Placenta(1991), 12, 277-284

Effects of Protein-calorie Malnutrition on Transplacental Kinetics of Aminoisobutyric Acid in Rats D. R. VARMA”” & R. RAMAKRISHNANb a Department of Pharmacologyand Therapeutics, and Nutrition and Food Science Center, McGill University,Montreal, Quebec, Canada H3G IY6 and b Department of Pediatrics and Medicine, Collegeof Physicians5 Surgeons, Columbia Universip, New York, NY 10032, USA ’ To whom correspondenceshould be addressedat: Department oj Pharmacology, McGill University, .?655 Drummond Street, Montreal, Que. Canada H3G I Y6 Paper accepted14.2.1991

SUMMARY In order tojnd out finefici~t tratispoti of amino acids contributes to a &crease in fetal weight during maternal malnutrition, we injected [t’C]- and [3H]-labelled aminoisobutyric acid (AIB), respectively, in the mother and itsfetuses and determined its transplacental kinetics on day 20 ofgestation in rats fed a 21 per cent (control) or a low (5per cent) protein diet. Rats fed a lowprotein diet consumedsign$cantly lessfood than did the rats fed a control diet and thus suffeedfrom protein-calorie malnuttition. A lowprotein diet led to a signaficant (P < 0.05) decrease in maternal andfetal volume of distribution ofAIB, a decrease in the clearance ofAIB from the mother to the jetus and an increase in the time required for the fetal plasma AIB concentration following maternal injection to exceed the maternal plasma AIB concentration. The clearance ofAIB from thefetus into the mother or to outside (e.g. amnioticfluid) was not altered byprotein deficiency. It is concluded that a decrease in the eficiency of the placenta to &liver amino acids to thefetus may be a contributingfactor infetalgrowth retardation during maternal protein malnutrition.

INTRODUCTION Association of maternal malnutrition with fetal growth retardation has been observed both in humans (Widdowson, 1977; Woods et al, 1979; Raman, 1981) and experimental animals (Young and Widdowson, 1975; Mulay, Varma and Soloman, 1982; Varma and Yue, 1983). Although a relative lack of nutrients essential for tissue building could be one reason, other changes associated with malnutrition, especially those in the placenta, may contribute to fetal 0143-4004/91/030277

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Placenta(1991), Vol. I.2

retardation. Malnutrition causes a decrease in placental size (Lechtig et al, 1975; Pivalizza et al, 1990) and alters its functions (Mulay et al, 1980). Many essential nutrients including amino acids are actively transported across the placenta from the maternal to the fetal side (Christensen, 1973; Hill and Young, 1973). Protein-calorie malnutrition has been shown to alter the placental transfer of dexamethasone (Varma and Yue, 1984), possibly involving an active transport mechanism (Varma, 1986). However, the influence of maternal malnutrition on the transplacental kinetics of amino acids has not been fully investigated. A recent study examining the effect of diet restriction on placental transfer of methionine in monkeys was inconclusive (Berglund et al, 1989). The present study measured the transfer of aminoisobutyric acid (AIB), a non-utilizable amino acid, in protein-calorie deficient rats. For this purpose [14C]AIB was injected into the mother and [3H]AIB into its fetuses; concentrations of both labels in serial maternal and fetal plasma samples permitted calculations of transfer rates of AIB in both directions across the placenta as independent parameters. Data suggest that the transport of AIB from the maternal to fetal side is less efficient in proteincalorie malnourished than in control rats. growth

MATERIALS AND METHODS

Virgin Sprague-Dawley rats (Charles River, St. Constant, Quebec, Canada) weighing 200225 g were fed ad libitum tap water and a 21 per cent protein (control) diet (pellets) of the following composition (g/kg): vitamin-free casein 23 l(21 per cent available protein), sucrose 5 19, corn starch 150, corn oil 50, mineral mixture 40, vitamin mixture 10 (Teklad Test Diets, Madison, Wisconsin, USA). On day 0 of gestation (presence of sperms in vaginal washing), rats were randomly divided to receive until day 20 of gestation the same control diet or an isocaloric 5 per cent protein diet which differed from the control diet in so far it contained 55 g/kg casein and 695 g/kg sucrose (Mulay, Varma and Solomon, 1982; Varma and Yue, 1983; Varma and Yue, 1984). Pregnant rats were housed individually in suspended steel cages and food intake was recorded. The study did not include controls pair-fed to rats receiving a low protein diet. Transplacental kinetics of aminoisobutyric acid (AIB) was determined on day 20 of gestation. Determination of transplacental kinetics All surgical interventions including injections and blood sample collections were done under brief periods (2-3 min) of ether anesthesia as previously described in detail (Varma and Ramakrishnan, 1985). [14C]AIB (100 nmol/kg) was injected into the tail vein of the mother. The abdomen was opened and [3H]AIB was injected through the uterine wall into the peritoneal cavities of all its fetuses; the sum of [3H]AIB injected in equal amounts into all fetuses was 1 mnol/kg maternal body weight. The abdomen was sutured and animals were reanesthetized at different times thereafter for sequential collection of maternal blood (0.2 ml) from the tail artery. Immediately after the maternal blood collection, one to two fetuses were removed starting from one ovarian end and decapitated for the collection of the trunk blood. The incised end of the uterus was ligated and the abdomen was sutured. The procedure was repeated at each sample collection time. We have previously shown that the plasma concentration of antipyrine in different fetuses at any given time did not differ, the concentration in fetal plasma was not altered by serial blood collections and rats resumed

C’anna,Ramakrishnan: Tramplacental Kinetics ofAIB

279

body weight gain if they were not sacrificed at the end ofthe last sample collection (Varma and Ramakrishnan, 1985). In the present series of experiments also blood samples were collected only once from a small number of control and protein-deficient rats at 1, 2, or 6 h after injections (one to two rats at each time-period); maternal and fetal plasma concentrations of AIB determined by this protocol were within range found when serial blood samples were collected. Both [14C]- and [3H]-labelled AIB in 25-50 ~1 maternal and fetal plasma were counted on a beta-scintillation counter (LKB 1219 Rackbeta). [14C]AIB and [3H]AIB were injected into the mother and its fetuses, respectively, in order to calculate clearances of AIB from the maternal-to-fetal and from the fetal-to-maternal side as independent parameters. An alternative approach to derive these kinetics requires producing steady-state plasma concentrations of the test agent in both the mother and its fetuses by constant infusion as can be done in catheterized sheep (Szeto, 1982). However, it is technically difficult to produce a steady-state drug concentration in the blood of fetal rats by constant infusion. Therefore, a model was developed to derive the same kinetics from non-steady plasma concentrations following bolus injections. The pregnant rat was assumed to behave as a two-compartment model with one maternal and one fetal pool exchanging with each other and each to outside (renal elimination in the case of the mother and net loss into the amniotic fluid in the case of its fetuses). AIB mass transported between the pools or to outside as a function of time can be estimated by multiplying its concentration, C, by the clearance, CL (volume of plasma totally cleared of the drug per unit time, expressed as ml/h/kg maternal body weight). Therefore, maternal plasma AIB concentration (C,) and fetal plasma AIB concentration (Cr) at any time can be described by the following equations (Varma and Ramakrishnan, 1985):

den -

CL,Cf

dt

dCr _ CL,&, -4

-

CL,C,

Vi - CL& Vf

(1) (4

where C, and Cr are, respectively, maternal and fetal plasma drug concentrations at any time, CLmfand CLf, are, respectively, placental clearances from mother-to-fetus and from fetusto-mother, CL, is the sum of maternal placental and non-placental clearances, CLf is the sum of fetal placental and non-placental clearances, and V, and Vf are, respectively, maternal and fetal volumes of distribution. Desired parameters were estimated by fitting the solution of the two-compartment model to the observed data using a non-linear least square approach. The computer program used for these calculations can solve a general pool model with multiple injections and computes the sum of exponential for the concentration in each pool for each injection. The program uses these exponentials to calculate model-predicted values at each observation time, which are then compared with the data. The program obtains improved guesses at model parameters by the Gauss-Newton method modified by Marquardt, which uses the sensitivity of the model-predicted values to the parameters. The iterative process used by the computer program terminated when no further improvement was observed in the last three iterations (Ramakrishnan, Leonard and Dell, 1984). The goodness-of-fit ofthese curves was indicated by a lack of significant deviations in plus and minus signs in relation to the curve on multiple runs and runs of residuals and the lack of significant weighted residual errors. Also the twocompartment open model fitted the data better than simpler models and there was no improvement if the model was made more complex. It was assumed that the absorption from

Placenta (I 991), Vol. 12

280

the fetal peritoneal cavities into the circulation was instantaneous and complete (Varma and Ramakrishnan, 1985). Statistics Differences between two means were compared by Student’s t-test and a probability of less than 0.05 was assumed to denote significant differences. Data are presented as means f s.e. Chemicals [14C]Aminoisobutyric acid (52.6 mCi/mmol) and [3H]aminoisobutyric acid (20 Wmmol) and scintillation fluid (Formula 963) were purchased from New England Nuclear, Boston, MA.

RESULTS A low protein (5 per cent) diet in place of the control, 21 per cent protein, diet caused a decrease in maternal and fetal body weights and in placental weights but did not affect the litter size (Table 1). Daily food consumption by rats on a low protein diet was significantly less that that by rats fed the control diet (Table 1). No obvious differences were noted in the behaviour of the two groups of rats during the blood sampling periods and animals moved freely during the interval between two sample collections. At all time-periods of blood collection, fetuses breathed spontaneously after caesarean removal and responded to touch. Significant quantities of aminoisobutyric acid (AIB) were transported across the placenta in both directions in control as well as in protein-deficient rats by 0.5 h after injections, the earliest time of measurement (Figure 1). In both groups of rats (Figure l), the fraction of the fetal dose of AIB that was transferred into the mother (CL) was smaller than the fraction of the maternal dose which was transferred into the fetuses (Q. Also, the maternal concentration of AIB following fetal injection appeared to attain a plateau after 4 h in rats fed a low protein diet but not in control animals (Figure 1). The maternal as well as fetal volume of distribution of AIB were significantly larger in control animals than the corresponding values in protein-deficient rats (Table 2), indicating a greater tissue uptake in the former than in the latter group of rats. Moreover, the volume of distribution of AIB in the fetus was significantly (P < 0.05) larger than in its mother (Table 2) indicating a relatively greater sequestration of AIB in fetal than in maternal tissues (Table 2). A higher clearance of AIB from the maternalto-fetal than from the fetal-to-maternal side in both control and protein-deficient rats Table 1. General

effects of a low protein diet in rats”

Parameters Dietary protein (per cent) haternal body weight, day 0 (9) Mean food intake (g/day) Maternal body weight, day 20 (g) Fetal body weight (g) Litter size Placental weight (mg)

Control 21 11 219 19.2 351 3.9 11.8 453

f 7 + 0.5 + 15 * 0.1 I!I 0.3 + 9

Protein-deficient 5 12 221 11.8 277 2.8 12.2 345

?z + f + * +

6 0.7’ 10” 0.05bb 0.3 8’

’ The low protein diet was fed ad libitum from days 0 to 20 of gestation; fetal values refer to day 20 of gestation. b Significantly (P < 0.05) different from the corresponding control value; data are means f se.

Ckm.

281

Ramaknshnan: TramplacentalKinetics ofAIB

1

I

&(b)

I*

loo-’

so -i

25 -

: z z \ 0 E a

“:--_1:

IO -

0 Time otter

injections

I

2

4

6

16

(h 1

Figure 2. Maternal and fetal plasma aminoisobutyric acid (AIB) concentrations in control (a) and protein-dehcient (b) rats on day 20 of gestation after intravenous injection of [‘~C]AIB(lOOmnol/kg) into the mother and intraperitoneal injection of [3H]AIB (1 nmol/kg maternal body weight) into its fetuses. Control and proteindeficient rats were fed ad libitum a 21 or 5 per cent protein diet, respectively, from days 0 to 20 of gestation. Serial blood samples were collected from the mother and one to two fetuses at time-periods shown on the abscissa. Concentrations in maternal and fetal plasma following maternal injections and fetal injections are, respectively, concentrations of [“C]AIB and [3H]AIB. C;, fetal concentration after maternal injection (0-U); C, maternal concentration after maternal injection (W-m); C$, fetal concentration after fetal injection (O-O); 6, maternal concentration after fetal injection (O-O). Data are means+s.e. from seven control and eight protein-deficient rats.

suggested that a greater amount of AIB was transported per unit time from the maternal to fetal side than in the opposite direction. However, the clearance of AIB from the maternal to fetal side (CL,r) was significantly greater in the control than in protein-deficient rats so that it took longer for the plasma concentration of AIB in the fetus to exceed that in the mother in the case of protein-deficient than in the case of control rats (Figure 1, Table 2). Protein deficiency did not alter the transfer rate of AIB from the fetus into the mother (CLr,) or from the fetus to outside (e.g. amniotic fluid, CL&.

DISCUSSION This study was performed in rats fed a control (21 per cent) and a low (5 per cent) protein diet ad libitum. Because rats fed a low protein diet consumed significantly less food than did the animals fed the control diet, they were deficient in both proteins and calories. Although we have previously shown that calorie restriction alone does not alter the pharmacokinetics and

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Table 2. Transplacental

Parameters

acid (AIB) in control and protein-deficient gestation Control 7 956

?I

&I (fig)

2337

Vf ww wnf

kinetics of aminoisobutyric

ww-4

$mo(Wk!&j C&gih) Time for gossover

(h)b

140 98 20 34 8.3 1.9

Protein-deficient

98

8 478

+ 192

1366

i + + f f + f

rats on day 20 of

22 18 3 8 2 0.16

55 48 11 22 7.3 3.4

f

81”

f 189” * f + * f f

9” 12” 5 4 2 0.19”

Control and protein-deficient rats were fed ad libitum a 21 or a 5 per cent protein diet, respectively, from days 0 to 20 of gestation. [14C]AIB (100 nmol/‘kg) was injected intravenously into the mother and [3H]AIB (1 nmol/kg maternal body weight) intraperitoneally in divided doses into all its fetuses. Abbreviations: V,, maternal volume of distribution; Vf, fetal volume of distribution; CL,r, placental clearance from mother to fetus; CL,, maternal non-placental clearance; CLf,, placental clearance from fetus to mother; CLf,, fetal non-placental clearance. a Significantly (P < 0.05) different from the corresponding control value. b Time when fetal concentration exceeded maternal concentration after maternal injection of AIB. Data are means f s.e.

metabolism of other drugs such as salicylates (Yue and Varma, 1982), effects of a low protein diet on transplacental kinetics of AIB might have been caused by protein-calorie rather than only by protein malnutrition. Plasma amino acids were not determined in this study. However, we have previously shown that a 5 per cent protein diet under identical conditions causes a significant reduction in maternal as well as fetal total plasma proteins (Varma and Yue, 1984) and albumin (Varma and Yue, 1983). No obvious differences in the behaviour of the control and protein-calorie malnourished rats were noted as a result of the invasive procedure used in this study and we have previously shown the suitability of this protocol in control rats (Varma and Ramakrishnan, 1985). Nevertheless, it is quite possible that surgery and anesthesia were more stressful to protein deficient than to control rats and this may have influenced our data. Placental transport of amino acids to the fetus is an active energy-dependent process and can operate against a concentration gradient (Christensen, 1973; Hill and Young, 1973; Smith et al, 1973). It is thus not surprising that the concentration of maternally administered aminoisobutyric acid (AIB) in the fetal plasma exceeded that in the maternal plasma as a function of time, as reported by others (Saintonge and ROSSO,1983). AIB can concentrate in tissues so that tissue to medium (or plasma) concentration is greater than unity (Smith et al, 1973). One would therefore expect the volume of distribution of AIB to be greater than total body water as was found in fetuses of both control and protein-deficient rats (Table 2). On the other hand, the concentration of AIB was less in fetal tissues of protein-deficient rats (as reflected by volume of distribution, Table 2) than of control animals; the reason for this is not clear from our data but could reflect a disproportionate decrease in amino acid concentrating tissue mass relative to total body weight. Results of this study indicate that the rate of transfer of AIB from the mother to the fetus (G&r) was significantly greater in control rats than in animals fed a low protein diet whereas the transfer in the opposite direction (C&J was not statistically different in the two groups of animals (Table 2). Our data do not exclude the possibility that differences in the transfer rate of AIB were influenced by the endogenous pool of amino acids, likely to be different in the two groups of animals (Morrison et al, 1961; Hill and Olsen, 1963). AIB shares a common

Varmu, Ramakrishnan: TransplacentalKinetic ofAIB

283

transport system with other neutral amino acids and competitive inhibition of transport of one by millimolar concentrations of another can occur (Gazzalo et al, 1972; Christensen, 1973). However, the normal amino acid concentrations of amino acids in rats are in micromolar range (Altman and Ditmer, 1974) and are expected to be even lower in rats fed a low protein diet. Thus, it is unlikely that amino acid transport system was saturated during the course of these studies and would have significantly affected transport of tracer amounts of AIB used in these experiments; moreover, if such an influence did exist it would reduce the rate of placental transfer of AIB to a greater extent in control than in protein-deficient rats. Since maternal protein malnutrition increased the time required for the fetal plasma AIB concentration to exceed the concentration in the maternal plasma (Table 2, Figure 1) and decreased the placental clearance of AIB from the maternal-to-fetal side (C&f), it would appear that the active transport mechanism for the delivery of AIB to the fetus was less efficient in protein-deficient than in control animals. This decrease in the transfer rate of AIB from the mother to fetuses in protein-calorie deficient rats may have been partly contributed by the reduced placental size; however, a greater decrease in the AIB transfer rate relative to the decrease in placental size suggests the involvement of other factors. In general, our data are in conformity with those of Jansson and Persson (1990) who reported that maternal to fetal transfer of AIB was significantly reduced in guinea pigs following intrauterine fetal growth retardation produced by uterine artery ligation. On the other hand, Young and Widdowson (1975) observed that 30 min after an injection of AIB, fetal tissues of guinea pigs fed a low-protein diet contained a greater fraction of injected AIB than did control fetal tissue; however, these workers did not determine the kinetics ofplacental transfer of AIB and therefore measurement ofwhole fetal concentration at a single time-period cannot represent the dynamics of transport. Our data clearly show that the fetal concentration of AIB continued to increase for 4-6 h after maternal injections. Several studies have found that placental size is directly related to fetal weight (Aheme and Dunnill, 1966; Lechtig et al, 1975). This relationship might be an expression of delivery of the nutrients to the fetus as exemplified by AIB in this study. If so, a contributing factor to the decrease in fetal size during malnutrition may be an impairment of the amino acid active placental transport process.

ACKNOWLEDGEMENT This study was supported

by a grant (MA-9726)

from the Medical Research

Council of Canada.

REFERENCES Aheme, W. and Dunn& M. S. (1966) Quantitative aspects of placental structure. 3ournulof Pathology and Bacteriblogy 91, 123-139. Alman, E. D. and Ditmer, D. S. (1974) BiologicalData Book, Vol. 3. Bethesda: Federation of American Societies for Experimental Biology, 1969 pp. Berglund, L., Gebremedhin, M., Lindberg, B., Lilja, A., Langstrom, B. & Lundqvist, H. (1989) Placental transfer of methionine during protein restriction in the Rhesus monkey studied by positron emission tomography. Placenta, 10,387-397. Christensen, H. N. (1973) On the development of amino acid transport systems. Federation Proceedings,32,19-28. Gazzola, G. C., Franchi, R., Saibene, V., Ron&, P. & Guidotti, G. G. (1972) Regulation of amino acid transport in chick embryo hean cells. Biochimiuzet BiophysiuzActa, 266,407-421. Hill, D. C. & Olsen, E. M. (1963) Effect of starvation and a nonprotein diet on blood plasma amino acids, and observations on the detection of amino acids limiting growth of chicks fed purified diets.3ournal ofNutrition, 79, 303-310.

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Hill, P. M. M. and Young, M. (1973) Net placental transfer of free amino acids against varying concentrations. Journal ofPhysiology,London, 235,409-422. Jansson, T. and Persson, E. (1990) Placental transfer of glucose and amino acids in intrauterine growth retardation: studies with substrate analogs in the awake guinea pig. Pediatric Research, 28,203-208. Lechtig, A., Yarbrough, C., Delgdo, H., Martorell, R., Klein, R.E. & Behar, M. (1975) Effect of moderate maternal malnutrition on the placenta. AmwicanJoumal of Obstetricsand Gynecology,123, 191-201. Morrison, A. B., McLaughlan, J. M., Noel, F. J. & Campbell, J. A. (1961) Blood amino acid studies III. Effects of amount and quality of dietary protein and length of test period on plasma free lysine levels in the rat. Canadian 3oumalofBiochemistry and Physioloa, 39, 1681-1686. Mulay, S., Vamm, D. R. & Solomon, S. (1982) Influence of protein-deficiency in rats on hormonal status and cytoplasmic glucocorticoid receptors in maternal and fetal tissues. Journal of Endocrinology,95,49-58. Mulay, S., Browne, C. A., Varma, D. R. SK Solomon, S. (1980) Placental hormones, nutrition, and fetal development. Federation Proceedings,39,261-265. Pivalizza, P. J., Woods, D. L., Sinclair-Smith, C. C., Kaschula, R. 0. C. & Pivatlizza, E. G. (1990). Placentae of light for dates infants born to underweight mothers at term: a morphometric study. Plucentu, 11,135-142. Ramalcrishnan, R., Leonard, E. F. & Dell, R. B. (1984) A proof of the occupancy principle and the mean transit time theorem for compartmental models. Mathematical Biosciences,68, 121-136. Raman, L. (1981) Influence of maternal nutritional factors affecting birthweight. American Journal of Clinical Nutrition, 34,775-783. Saintonge, J. & ROSSO,P. (1983) Placental blood flow and transfer ofnutrient analogues during normal gestation in the guinea pig. Pkzcentu,4,3 l-40. Smith, C. H., Adcock, E. W., Teasdale, F., Meschia, G. & Battaglia, F. C. (1973) Placental amino acid uptake: tissue preparation, kinetics, and preincubation effect. AmericanJournal ofPhysiology,224, 558-564. Szeto, H. H. (1982). Pharmacokinetics in the ovine maternal-fetal unit. Annual Review of Pharmacology,22,221243. Varma, D. R. (1986) Investigation of the maternal to foetal serum concentration gradient of dexamethasone in the rat. BritishJournal ofPhannacolo~, 88,815-820. Varma, D. R. & Yue, T. L. (1983) The influence of maternal protein deficiency on the placental transfer of salicylate in rats. British journal ofPharmaology, 78,233-238. Varma, D. R. & Yue, T. L. (1984) Influence of protein-calorie malnutrition on the pharmacokinetics, placental transfer and tissue localization of dexamethasone in rats. BritishJournal ofPharmmology, 83,131-137. Varma, D. R. & Ramalcrishnan, R. (1985) A rat model for the study of transplacental pharmacokinetics and its assessment with antipyrine and aminoisobutyric acid.3oumal ofPhannacologica1Methods, 14,61-74. Widdowson, E. M. (1977) Prenatal nutrition. Annals of New York Academy of Sciences, 300,188-196. Woods, D. L., M&n, A. F., Heese, H. D. V. & Schalkwyk, D. J. V. (1979) Maternal size and fetal growth. South African MedicalJournal, 56,562-564. Young, M. & Widdowson, E. M. (1975) The influence of diets deficient in energy, or in protein, on conceptus weight, and the placental transfer of a non-metabolisable amino acid in the guinea pig. Biologyof the Neonate, 27, 184-191. Yue, T. L. & Varma, D. R. (1982) Pharmacokinetics, metabolism and disposition of salicylates in protein-deficient rats. DrugMetabolism and Disposition, 10, 147-152.

Effects of protein-calorie malnutrition on transplacental kinetics of aminoisobutyric acid in rats.

In order to find out if inefficient transport of amino acids contributes to a decrease in fetal weight during maternal malnutrition, we injected [14C]...
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