PEDRO ROSSO ANDEDWARD NORKUS Institute of Human Nutrition, College of Physicians and Surgeons, Columbia Presbyterian Medical Center, 630 West 168th Street, New York, New York 10032 ABSTRACT Transfer of ascorbic acid and/or its derivatives from maternal blood into the fetus was studied during the last week of gestation in the rat. Rats were injected intravenously with [l-14C]-ascorbic acid and the rate of transfer estimated by the concentration and content of label present in placentas and fetuses. At all times studied the concentration of label in the placenta was greater than in the fetus. The highest capacity of the placenta to concentrate label was found on day 15 decreasing to a low at day 19 and again increasing up to day 21. While in the fetuses, the con centration of label per gram of tissue remained remarkably constant throughout the study. The quantity of labeled compounds transferred into the fetus per gram of placental tissue increased between day 15 and day 21 of gestation. J. Nutr. 106: 767-770, 1976. INDEXING KEY WORDS placental transfer
prenatal
Beginning with the observations by Wahren and Rundquist (1) and Braestrup (2), numerous authors have demonstrated that plasma levels of ascorbic acid in humans are higher in the fetus than in the mother. A similar finding has been reported by Raiha in guinea pigs (3). These in creased fetal plasma levels have been ex plained as due to: fetal synthesis of the vitamin (4); different rates of utilization between fetus (5);within and, thea "selective mother retention"andmechanism placenta (6). The work done by Raiha in guinea pigs (3), while unable to elucidate the mechanisms that maintain the fetalmaternal gradient, indicated that only the oxidized form of ascorbic acid (dehydroascorbic acid) readily crosses the placental barrier. Our current understanding of placental transport mechanisms would suggest that the existence of the fetal-maternal gradient probably reflects an active transfer mech anism for ascorbic acid. However, data directly verifying such active transfer are not yet available. Further, data dealing
ascorbic
acid metabolism
with the rate of transfer of ascorbic acid across the placenta at different times throughout gestation are also unavailable. It is known that ascorbic acid concen tration is highest in tissues with a high rate of metabolism (7, 8, 9). It seemed possible then, that the fetal ascorbic acid requirement could change as both the fetal growth rate and metabolic rate changes, and that this change in fetal requirement could be paralleled by changes in placental transfer. For these reasons we have studied the pattern of ascorbic acid transfer at dif ferent times during normal gestation in the rat. Although the rat is able to synthesize ascorbic acid, the enzymes necessary for this synthesis are not present in the fetus before day 20 of gestation and adult levels are not attained until the second week of postnatal life (10). Thus, the fetus is de pendent on placental transfer of ascorbic acid to meet its requirements. Received for publication July 28, 1975. 1 This work has been partially supported by grants from Hoffman Lu-Uoche Co., Nutley, N.J. and the Nutrition Foundation. 767
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Prenatal Aspects oÃ-Ascorbic Acid Metabolism in the Albino Rat1
768
PEDRO ROSSO AND EDWARD NORKUS METHODS
2 5,0001— T
20,000
£
15,000
a. o Plasma I 0,000
a. o
Placenta 5,000 Fetus
I 10
I 20
I 30
TIME
40
50
60
(min.)
Fig. 1 The concentration of label in maternal plasma, (cpm/ml) placenta and fetus (cpm/g) following injection of [1-"C] ascorbic acid into the maternal circulation. Values represent the mean ±SE of samples taken from four animals at day 20 of gestation.
Samples of maternal blood, one placenta and one fetus were removed from each ani mal immediately prior to the injection of the radioactive compound to be used as zero time samples. Samples were subse quently obtained at 10, 20, 40 and 60 min utes after injection. Only those animals having a minimum of seven fetuses were included in the study. Fetuses and placen tas were kept at 0 to 4°until they could be weighed and subsequently frozen. Sam ples were subsequently thawed out and whole fetuses and placentas were homog enized 1:4 (w/v) in distilled water. Radio activity was determined in 0.5 ml aliquots of the homogenates following the proce dure reported previously (11). RESULTS As shown in figure 1, the concentration of label from ascorbic acid in maternal plasma decreased with time. This clearance probably reflects redistribution of this sub stance into other maternal compartments, maternal metabolism and excretion and transfer of ascorbic acid to the fetus. A greater concentration of labeled ascorbic acid was present in the placenta than in the fetus throughout the experiment. Fetal concentration increased steadily with time. By contrast placental concentration of label did not show a well defined pattern, al though, with the exception of the values from day 17 the concentration of label at 60 minutes tended to be lower than those at 10 minutes. The concentration of label from ascorbic acid in fetal tissues remained remarkably constant from day 15 through day 21 of gestation (fig. 2), indicating that the same proportional amount of ascorbic acid was being transferred to the fetus throughout pregnancy. The placental concentration of label on the other hand tended to decrease be tween days 15 and 19 and increased sig nificantly between days 19 and 21 of pregnancy. As shown in figure 3, the amount of label 1Camm Research Institute, Wayne, N.J. 8Since In the rat ascorbic acid Is rapidly metab olized any reference to ascorbic acid transfer made In the text Implies transfer of label reflecting ascorbic acid and/or Its metabolic derivatives. 4Urethane, Sigma Chemical Company, St. Louis, Mo. "New England Nuclear, Boston, Mass.
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/767/4768838 by Tulane University Medical Library user on 19 January 2019
Sprague-Dawley rats (Holtzman strain) fed, ad libitum, a commercial laboratory diet,2 caged individually and maintained under constant temperature and 12 hour alternating, light-dark periods were used for the study. The animals were mated overnight and the presence of spermatozoa in the vaginal smear determined their in clusion in the study. This time was desig nated day one of pregnancy. Transfer of ascorbic acid3 was mea sured by a method similar to the one described previously for glucose and amino acids (11). On days 15, 17, 19, 20 and 21 of pregnancy, animals were chosen for study. The rats were anesthetized by sub cutaneous injection of ethyl carbamate * (200 mg/100 g body weight) followed by administration of 1 /^Ci/100 g body weight of L-[l-14C]ascorbic acid (3-4 mCi/ mmole).5 The ascorbic acid was dissolved in physiological saline immediately before injection into the maternal femoral vein.
7(59
PRENATAL ASCORBIC ACID METABOLISM 20,000l-
IL O
10,000
Placenta
5,000
»Fetus I 15 16 17 18 19 20 21 GESTATION AL AGE (days)
Fig. 2 The concentration of label (cpm/g) in fetus and placenta 10 minutes after injection (I.V.) of [l-uC]ascorbic acid into the mother at different gestational ages. Values represent mean ± SE from four animals. Differences between mean values of the placenta are significant only between days 19 and 21 (P
prenatal
Beginning with the observations by Wahren and Rundquist (1) and Braestrup (2), numerous authors have demonstrated that plasma levels of ascorbic acid in humans are higher in the fetus than in the mother. A similar finding has been reported by Raiha in guinea pigs (3). These in creased fetal plasma levels have been ex plained as due to: fetal synthesis of the vitamin (4); different rates of utilization between fetus (5);within and, thea "selective mother retention"andmechanism placenta (6). The work done by Raiha in guinea pigs (3), while unable to elucidate the mechanisms that maintain the fetalmaternal gradient, indicated that only the oxidized form of ascorbic acid (dehydroascorbic acid) readily crosses the placental barrier. Our current understanding of placental transport mechanisms would suggest that the existence of the fetal-maternal gradient probably reflects an active transfer mech anism for ascorbic acid. However, data directly verifying such active transfer are not yet available. Further, data dealing
ascorbic
acid metabolism
with the rate of transfer of ascorbic acid across the placenta at different times throughout gestation are also unavailable. It is known that ascorbic acid concen tration is highest in tissues with a high rate of metabolism (7, 8, 9). It seemed possible then, that the fetal ascorbic acid requirement could change as both the fetal growth rate and metabolic rate changes, and that this change in fetal requirement could be paralleled by changes in placental transfer. For these reasons we have studied the pattern of ascorbic acid transfer at dif ferent times during normal gestation in the rat. Although the rat is able to synthesize ascorbic acid, the enzymes necessary for this synthesis are not present in the fetus before day 20 of gestation and adult levels are not attained until the second week of postnatal life (10). Thus, the fetus is de pendent on placental transfer of ascorbic acid to meet its requirements. Received for publication July 28, 1975. 1 This work has been partially supported by grants from Hoffman Lu-Uoche Co., Nutley, N.J. and the Nutrition Foundation. 767
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/767/4768838 by Tulane University Medical Library user on 19 January 2019
Prenatal Aspects oÃ-Ascorbic Acid Metabolism in the Albino Rat1
768
PEDRO ROSSO AND EDWARD NORKUS METHODS
2 5,0001— T
20,000
£
15,000
a. o Plasma I 0,000
a. o
Placenta 5,000 Fetus
I 10
I 20
I 30
TIME
40
50
60
(min.)
Fig. 1 The concentration of label in maternal plasma, (cpm/ml) placenta and fetus (cpm/g) following injection of [1-"C] ascorbic acid into the maternal circulation. Values represent the mean ±SE of samples taken from four animals at day 20 of gestation.
Samples of maternal blood, one placenta and one fetus were removed from each ani mal immediately prior to the injection of the radioactive compound to be used as zero time samples. Samples were subse quently obtained at 10, 20, 40 and 60 min utes after injection. Only those animals having a minimum of seven fetuses were included in the study. Fetuses and placen tas were kept at 0 to 4°until they could be weighed and subsequently frozen. Sam ples were subsequently thawed out and whole fetuses and placentas were homog enized 1:4 (w/v) in distilled water. Radio activity was determined in 0.5 ml aliquots of the homogenates following the proce dure reported previously (11). RESULTS As shown in figure 1, the concentration of label from ascorbic acid in maternal plasma decreased with time. This clearance probably reflects redistribution of this sub stance into other maternal compartments, maternal metabolism and excretion and transfer of ascorbic acid to the fetus. A greater concentration of labeled ascorbic acid was present in the placenta than in the fetus throughout the experiment. Fetal concentration increased steadily with time. By contrast placental concentration of label did not show a well defined pattern, al though, with the exception of the values from day 17 the concentration of label at 60 minutes tended to be lower than those at 10 minutes. The concentration of label from ascorbic acid in fetal tissues remained remarkably constant from day 15 through day 21 of gestation (fig. 2), indicating that the same proportional amount of ascorbic acid was being transferred to the fetus throughout pregnancy. The placental concentration of label on the other hand tended to decrease be tween days 15 and 19 and increased sig nificantly between days 19 and 21 of pregnancy. As shown in figure 3, the amount of label 1Camm Research Institute, Wayne, N.J. 8Since In the rat ascorbic acid Is rapidly metab olized any reference to ascorbic acid transfer made In the text Implies transfer of label reflecting ascorbic acid and/or Its metabolic derivatives. 4Urethane, Sigma Chemical Company, St. Louis, Mo. "New England Nuclear, Boston, Mass.
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/767/4768838 by Tulane University Medical Library user on 19 January 2019
Sprague-Dawley rats (Holtzman strain) fed, ad libitum, a commercial laboratory diet,2 caged individually and maintained under constant temperature and 12 hour alternating, light-dark periods were used for the study. The animals were mated overnight and the presence of spermatozoa in the vaginal smear determined their in clusion in the study. This time was desig nated day one of pregnancy. Transfer of ascorbic acid3 was mea sured by a method similar to the one described previously for glucose and amino acids (11). On days 15, 17, 19, 20 and 21 of pregnancy, animals were chosen for study. The rats were anesthetized by sub cutaneous injection of ethyl carbamate * (200 mg/100 g body weight) followed by administration of 1 /^Ci/100 g body weight of L-[l-14C]ascorbic acid (3-4 mCi/ mmole).5 The ascorbic acid was dissolved in physiological saline immediately before injection into the maternal femoral vein.
7(59
PRENATAL ASCORBIC ACID METABOLISM 20,000l-
IL O
10,000
Placenta
5,000
»Fetus I 15 16 17 18 19 20 21 GESTATION AL AGE (days)
Fig. 2 The concentration of label (cpm/g) in fetus and placenta 10 minutes after injection (I.V.) of [l-uC]ascorbic acid into the mother at different gestational ages. Values represent mean ± SE from four animals. Differences between mean values of the placenta are significant only between days 19 and 21 (P
