Pflfigers Arch. 356, 267--275 (1975) 9 by Springer-Verlag 1975
The Transport of D-Glucose, T,-Glucose and D-Mannose across the Isolated Guinea Pig Placenta* ** ]~. Schr5der, H.-P. Leichtwcil~, and W. Madee Abteilung fiir experimentelle Medizin der Universit~ts-Frauenklinik und -Poliklinik Hamburg, Germany Received December 27, 1974 Summary. Studies were carried out on the transport of all-D-glucose, laC-Lglucose and aH-D-mannose across the isolated artificially perfnsed guinea pig placenta and the inhibition of these transports by phloretin was investigated. 1. In 10 out of 13 placentas the transfer of D-glucose exceeds that of L-glucose. 2. In 10 out of 13 placentas the transfer of ])-mannose exceeds that of T.-glucose. 3. Phloretin (1 • 10-a M/l) decreases the transport of ])-glucose, and of ])mannose, it does not decrease the transport of L-glucose. The inhibition is not complete. The characterization of the washing-out curves permits the assumption that some placentas (6 out of 21) cannot discriminate between D- and L-hexoses, because a non specific transfer (simple diffusion) covers the specific one. Key words: Placental Transport -- D-Glucose -- ])-Mannose -- T.-Glucose Phloretin -- Washing-Out Curves. -
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Chinard et al. (1955) ascertained that glucose is transferred from the maternal to the fetal side and vice versa of the rhesus monkey placenta corresponding to the arterial difference of glucose concentration. Leiehtweiss and SehrSder (1971) could demonstrate, that the maternofetal and the fetomaternal transport rates are equal in the hemochorial placenta of the guinea pig. From experiments performed b y Hugger et al. (1951) on the sheep placenta, Widdas (1952) concluded, that the transfer of glucose across this epitheliochorial placenta cannot be explained sufficiently b y simple diffusion and took into consideration the possibility of a glucose carrier. I n 1973 Young found a limitation of the glucose transfer in spite of increasing maternal blood glucose concentration in the guinea pig placenta. Similar results, with some evidence for an uphill transport by eounterflow for glueose/galactose formerly were presented b y Ely (1966). The above findings are hints for a specific mechanism of transport of sugars, especially of hexoses, across the placenta. As far as we know, no * Supported by the Deutsche Forsehungsgemeinsehaft. ** We are thankfully indebted to Bayer for Bay a 1040 (Nifedipin).
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direct i n v e s t i g a t i o n s considering a (stereo-)specifie t r a n s p o r t system for hexoses h a v e b e e n carried out. This p a p e r therefore presents e x p e r i m e n t s o n t h e m a t e r n o f e t a l t r a n s f e r of D-glucose, L-glucose a n d D-mannose across t h e fully isolated g u i n e a pig p l a c e n t a .
Methods The placentas of guinea pigs at the end of pregnancy are fully isolated and artificially perfnsed on both sides according to previous publications (Leichtweiss and SchrSder, 1971, 1972). The original technique is changed so far as the complete preparation procedure is performed in Ringer's solution of 37~C. Perfusion is mainrained at a constant flow rate by means of piston pumps (modified Perfusor | Braun), inflow pressures range between 10 and 30 mmHg (measured in an a. umbilicalis) at a flow rate of ca. 1 ml • min-1 • g-1. The perfnsion medium is saturated with 02 and heated up to 37~C. I t consists of: NaC1 140, KC15.0, CaC120.5, NaHCO8 5.0, Na-acetate 5.0, Na-malate 0.1, guanosine 0.25, sodium-2oxy-glutarate 0.05 (mMfl) and albumin 1 g/1. The maternal solution contains 5 mlK/1D-glucose; pH is adjusted to 7.4 by 1 N HC1 ( ~ 0.1 raM/l) after oxygen addition. I n experiments concerning the effect of phloretin pluronic F 108| (Wyoming Corp.) is added (2 g/1 in experiments No. 17--21). Venous outflow is measured by electromagnetic flowmeters (Liepelt, Hamburg) or by drop recorders (SchrSder, 1974). Placentas that show different venous outflow rates are refused. During ~he artificial perfusion the isolated placenta lies in a bath of Ringer's solution (37~C). Contractions of the uterine nmscles that may lead to separation of the placenta are suppressed by admixture of l~ffedipin (Bayer) to the bath (0.2 rag/ 100 ml). In the course of each experiment venous samples are taken simultaneouslyfrom the maternal and fetal side at intervals of 2 rain; the flow rates are constant. In 5 experiments phloretin is added after 20 rain in its most efficient concentration of 1 • 10-a IVI/1, as found in 3 preliminary experiments. I t is the highest concentration attainable in the perfusionmedium in the presence of pluronic (2 g/l), too. I n experiments No. 17--21 D-glucose is measured enzymatically by the hexokinase reaction. I n some experiments washout curves are obtained as follows: the maternal inflow and outflow is stopped and 10 samples of equal volume are gathered at 25 sec intervals at the fetal outflow. Preparation time normally lasts 45 rain, samples are taken for additional 25--40 rain. The labelled hexoses (Amersham Buchler, Braunschweig) are added solely to the perfusion medium on the maternal side. Simultaneously are offered: 8H-Dglucose and laC-L-glucose (8 experiments) and 3H-D-mannose and l~C-L-glucose (13 experiments). The activity of two aliquots of the samples (0.2 ml into 10 ml Instagel| Packard Instruments) is measured in a Triearb scintillation counter (Packard Instruments, model 3380) in standard channels for 8H and 1~C. Conversion to 3H- and laC-aetivity in a sample is performed according to Vetter and Veall. We have verified the computation using samples with known contents of either isotope. The rate of transfer is calculated as the fraction (in percent) fetal venous activity: maternal arterial activity. Calculation of the balance shows that the amount of hexose delivered by the maternal side equals the amount of the same sugar received at the fetal side of the placenta.
PlacentM Transfer of ])-Glucose, L-Glucose and ])-Mannose
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To analyze a curve of washout its ten points are interpolated logarithmically and the data points obtained are punched (MincM 621, Dietz). By means of a computer programm (Dell et a/., 1973) the exponential functions are calculated that are necessary to describe the observed curve of washout (IBM 370, computation center of DESY, Hamburg).
Results Tables I a n d 2 summarize the results of 21 experiments. The rates of transfer for D-glucose, D-mannose a n d L-glucose are declared as the m e a n o f n samples ~= s t a n d a r d deviation. The transfer rate is calculated as the percentage of the fetal venous concentration (or counts/rain) of the m a t e r n a l arterial concentration of the respective hexose. Since flow rates on the fetal a n d maternal side of the placenta are the same and constant (3.2 ml/min) the ratio m a y be regarded as the transfer rate in relation to the m a t e r n a l load (transfer in ~ of load). Table 1 renders 16 experiments concerning the simulataneously measured transfer of ])-glucose and L-glucose (experiments 1--8) and of ])-mannose a n d L-glucose (experiments 9--16). I t is obvious, t h a t in m o s t experiments ])-glucose a n d ])-mannose cross the placental membranes m u c h easier t h a n L-glucose. The criteria for a "discriminating" placenta ~re:
a) a significant difference between the transfer rates for ] ) - a n d L-hexoses (P ~ 0.05, S t u d e n t ' s t-test) b) This difference should be greater t h a n 10~ Thus the following 6 placentas do n o t discriminate: placentas No. 6 a n d 7 show no difference between ])- a n d L-glucose, so does No. 15 between ])-mannose and Lglucose; l~o. 8, 13 a n d 14 show only small differences between the transfer rates of the respective hexoses. Table 1. Transfer rates (percent of load), means of n samples -~ SD. No: number of experiments. Load (all-D-glucose, 14C-L-glucose, 3H-D-mannose): activity (counts/min) • rain-~ No. I)-glucose
n
L-glucose
No.
D-mannose
n
L-glucose
1. 2. 3. 4. 5. 6. 7. 8.
(9) (10) (5) (5) (5) (8) (10) (15)
19.2 4- 0.96 11.9 4- 0.7 14.4 ~ 0.6 30.3 :E 0.4 36.3 ~ 0.73 54.1 ~: 2.4 62.3 4- 9.1 54.4 • 2.5
9. 10. 11. 12. 13. 14. 15. 16.
41.2 4- 1.3 32.9 ~: 2.0 45.4 ~: 2.3 35.9 4- 1.0 21.4 4- 5.9 5.3 4- 1.3 66.2 • 4.8 50.6 -V 2.5
(10) (9) (10) (8) (10) (10) (10) (7)
5.9 ~: 0.6 9.9 4- 0.7 13.3 ~: 0.9 2.0 ~: 0.6 17.3 ~: 4.4 1.2 4- 0.1 66.9 ~ 5.7 6.1 ~: 2.1
43.5 ~- 3.6 60.8 • 2.8 52.3 -V 1.5 58.8 4- 0.7 50.6 4- 1.1 54.7 4- 2.4 62.3 4- 3.0 62.7 4- 2.9
In brackets: number of samples.
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Placental Transfer of D-Glucose,L-Glucose and D-Mannose
271
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oad
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Fig.1. Experimen~ No.18. Ordinate: concentration (percen~ of load) of ])-glucose, D-mannose and L-glucose in fei~al perfusate. Abscissa:! perfusion ~ime
Table 2 lists the results of 5 experiments that were carried out for studying the transfer of ])-glucose, 3H-D-mannose and 14C-L-glucose simultaneously before (a) and after (b) addition of phloretin to the perfusion medium (1 • 10-3 M/l). I n these experiments D-glucose concentration is measured enzymatically. All placentas discriminate between the D- and L-hexoses. Phloretin inhibits the transport of D-glucose and D-mannose, whereas the passage of L-glucose increases slightly. The effect of phloretin is small in No. 19. In this placenta the ability for discrimination between I)- and L-sugars is weak, too, the permeability for L-glucose is comparatively high. Fig. 1 illustrates the course of a typical experiment (No. 18). The transfer rates (as defined above) are plotted against the perfusion time. After addition of phloretin (1 • -3 M/I; arrow) the transport of the ])-sugars decreases rapidly, whereas the transfer of L-glucose increases slightly, and the maximum concentration of phloretin does not evoke complete inhibition as compared with the L-glucose transfer, which is still smaller. Fig. 2 shows curves of washing out of D-mannose and L-glucose of the placentas No. 9--11, which do and of No. 13 and t5, which do not discriminate between both sugars. The activity for either isotope of a sample is plotted as the fraction of the first sample on a semilogarithmie scale.
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,
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Fig. 2. Washout-euzves of D-maz~ose and L-glucose. Abscissa: time (see). Ordinate:
amount of marmose resp. glucose in a sample as the fraction of the first sample. Symbols for L-glueose: ~, D, .; for D-mannose: x, =, o; Straight lines (broken: D-mannose) represent the two exponential functions whose sum fits the washoutcurves best (mean of 3 (top) resp. 2 (bottom) experiments). Top: placentas discriminating between D-mannose and L-glucose (No.9,10,11). The following pairs of symbols belong to one experiment: Ax, ==, . o . K 1 and K s are the respective rate constants of the fast and slow component. Bottom: two placentas with the same transfer-rate for D-mannose and n-glucose (No.13,15). Pairs of symbols belonging to one experiment: Ax, .o The straight lines shown represent the mean of the exponential functions, the sum of which fits the observed curves best. The curves differ especially in their second, slow component. The curves of washing-out of D/Lglucose are similar to the curves shown here.
Discussion The results show that D-glucose is transferred across the hemochorial placenta of the guinea pig much easier than L-glucose. The transport of D-mannose exceeds the L-glucose transfer as well. The findings "agree with those reported b y Folkart e t a l . (1960), who ascertained a higher disappearance rate of D-xylose than of L-xylose out of the fetal perfusion
Placental Transfer of ])-Glucose, T.-Glucoseand D-Mannose
273
medium during artificial perfusion of the fetal side of the guinea pig placenta in situ. I t is not sufficiently ruled out, whether D-glucose and D-mannose make use of a common transport system. The differences between the D-hexoses and L-glucose seem to be ahke (Table 1), but Table 2 illustrates that the transfer rate of D-glucose is greater than that of D-mannose. Different transfer rates do not necessarily mean that different transport systems exist. The difference may be due to a competitive inhibition of D-mannose transfer b y D-glucose, because in our experiments the concentration of D-glucose (5 mM/1) exceeded that of D-mannose (0.2 ~ / 1 ) . The results concerning the effect of phloretin are in favour for a common transport system. Phloretin (1 • 10-S M/l) decreases the transfer rates of D-glucose and Dmannose (cf. Table 2), whereas the transport of L-glucose has a tendency to increase. (After reaching a maximum of inhibition b y phloretin the transfer of D-hexoses increases slightly again. The increase is a parallel of t h a t of L-glucose. This effect is independent of phloretin; it m a y be due to pluronic, perfusion time or other unknown factors. That is why in Table 2 only 5 samples after addition of phloretin are taken into account). The inhibiting effect of phloretin on the carrier mediated transport of D-monosaecharides across the membranes of ery6hrocy~es is well known (Rosenberg and Willbrand, 1957; Bower and Widdas, 1958; Le Fevre, 1961). The remaining difference between D-hexoses and L-glucose after addition of phloretin (Table 2) demonstrates that the inhibition of transfer is not complete. In the experiments of Ely (1966) phloretin had no effect on the transport of glucose in the guinea pig placenta. This may be due to the low concentrations of phloretin in the perfusion medium or to a very high unspecific diffusion of D-glucose. Considering the time course of L-glucose diffusion as mentioned above it is possible that an inhibition of the transfer remains undetected because of the increase of simple diffusion of D-glucose at the same time. We do not know, why some of the guinea pig placentas show a very high diffusion rate for L-glucose (cf. experiments 4--8, 15, 19), but the fact has to be taken into consideration and may explain, that in former experiments we did not reach a limitation of glucose transfer at great arterial concentration differences (Leiehtweiss et al., 1974) as Krauer et al. did (1973). Our investigations on the effect of phloretin on the transfer of Dhexoses and L-glucose make it probable that the absent or weak ability of 19 Pflfigers Arch., Vol. 856
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discriminating between I)- a n d L-hexoses is due to an increased unspecific diffusion. A n o t h e r s u p p o r t for this opinion is discussed below. As in spite of a great permeability o f the placental m e m b r a n e s the absolute rate of transfer m a y be low according to a small area of exchange (of. Table 1, experiment No. 14), the a m o u n t of transfer c a n n o t settle the question whether the failure of discrimination between D- a n d L-hexoses is due to a great (additional diffusion) or a small (loss o f a carrier system) permeability. Thus the washing-out curves m a y give some additional information w h y some placentas did n o t discriminate. Comparing the curves of placentas with a n d w i t h o u t the ability for discrimination it is obvious t h a t the curves of the latter are steeper. The analysis shows t h a t the curves consist of two c o m p o n e n t s a n d t h a t the different characterization can be referred to differences of the slower component. The ratio of the rate constants K (Fig.2) of the fast c o m p o n e n t equals unity, the rate constants of the slower p a r t are related as 1 : 2 : 3 (T,-glucose: K = - - 0 . 1 2 rain-l; D-mannose: K = - - 0 . 2 7 rain-l; n o n discriminating placentas : K = -- 0.38 rain-l). The fast c o m p o n e n t m a y indicate the velocity of w a s h - o u t (that is the flow rate), therefore the fast p a r t in all curves is almost identical. The second, slower component, however, m a y be d e t e r m i n e d b y the permeability o f the membrane. Thus a greater rate c o n s t a n t would indicate a higher permeability.
Rr Bower, F., Widdas, W. F. : The action of inhibitors on the facilitated hexose transfer system h erythrocytes. J. Physiol. (Lend.) 141, 219--232 (1958) Chinard, F.P., Danessiono, V., Hartma~n, W.L., Hugger, A. St. G., Paul, W., Reynolds, S. R.M.: The transmission of hexose across the placenta in the human and the rhesus monkey. J. Physiol. (Lond.) 182, 289--303 (1956) Dell, R. B., Seiacca, R., Liebermann, K., Case, D. B., Cannon, P. J.: A weighted least-squares technique for the analysis of kinetic data and its application to the study of renal 133 Xenon washout in dogs and man. Circular. Res. 83, 71 (1973) Ely, P. A.: The placental transfer of hexoses and polyols in the guinea pig, as shown by umbilical perfusion of the placenta. J. Physiol. (Lond.) 184, 255--271 (1966) Folkart, G. R., Dancis, J., Money, W. L.: Transfer of carbohydrates across guinea pig placenta. Amer. J. Obstet. Gynec. 80, 221--223 (1960) Hugger, A. St. G., Warren, F. L., Warren, 1~. V. : The origin of the blood fructose of the foetal sheep. J. Physiol. (Lond.) 118, 258--275 (1951) Krauer, 1~. J., Joyce, J., Young, 1VL: The influence of high maternal plasma glucose levels and maternal blood flow on the placental transfer of glucose in the guinea pig. Diabetologia 9, 453--456 (1973) Le l~evre, P. G. : Sugar transport in the red blood cell: structure activity relationship in substrates and antagonists. Pharmacol. Rev. 18, 39 (1961) LeichtweiB, H. P., SchrSder, H.: Untersuchung fiber den Glukosetransport durch die isolierte, beiderseits kiinstlich perfundierte !Vfeerschweinchenplazenta. Pfliigers Arch. 825, 139--148 (1971)
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LeichtweiB, H . P . , SchrSder, H., l~Iadee, W.: Inhibition and discrimination of transport of ])-glucose across the isolated guinea pig placenta. Pflfigers Arch. 847, 1%35 (1974) Rosenberg, T., Wilbrandt, W.: Strukturabh~ngigkeit der Hemmwirkung yon Phlorezin and anderen Phlorezinderivaten auf den Glucosetransport durch die Erythrozytenmcmbranen. Helv. physiol, pharmacol. Acta 15, 168--176 (1957) Schr5der, H. : An easy-to-build digital flow meter (drop-recorder). Pfliigers Arch. 850, 97 (1974) SehrSder, H., Stolp, W., Leichtweil3, H. P. : Measurement of Na+ transport in the isolated, artificially perfused guinea pig placenta. Amer. J. Obstet. Gynec. 114, 51 (1972) Vetter, H., Vecall, N.: Radioisotopen-Technik in der klinischen Forschung und Diagnostik. Miinchen-Berlin: Urban & Schwarzenberg 1960. Widdas, W. F. : L-lability of diffusion to account for placenta glucose transfer in the sheep and consideration of the kinetics of a possible carrier transfer. J. Physiol. (Lend.) 118, 23--39 (1952) Prof. Dr. reed. H.-P. Leichtweil~ W. Madee Dr. med. H. SchrSder Universif~ts-Frauenklinik und -Poliklinik Abteilung ffir experimentelle Medizin D-2000 Hamburg 20 Martinistr. 52 Federal Republic of Germany
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