ELSEVIER

tile Sciences, Vol. 64, No. 26, PP. 243!XI450,1%9 Copyright 0 1999 Elwvier science Inc.

Printed in the USA. All rights resewed O&24-32QS/W/S-see front matter

PI1 s0024-3205(99)00201-5

IMPACT OF A CHOLESTEROL ENRICHED DIET ON MATERNAL AND FETAL PLASMA LIPIDS AND FETAL DEPOSITION IN PREGNANT RABBITS

Alam Montoudis’, Lucie Simoneau’, Louise Brissette’, Jean-Claude Forest*, Roland Savard’ and Julie Lafond’,3*4.

‘Dkpartement des Sciences Biologiques, UniversitC du Qu&ec & Montrkal, Mont&al, Canada, ‘Facultk de Mkdecine, Universitt Laval, Qu&ec, Canada. 3This work was supported by grants from FCAR. 4Address all correspondence to: Prof. Julie Lafond, Universitt du Qdbec d Montreal. Dkpartement des Sciences Biologiques, C.P. 8888, Succursale (Kentre-Ville)), Mont&al (Qu&ec), Canada, H3C 3P8, Fax: (514) 987-4647, E-mail: lafond.julie@uqam.ca

(Received in final form March 9, 1999)

Summary

Pregnancy is associated with a hypercholesterolemic and a hyperlipidemic state. The totality of the essential fatty acids and 50% of the lipids needed by the fetus are transferred by the placenta from the maternal circulation. The hypothesis of this study is that an augmentation of the maternal plasmatic cholesterol is modifying the fetal lipids accumulation and development during rabbit pregnancy. To demonstrate the impact of a cholesterol enriched diet on plasma lipids during rabbit’s pregnancy and on their fetus, we have established two groups: control and hypercholesterolemic rabbits (fed with a 0,2% cholesterol diet). Blood samples were collected before mating and at each trimester of pregnancy for analysis of lipid fractions and their lipoproteins. Plasma analysis shows that starting the 10” day of pregnancy the concentration of total-cholesterol and lipoproteins decreases for both groups . We have demonstrated that for the hypercholesterolemic group, concentrations of total-cholesterol (63 1%) and lipoproteins are significantly higher at the end of pregnancy than those for the control group. For both groups, after 20 days of pregnancy, triglycerides metabolism was biphasic showing a significant increase followed by a diminution in their concentration. In both groups, f?ee fatty acids increases significantly at the end of the pregnancy (537.5% for the control group and 462.5% for the hypercholesterolemic group). Furthermore, the offsprings of hypercholesterolemic dams manifest a lower birth weight (15.5%) than those of control group. Our results demonstrate that a cholesterol enriched diet modifies greatly the fetal development and lipid metabolism during rabbit’s pregnancy. These modifications could be u&X for the understanding of the interaction between diet and fetal development in rabbit and probably during human

pregnancy.

Key Words: gestation, rabbit, dam, diet, placenta, offspring, fetal development, lipids

2440 Cholesterol Diet during Rabbit Gestation Vol. 64, No. 26,1!B9

The placenta cannot synthetize the essential fatty acids and sufficient amounts of the other free fatty acids (FFA). Thus, the fetus is depending on the FFA supplied from the maternal circulation (1). During normal pregnancy, according to the fetus’s needs, the placenta transfers up to 50% of the lipids and 100% of the essential FFA provided by the maternal circulation (2). The fetal development depends, in part, on appropriate accumulation of essential FFA such as

arachidonic, linoleic and docosahexanoic acids (3). A deficiency in dietary essential o-6 FFA during pregnancy compromises somatic fetal development (4), while a deficiency in dietary essential o-3 FFA contributes to a poor development of the mammalian nervous system in newborns (5, 6). Zn viva, linoleic acid is preferentially transported in a saturable way by the placental syncytiotrophoblast brush border membrane and at a slower rate by basal plasma membrane (7). These findings reveal that maternal diet plays a important role in fetal development.

FFA, being hydrophobics, are associated with carriers in blood circulation. Albumin is the principal FFA carrier (8) but fetuin is also believed to play a role at the level during pregnancy (9). Fetuin is a more efficient carrier of FFA since it carries 50 times more FFA than albumin (10). Triglycerides (TG) are unable to cross the placenta (1 l), they must be hydrolyzed before reaching fetal circulation (12). Lipoprotein lipase (LPL), an enzyme that hydrolyzes very low density lipoprotein enriched in triglycerides (VLDL-TG), is found near the syncytiotrophoblast brush border membranes of guinea pig (13). Also, at the end of pregnancy, LPL activity rises 11 times in the guinea pig (13). The released free fatty acids are used for the development of the fetus or are esterified by the fetal liver to produce TG stocks for further use (14). At parturition, the concentrations of total cholesterol (TC) and lipoproteins (low density lipoprotein (LDL) and high density lipoprotein (HDL)) are higher in pregnant then in non-pregnant women (15). In rabbits, cholesterol and LDL concentrations diminish markedly (16, 17) starting the 10th day of pregnancy (16) reaching lower levels than those observed before mating. This is associated with a higher level of estrogen (18) that up-regulates LDL receptor (LDLr) expression (18) and leads to a higher turnover of LDL in rabbit (17). LDL-cholesterol (LDL-C) is believed to be used for steroid synthesis (cortisone, progesterone) which is required for adequate fetal development (18).

The main hypothesis of this work is that during rabbit’s pregnancy, an augmentation of the maternal plasmatic cholesterol level induced by an enriched cholesterol diet, would modify the fetal lipids accumulation and development. To verify this hypothesis, we have analyzed the effects of an enriched cholesterol diet on various plasma parameters of dams and their offsprings (FFA, TG, glycerol, albumin, TC, LDL-C. and HDL-cholesterol (HDL-C)) and the effect of this diet on fetal development.

Materials and methods

Experimental animals New Zealand’s adults white virgin females rabbits weighting 3-4 Kg were purchased from Charles River (Saint-Constant, Canada). During an initial two weeks equilibrium period, all animals have been fed on a standard rabbit’s Chow diet (Ralston Purina, Drummondville, Canada) and water ad libitum. To conduct this study, female’s rabbits were divided in two groups. A control group in which rabbits were fed with a standard rabbit’s Chow diet (control diet) (150 g/day) during all their 30 days of pregnancy. This control group follow-up began on the mating day (day 0 until day 30). The other group was fed with a standard rabbit’s Chow diet (150 g/day) enriched with 0.2% (w/w) cholesterol (enriched cholesterol diet), for seven days before mating (-7 days) and throughout the pregnancy (30 days). It has been established that an enriched cholesterol diet of 0,2% was adequate to induced an hypercholesterolemic state on rabbits (19). We made the cholesterol enriched diet by adding 2 g of cholesterol (ICN) to 1 Kg of

Vol. 64, No. 26,199!J Cholesterol Diet during Rabbit Gestation 2441

rabbit CHOW powder and then reconstitute as pellets. All animals were fed concurrently under similar conditions. Thus, for the enriched cholesterol diet’s group, the follow-up began seven days before mating (first day of enriched cholesterol treatment) and ended at parturition, 37 days later. A non-pregnant group of rabbits, fed with an enriched cholesterol diet, was also established in order to determine the effect of the enriched cholesterol diet on lipids metabolism and their follow-up began also at the first day of the enriched cholesterol diet’s treatment (-7 days), and lasted 37 days.

Drugs and materials Cholesterol was purchased from ICN (Aurora, United States), Acepromazine maleate (Atravet) and Ketamin chlorhydrate were purchased from Ayerst (Montreal, Canada). Xylazin was obtained Erom Bayer (Etobicoke, Canada) and Euthanyl was bought f?om MTC (Cambridge, Canada). The entire enzymatic kits assay were obtained f?om Boehringer-Mannheim (Laval, Canada). All enzymes and chemicals used for the glycerol assay were purchased from Sigma (St-Louis, United-States).

Experimental methods Maternal blood was collected by an ear artery puncture, following the subcutaneous injection of acepromazine maleate (Atravet) (0.6 n&Kg), before mating and at each third of pregnancy. An additional blood sampling was done on the rabbits determined to be part of the hypercholesterolemic group prior the beginning of the cholesterol enriched diet. Following the blood clothing at room temperature, plasma was kept at -80°C until analysis. The last blood collection was done 30 days after mating by an intracardiac puncture, following a subcutaneous injection of Atravet (0.6 mg/Kg) and an intramuscular injection of Ketamin chlorhydrate (40 mg/Kg) and of Xylazin (5 mg/Kg). After this puncture, the dams were killed by a lethal intracardiac injection of Euthanyl (0.5 ml/Kg), then their abdomen was opened. Both horns of the uterus were excised and the offspring was removed from the uterus with their intact placentas. Blood was obtained from the offsprings by cardiac puncture and they were killed by an intracardiac injection of Euthanyl (0.5 ml/Kg). Macroscopic observations were made on rabbit’s livers and placentas and the offsprings weights were evaluated. Half of the offsprings was used to quantie brain’s lipids and the other half was used to measure total lipids.

Lipid mass For the fetal brain’s lipids mass determinatiolh each brains were homogenized in 10 ml of methylene chloride using a poly-tron for 1 min and then decanted so the organic phase could be recovered. Three extractions were made and the 3 extracted phases were pooled, filtered on anhydrous sodium sulfate and evaporated at room temperature. The offsprings were homogenized for 10 min in 75 ml of methylene chloride containing 25 g of a hydrous sodium sulfate, and the homogenates were left at room temperature for 10 hours. Then, an additional 75 ml of methylene chloride solution was added to the homogenate, mixed and filtered in order to measure the totality of the lipids mass. Two other extractions were made with 75 ml of methylene chloride. A fraction of those 3 extractions pooled together was evaporated at room

temperature and was weighed (20).

Lipid assays The following analysis were done in duplicate on the females and offsprings plasma’s: FFA, TG, TC, HDL-C. and albumin. These assays were made by enzymatic and calorimetric reactions (21) using Boehringer-Mannheim kits and were all done by autoanalyzer, except for the FFA assay. The LDL were quantified using a modified formula of the one developed by Friedewald et al. (22). This formula is correlated with the other lipids already quantified which is LDL-C. = TC - (HDL-C. + (0.46*TG)). Glycerol was assayed in duplicate by bioluminescence (23) using an

2442 Cholesterol Diet during Rabbit Gestation Vol. 64, No. 26,1999

enzymatic cascade involving the production of nicotinamide adenine dinucleotide, reduced form (NADH) from glycerol transformation NADH was then measured using flavine mononucleotide (FMN), oxydoreductase and luciferase through bioluminescence and was expressed in uM. Statistical analysis All results are expressed as means f (SE). We compared the different parameters for every third of gestation within both groups with a two tailed non-paired t-test. Also, we did the same comparison between both groups with a two tailed non-paired t-test. The acceptable level of significance was p < 0.05 for all the comparisons of the study.

Results

We used an enriched 0.2% cholesterol diet, because a preliminary study showed that a enriched 2% cholesterol diet was not compatible with gestation (miscarriage after 10 days of gestation). Macroscopic observations have demonstrated that placentas from dams fed with an enriched cholesterol diet (0,2%) have an important lipids accumulation, when compared with the control group (fig. 1). The placentas from the hypercholesterolemic dams were lightly colored and their surface presented distinct lobules, while normal placentas had a dark red color and presented a smooth texture (fig. 1).

1

Fig. 1 Illustration of a placenta from 1) a control rabbit and 2) an hypercholesterolemic rabbit.

For of the control and the hypercholesterolemic dams before mating, their average weights were not significantly different and were 3675.7 + 133.7 g (n=7) and 3880.9 f 69.2 g (n=7), respectively. Also, the average weights of the placentas from the control and the hypercholesterolemic dams were not significantly different and respective weights were 3.48 k 0.12 g (n=56) and 3.43 f 0.12 g (n=51). Moreover, the average number of offspring per dam of the control and the hypercholesterolemic group was not different, their number were respectively 8.00 f 1.01 offspring/ dam (n=56) and 7.43 + 0.75 offspring/ dam (n=51). In contrast, the average weights of the offsprings of the hypercholesterolemic dams (n=51) was significantly lower (15.5%) than the average weights of the offsprings of the control dams (n=56) (table 1).

First, FFA, TG, glycerol, albumin TC, LDL and HDL were measured in non-pregnant and non- treated rabbits in order to establish reference values (table 1). Thereafter, those parameters were

Vol. 64, No. 26,1999 Cholesterol Diet during Rabbit Gestation 2443

measured during pregnancy for both groups (control and hypercholesterolemic) and for their term offsprings, Table 1 shows an important increase of FFA at the end of pregnancy for both groups (fig. 2~). However, FFA concentration rises at the 20” day of pregnancy for the hypercholesterolemic group while this increase was observed only at the end of the pregnancy for the control group.

TABLE I Lipids and their lipoproteins and corporal weights of non-pregnant rabbits (reference values), dams (at 30 days of pregnancy) and offsprings from control and hypercholesterolemic rabbits.

The reference values (mean f SE) represent the concentration of lipids and lipoproteins in non-pregnant females rabbits (n=21) fed with a standard rabbit’s Chow (15Og/ day). Values for the offsprings and the dams (mean f SE) (Offsprings: n=56 from controls dams and 51 from hypercholesterolemic dams; dams: n= 7 controls and 7 hypercholesterolemic). * ~~0.05 compared with the offspring of control dams; n ~~0.05 dams at 30 days of pregnancy vs offsprings in both groups; 1 p ~0.05 hypercholesterolemic dams at 30 days of pregnancy vs control dams at 30 days of pregnancy. [FFA (Ike fatty acids), HDL-C (high density lipoprotein-cholesterol), LDL-C (low density lipoprotein-cholesterol), TC (total- cholesterol) and TG (triglycerides)].

For both groups, those FFA levels are significantly higher than those observed at the beginning of pregnancy. The concentrations of plasma’s FFA for both non-pregnant groups were not modified by the diet (30 or 37 days) (table 1 and fig. 2~). Overall, this indicates that only pregnancy have an effect on FFA levels. The FFA concentrations of the offsprings are by far lower than the level observed at the end of pregnancy for the dams of both groups studied (table 1). Furthermore, FFA concentrations of the offsprings are significantly lower in the hypercholesterolemic group.

The TG profile during the pregnancy can be subdivided in two phases in the control group; an increase on the 20’ day of pregnancy followed by an important reduction on the 30* day (fig. 2b). The TG level in the non-pregnant group fed with an enriched cholesterol diet was constant. For the hypercholesterolemic pregnant group, the increase at the second third was not significant. However, TO decreased in a significant way at the end of pregnancy. It can be concluded that the cholesterol enriched diet does not affect greatly maternal TG levels during pregnancy. The offsprings concentrations of TG are higher than those observed for the dams of both groups at the end of pregnancy (table 1). Moreover, levels of TG of the offsprings of the

2444 Cholesterol Diet during Rabbit Gestation Vol. 64, No. 26,1!?99

hypercholesterolemic group are higher than those observed for the offsprings of the control dams.

The plasma concentration of glycerol begin to increase on the 20’r’ day of pregnancy for the control group in comparison with the reference value (fig. 2~). This concentration reaches 13.061 f 2.004

a.

Expsrhmatal days

d.

Experlmmbf days Expelimental day6

Fig. 2

Effects of a diet containing 0,2% of cholesterol on the plasma a) free fatty acid, b) triglyceride, e) glycerol and d) albumin levels during pregnancy of rabbits. The cholesterol enriched diet was introduced 7 days before mating. The values represent the mean f SE of the different parameters cited above in pregnant rabbits of the control group ((n) n=S) and feed with a cholesterol enriched diet ((83) n=6) and in the non-pregnant rabbits group feed with a cholesterol enriched diet ((D) ~6). * ~~0.05 compared to the first day of mating for the same group; 1 ~~0.05 non-pregnant rabbits vs hypercholesterolemic dams; &fi ~~0.05 non- pregnant rabbits vs both groups of pregnant rabbits; n ~~0.05 20 vs 30th day of pregnancy for the same group; 5 ~~0.05 both groups of pregnant rabbits.

uM at the end of pregnancy for the control group (table 1). As opposed to . . . .

the nypercholesterolemlc group, the concentration of glycerol does not vary during the pregnancy and reaches 8.129 f 0.617 uM at term (fig. 2~). Then, at the end of the pregnancy for these rabbits, the plasma ~oncen~tion in glycerol is signi~~~tly lower then the one found at the end of pregnancy of the control group (fig. 2~).

Vol. 64, No. 26,1999 Cholesterol Diet during Rabbit Gestation 2445

The level of albumin fell below the one observed on the IO* day of pregnancy of the control dam group. A similar decrease was observed only on the 20’ day of pregnancy for the hypercholesterolemic group (fig. 26). For both groups, this diminution was greatly marked at the end of the pregnancy to reach half of the concentration before mating. The albumin concentration of the non-pregnant group did not change throughout the treatment indicating that a cholesterol enriched diet does not affect their level. The lcvcls of albumin in both offsprings groups are similar to those observed for their dams at the end of pregnancy and there is less albumin found in the offsprings from hypercholesterolemic dams (table 1).

For the control group, on the 20* day of pregnancy, the level of TC and LDL fell below the concentrations observed before mating (fig. 3a and 36). The dams of the hypercholesterolemlc group show an increase in TC and LDL between the beginning of the cholesterol enriched diet and the mating (fig. 3a and 36). The maximal value observed in this group is reached at the fust third of pregnancy. On the 20* day of pregnancy, an important decrease of the TC and LDI. levels is observed, leading, at the end of pregnancy, to significantly higher concentrations in the hypercholesterolemic group than in the control group. A different pattern is observed for the non-pregnant treated group, as TC and LDL concentrations progressively increase throughout the diet. Thus, adding cholesterol to the diet severely increases TC and LDL-C. in rabbit serum as expected and leaves the dams with significantly higher levels of TC and LDL-C. throughout pregnancy. The levels of both TC and LDL-C. are higher in the offsprings of both groups compared with the dam’s levels (table 1). Moreover, the levels of TC and LDL-C. were higher for the offsprings from the hypercholesterolemic group than for those of the control group (table 1 ).

For the control group, the level of HDL-C. during pregnancy decreases starting the 20th day of the pregnancy in comparison with the level observed before mating (fig. 3~). For the hypercholesterolemic group, this decrease is significant only at the end of pregnancy and this value is significantly higher than the one obtained for the control group. Also. pregnancy affects HDL-C. by lowering levels in the control dams at the end of pregnancy. However, the level of HDL does not vary throughout the cholesterol diet of the non-pregnant group. Thus, the addition of cholesterol during rabbit’s pregnancy has no effect of the concentration on HDL-C. The offsprings’s level of HDL is similar to the one observed at the end of pregnancy for both rabbits groups (table 1). However, for the offsprings of hypercholesterolemic group, the HDL concentration is higher than the one observed for the offsprings of the control group (table 1).

There were no significant differences observed between the total lipid content of the offsprings of the control group (1.81 f 0.21 g, n=7) and of the hypercholesterolemic group (1.32 * 0. IO g. n=5). Also, we observed no significant differences between the brain lipid content of the control offsprings group (3.54 & 0.16 mg, n=15) and of the hypercholesterolemic group (4.17 i 0.38 mg, n=23).

Discussion

In this study, we observed a marked elevation of FFA at the end of pregnancy for both control and hypercholesterolemic rabbits. Some authors have demonstrated (13. 24, 25) similar results for rabbits, primates and guinea-pigs, but these animals were fed with a standard Chow diet . The FFA are essential for the synthesis of phospholipids, sphingolipids. cholesterol esters and TG. The fact that this increase is similar for both pregnant groups in our study, and since this increase of FFA is not observed in the non-pregnant group, we believe that the elevation of FFA is a consequence of the pregnancy state and is not associated with cholesterol absorption. In contrast. the very low concentrations of FFA observed in newborns from pregnant rabbits of the control

2446 Cholesterol Diet during Rabbit Gestation Vol. 64, No. X,1999

b. 10

s 1 wll

E a §

Fii. 3

Effects of a diet containing 0,2% of cholesterol on the plasma A) total cholesterol B) LDL-C. and C) HDL-C. levels during rabbit’s pregnancy. The cholesterol enriched diet was introduced 7 days before mating. The values represent the means f SE of the different parameters cited above in pregnant rabbits of the control group ((.) s8) and fed with a cholesterol enriched diet ((m) n=6) and in the non-pregnant rabbits group also fed with a cholesterol enriched diet ((0) s6). * ~~0.05 compared to the fust day of mating for the same group; u ~~0.05 20ti vs 30ti day of pregnancy for the same group; 9 ~~0.05 both groups of pregnant rabbits; # ~~0.05 beginning of the treatment (-7 days) vs just before mating; lp<O.O5 non-pregnant rabbits vs the control group of pregnant rabbits; flpcO.05 non-pregnant rabbits vs both groups of pregnant rabbits.

Vol. 64, No. X,1999 Cholesterol Diet during Rabbit Gestation 2441

group confirmed the results obtained by Her&field and Nemeth (26) in guinea pig fed with standard Chow diet. They correlated this low level of FFA with the very high lipids metabolism of the fetus in order to allow its development and growth. Also, our study shows that under enriched cholesterol (0,2%) diet, the fetal FFA concentration was significantly lower. All this together strongly confirms that the placenta plays a major role in FFA exchange.

In human the decrease of albumin’s concentration is observed throughout pregnancy (14) and is not linked to hypercholesterolemia. Effectively, in the non-pregnant hypercholesterolemic rabbits no change in plasma albumin’s concentration has been observed throughout enriched cholesterol diet (0,2O). It has been established that on the 21 day of rabbit’s pregnancy, plasma volume increased about 50% (17). This augmentation could be in part responsible for the slightly decrease of albumin (10%) observed on the 20’ day of pregnancy. However, our results show that there has been an increase of the albumin synthesis during rabbit’s pregnancy since the diminution observed on the 20” and 30* days of pregnancy cannot be directly correlated with thii hemodilution. Since albumin is highly associated with the FFA transport, we hypothesize that other proteins, involved in FFA circulation, could be synthesized in order to transport the FFA during pregnancy.

This study shows an significant increase of TG up to the 20” day of pregnancy in rabbits fed with a control diet, which was followed by a decrease in the last trimester of pregnancy. A previous study (17), using pregnant WHHL (Watanabe Heritable Hyperlipidemic) rabbits, demonstrated that any modulation in the TG metabolism was associated with hypercholesterolemia or LDL-C. metabolism, and that the TG increase was linked to pregnancy. Gur present study confii their results, since we show that the TG levels in the non-pregnant rabbits fed with a enriched cholesterol diet (0,2%) remained unchanged. Then, the plasma TG concentrations measured for the offsprings are significantly higher than those observed for dams at the end of the pregnancy for both groups. In 1973, Szabo et al. (14) demonstrated that this difference could be the consequence of an esteritication of FFA originating from the hydrolysis of maternal TG by the human placental LPL. This esterification takes place in the fetal liver which esterifies 83% of the FFA in TG, compared to 35% for the maternal liver (13). This can explain the low concentrations of FFA and the high concentrations of TG in the plasma of the offsprings compared to the dams in our study. In contrast, significant high concentrations of TG were observed in offsprings providing horn dams fed with an enriched cholesterol diet (0,2%).

The elevation of glycerol reported at the end of pregnancy could be a consequence of an increase of the TG lipolysis. This lipolysis leads to an important diminution of the TG between the 20” and 30” days of pregnancy, in the control group. Glycerol, which is a lipolytic activity’s indicator, is released by the adipocyte celIs during lipolysis (27). In the hypercholesterolemic group, the concentration of glycerol throughout the pregnancy remained constant. However, it is important to recall that at the end of the rabbit’s pregnancy the plasma volume increase greatly (17) then it indicates that the lipolysis level was increased, but to an inferior level that the one observed in the control group. This lower lipolysis rate could be explained by the fact that those rabbits have VLDL highly enriched in cholesterol and poorly in TG (28). Thus, the low quantity of VLDL-TG could lead to a reduction of the lipolytic substrate, having for consequence a low lipolysis level showing the constancy of the plasma’s concentrations of glycerol throughout the pregnancy.

In the non-pregnant rabbits group, the TC and LDL-C. concentrations increased in a linear way with a regression coefficient (3=0.995) throughout the treatment of the enriched cholesterol diet (0,2%) for the non-pregnant rabbits group. In both pregnant rabbits groups, the fall of TC and LDL-C. observed begin on the lO* day and lasted until the end of pregnancy in comparison with the reference values. Similar results were observed in some studies with pregnant rabbits (16, 29) and WHHL rabbits (17) fed with a standard Chow diet. This means that the reductions of TC and

2448 Cholesterol Diet during Rabbit Gestation Vol. 64, No. 26,19!B

LDL-C. are related to the pregnancy and not to the enriched cholesterol treatment (0,2%). In addition, the concentrations of TC at the end of the pregnancy for the treated group with the enriched cholesterol diet are higher than for control group of pregnancy rabbits. As the profile of LDL-C. resembles more the TC profile than HDL-C. profile, the TC profile would reveal much more the effects of pregnancy and diet than the HDL-C. does. The TC and LDL-C. decrease could be related to the increase of estrogen observed at the end of pregnancy (17, 18) since it induces LDLr expression. This increase of LDLr was observed with WHHL rabbits. where the amount of LDLr increases from 0.5 4 0.2 ng/mg of proteins before mating to 6.0 + 0.7 ng/mg of proteins on the 21% day of pregnancy (17). This high level of cholesterol uptake could be useful for the synthesis of steroid hormones during pregnancy, since there is a correlation between the rise of progesterone’s concentration and the reduction of plasma’s cholesterol (18). According to

this study, cholesterol (in particular the LDL fraction) could be used for the synthesis of progesterone in the corpus lutea of the rabbit ovaries (18).

In our study, most of the placentas of the hypercholesterolemic group of pregnant rabbits displayed an important quantity of lipids. Since the placenta is the main site of nutritional exchanges between maternal and fetal circulations (30), the offsprings’s low birth weight from hypercholesterolemic pregnant rabbits could be related, in part, to this placental lipids accumulation. This low birth weight is confvmed by our research. The fact that neither the average dams weight (before treatment and gestation), the average number of offspring per dam or the average placental weight have been influenced by the hypercholesterolemic diet. In that matter, Bastiaanse et al. (31) showed that an excess of cholesterol in cellular membranes modifies the ionic transport and inhibits some ATPase activities (example Ca2+-ATPase). Because, we have already demonstrated that the arachidonic acid transport is dependent on ATP and sodium linked in brush border and basal plasma membranes of syncytiotrophoblast (32), and that calcium transport is dependent on ATP in human and guinea pig placental basal plasma membrane (33), we hypothesis that these mechanisms are impaired and lead to the fetal low birth weight of the offsprings from hypercholesterolemic dams. This is confirmed by the fact that this fatty acid is essential for the biogenesis of cellular membranes (kidney, cervical, spinal nerve) and is an important precursor of eicosanoids (prostaglandins, thromboxanes and leukotriens). It is known that these lipids have numerous physiological tinctions (increase lipolysis, bone resorption, bronchial tonus etc.) (34) and are essential to the fetal development (somatic (FFA o- 6) and for the central nervous system (FFA o-3)) (4,5,6).

In conclusion, our research has demonstrated that rabbits became hypercholesterolemic by being one week on a cholesterol (0,2%) enriched diet and that the lipids metabolism is different from the standard diet group. Furthermore, we showed that this induced hypercholesterolemia has an important effect on TG, glycerol, TC and LDL-C., but has no effect on FFA, albumin and HDL- C. metabolism during the rabbit’s pregnancy. However, for the pregnant rabbits fed with an enriched cholesterol (0,2%) diet, the increase of the lipolysis was not observed (basal level of plasma glycerol) in comparison with the control pregnant rabbits (high level of plasma glycerol). We have also confrmed that the concentrations of TC and lipoproteins decrease with the progression of pregnancy. This reduction may coincide with an increase of the estrogen level on the 10” day of pregnancy (18), which could induce LDLr expression (17). Moreover, the offsprings weights from hypercholesterolemic dams are significantly lower (15.5%) than that from control dams. These low birth weights could be explained by the hypercholesterolemia during the pregnancy of dams, since their placentas showed an excessive lipids accumulation. This state could lead to a reduction of essentials FFA and ions transport from the maternal to the fetal circulations (31, 32). These fetal lipids modifications, caused by an enriched cholesterol diet, could have an important impact on the future health of the fetus. We believe that our results could be linked to the human physiology disease. However, we think that an increase of circulating plasma lipids could equally has an important harmtill impact on the transport of fetal

Vol. 64, No. 26,1999 Cholesterol Diet during Rabbit Gestation 2449

essential nutrients, which could be lied to different pathologies, such as in&a-uterine growth retardation, h~rlipide~c states or maternal diabetes. Thus, the newly knowledge stated in this paper showed the harmi%11 effect of a high choiesterol diet on the fetal development.

Acknowledgments

The authors wish to thank M. Jean-Claude ThCriauit from clinical biochemistry service of St- Franqois d’Assise Pavillon of CHUQ for the technical assistance (blood analysis from the dams and the offsprings). This work was supported by grants &om FCAR (J. Lafond) and 1JniversitC du Quttbec B Mont&al. Alain Montoudis is a recipient of a studentship from FCAR-FRSQ (Q&xc).

References

1. L. HUMMEL, A. SCHWARTZE, W. SCHlRRME%STER and H. WAGNER Acta Bio!. h/fed. Ger. 35 1635-1641 (1976)

2. RA. COLEMAN, Fed. Proc. 45 2519-2523 (1986) 3. S.C. CUNNANE and Z.-Y. CHEN, Am. J. Physiol. 262 R&R13 (1992) 4. A.E. HANSEN? M.E. HAGGARD, A.N. BOELSCHE, D.J.D. ADAM and H.F. WEISE. J.

Nutr. 66 565-576 (1958) 5. M. LAMPETY and B. WALKER, J. Nutr. 106 86-93 (1976) 6. M. AUGER W.E. CONNOR, D.S. LIN, L. BARSTAD and S. LUCK.. Proc. Natl. Acad.

Sci. USA 83 4021-4025 ( 1986) 7. J. LAFOND, L. SIMONEAU, R. SAVARD and M-C. GAGNON. Eur. J. Biochem. 226 707-

713 (1994) 8. J. NAVAL, M. CALVO, J. LABORDA, P. DUBOUCH, M. FRATN, J.M. SALA-TREPAT

and J. URIEL, J. Biochem. 111 649-654 (1992) 9. T.T. PUCK, A. WALDREN and C. JONES, Proc. Nat. Acad. Sci. USA 59 192-199 (1968) 10. A. J. CAYAT’IE, L. KUMBLA and M.T.R. SUBBlAH, J. Biol. Chem. 265 5883-5888 (1990) 11. J.P. STIRS, D. HULL, M. SILVER, A.L. FOWDEN, J. OUSEY and P.D.

ROSSDALE, Placenta 15 857-872 (1994) 12. M.C. ELPHICK and D. HULL, J. Physiol. 264 75 l-766 (1977) 13. C.R THOMAS, C. LOWY, R.J. ST-HILAIRE and J.D. BRUNZELL, Fetui nutrition In

~~etabo~~s~ and j~~~~~o~, RK. Miller and H.A. Thiede (Eds), t35- 148, Plenum Press. New York, New York (1984)

14. A.J. SZABO, R. De LELLIS and R.D. GRIMALDI. Am. J. Obstet. Gynecol. 115 257-262 (1973)

15. A.-N. CMANG, M.-L. YANG, J.-H. HUNG, P. CHOU, S.-K. SHYN and H.-T. NG. Life SC. 56 2367-2375 (1995)

16. D.W. QUIG and D.B. ZILVERSMIT, Proc. Sot. Exp. Biol. Med. 182 386-392 (1986) 17. M. SMOMI, T. IT0 and Y. WATANABE, Biochim. Biophys. Acta 917 92- 100 (1987) 18. K. KRIESTEN and U. ~WS~, Comp. B&hem. Physiol. 90A 413-420 (1988) 19. A.H.M. TERPSTRA and A.C. BEYNEN, Camp. Biochem. Physiol. 77B 523-528 (1984) 20. J.E. McNEAL, J. AOAC 58 1182 (1975) 21. C.A. ALLAIN, L.S. POON, C.S.G. CHAN, W. RlCHMOND and P.C. FU, Clin. Chem. 20 470

(1974) 22. W.T. FRIEDEWALD, R.I. LEVY and D.S. FREDERICKSON, Clin. Chem. 18 499-502

(1972) 23. H. KATHY, S. FRIEDHELM and S. BERND, Clin. Chim. Acta 120 295-300 (1982) 24. D. HULL and M. ELPHICK, Placental trmsfer. G.V.D. Chamberlain and A.W. Wilkinson

(Eds), 159-l 65, Pittman Medical, New York (1979) 25. L. HUMMEL, R SCHIRRME ISTER and T. ZIMMERMAN, Biophys. Biochim. Acta 42 143-

2450 Cholesterol Diet during Rabbit Gestation Vol. 64, No. 26,1!?39

145 (1983) 26. M.S. HERSHFIELD and A.M. NEMETH, J. Lipid Res. 9 460-468 (1968) 27. P. BJORNTORP and J. OSTMAN, Adv. Metab. Disord. 5 275-327 (1971) 28. S. CHANG and J. BORENSZTAJN, Biochem. J. 293 745-750 (1993) 29. P.T. KOVANEN, J.L. GOLDSTEIN and M.S. BROWN, J. Biol. Chem. 253 5 126-5 132 (1978) 30. S.H. LEWIS and K. BENIRSCHKE, Histology for Pathologists, chapter 43. S.S. Stemberg

(eds), 835-863, Raven Press, New York (1992)

3 1. E.M.L. BASTIAANSE, KM. HOLD and A. VAN DER LAARSE, Cardiovasc. Res. 33 272- 283 (1997)

32. F. MOUKDAR, Mdmoire de ma&rise, DCpartement des Sciences Biologiques, UniversitC du Qdbec g Mont&al ( 1996)

33. J. LAFOND, M. LECLERC and M.G. BRUNETTE, J. Cell Physiol. 148 17-23 (1991) 34. A.J.R HABENICHT, P. SALBACH and U. JANSSEN-TIMMEN, Platelet-dependent vascular

occlusion. G.A. Fitzgerald, L.K. Jennings and C. Patron0 @is), 237-246, Ann. NYAcad. Sci. 74 ( 1994)