. This caffeine accumulation may potentially produce a toxic effect if the mother has toxic caffeine plasma concentrations. However , this metabolical specificity in the fetus could not explain the difference in fetal cerebrum between W-C and C-C. How may the different concentration of fetal cerebral caffeine between W-C and C-C be explained by the maternal caffeine metabolism or kinetics? In our study , the molar concentration ratio of theophylline to caffeine at 6 and 8 hours was increased in both W-C and C-C to similar levels. This result may be explained by the stimulatory effect of caffeine administered twice daily for only 3 days on drug metabolizing activities which lasted for only 24 hours after the last dose in male mature rat [16, 17]. According to the dog study by Axelrod et al [18], caffeine is distributed in tissues in approximate proportion to their water content. From this view point , the much decreased apparent volume of distribution of caffeine in the W-C group compared to the other three groups is reasonable. The low value of the volume of distribution in maternal plasma may result in the high concentration of caffeine in mother and fetus , although explanation of this phenomenon must await further experiments. Recently there have been several studies on the effect of pregnancy on pharmacokinetics of caffeine for estimating the caffeine effect on the fetus in man and animals [l , 19-21]. Delayed elimination of caffeine in pregnant women [1, 19] or a greatly increased half-life of elimination of caffeine [20] could produce the side effects of coffee in pregnancy at a lower rate of consumption than in the nonpregnant state. These findings may prompt further study to assess the pharmacokinetic changes of caffeine in pregnant women and fetus or neonate and the possibility of its adverse effect.
Acknowledgments This study was partly supported by Grant No 83-05 from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan.
10.
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