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Differential transplacental binding of valproic acid: influence of free fatty acids

Differential transplacental binding of valproic acid: influence of free fatty acids
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  Br. J. clin. Pharmac.  1984), 17, 759-762 Differential transplacental binding of valproic acid: influence of free fatty acids F. ALBANI', R. RIVA  , M. CONTIN , A. BARUZZI', M. ALTOMARE2, G. P. MERLINI:   E. PERUCCA4 Institute of Neurology , University of Bologna and Institutes of Obstetrics and Gynaecology2, MedicalPathology I:, and MedicalPharmacology4, University ofPavia, Italy. The unbound fraction of valproic acid (VPA) was found to be significantly lower in cord serum  6.0 + 0.8 ) than in maternal serum collected before oxytocin  12.2 + 2.7 ) or after delivery  9.9 + 2.3 ). The difference was probablydue to the concentrationof free fatty acids  acting as displacing agents) being higher in maternal serum. The transplacental binding gradient explains the clinical observation that total VPA levels at delivery are higher in the newborn than in the mother. Keywords valproic acid free fatty acids transplacental binding Introduction A number of investigators have reported that valproic acid (VPA) levels are higher in umbil- ical cord or neonatal serum than in maternal serum (Dickinson etal., 1979; Froscher et al., 1981; Ishizaki et al., 1981; Nau et al., 1981; Kaneko et al., 1983). Possible mechanisms pro- posed to explain this finding include  1) the pos- sibility of the foetus acting as a deep compart- ment  Dickinson etal., 1979; Kaneko et al., 1983),  2)the presenceof an active transport of VPA across the placenta (Nau et al., 1981; Kaneko et al., 1983) and/or  3) more extensive protein binding of the drug in foetal as compared withmaternal blood (Nau et al., 1981). Since only the free fraction is pharmacologically active, theexistence of a transplacental binding gradient, such as suggested in preliminary observations by Nau et al.  1981), will have important implications in the interpretation ofthe observed differences in total serum VPA levels betweenmother andnewborn. The present study was designed to compare the plasma proteinbindingof VPA in maternal and cord blood in vitro. Since the free fraction of VPA is highly dependenton the level of albumin and free fatty acids (FFA) (Perucca et al., 1981; Riva et al., 1982,1984; Albani et al., 1983) we also considered it of interest to examine the transplacental gradient of these compounds and their possible contribution to the drug binding differences betweenmotherand newborn. Methods As oxytocin has been reported to alter the plasma protein binding of certain drugs (Hamar   Levy, 1980), blood was collected from each of five healthy women on two occasions:  a) im- mediately before administration of oxytocin  i.e. 20-30 min before delivery)  Syntocinon i, Sandoz) and  b) immediately after delivery. All women had a normal gestation and an unevent- ful delivery and were free of medications  other than oxytocin) at the time the blood was collec- ted. Mixed  arterial-venous) umbilical cord blood wasobtained immediately after birth. In order to prevent spurious binding results caused by in vitro release of FFA  Albani et al., 1983) the serum wasseparated within 1 h and stored at -20°C until analysis. Drug spiking was per- formed by evaporating to dryness a solution of sodium valproate in methanol  50 mmol/l) in glass stoppered tubes prior to additionofthe serum. The protein binding of VP was assessed by using a rapidequilibrium dialysis technique 759  760 F. Albani et al. free from in vitro alterations of FFA at a temper- ature of 37°C (Riva et al, 1982). Values of free fractionrefer to the ratio between the drug con- centration in the buffer and serum dialysates at equilibrium. Valproic acid was measured in buffer and serum dialysates by g.l.c. (Riva et al., 1982). Serum albumin and FFA in drug-free serum aliquots were determined according to Laurell (1966) and Duncombe  1964) respectively. Statistical analysis was made by usingthe Student s t-test for paired data. Correlations were examined by regression analysis. 20.0 ..- 16.0   co < 8.0 a)4)   4.0- IL   Results a A Av v   vv v AA   0 400440 480 520560 600 Albumin concentration  pmol/l) 20.0 r The concentration of VPA (mean + s.d.) in the serum compartment of the dialysis cell at equil- ibrium was 527 + 62 ,umol/l  pre-oxytocin mater- nal samples), 493 ± 55 ,umol/l  post-delivery maternal samples) and 500 ± 55 umol/l(cord serum). Therefore estimatedvaluesof free frac- tionrefer to a very similar total concentration in all samples, thus avoiding the interpretative problems related to the concentration depen-dent nature of VPA binding (Riva et al., 1982). As summarized in Table 1, the free VPA frac- tion was much greater in maternal than in cord serum (P < 0.02). There was also an appreciable difference between the two maternal samples, with a mean 23 decrease in freefraction in the sera collected after delivery (P < 0.05). Serum albumin levels tended to be greater in umbilical serum but the magnitude ofthe differ- ence was small, with a wideoverlapping between maternal and cord values  Figure la). The maternalsamples with thelowest serum albumin were those collected after delivery, in spite of the fact thatthese samples showed a VPA binding greater than that observed before oxytocin. Asshown in Table 1, FFA were markedly more elevated in maternal  especially in the samples collected beforeoxytocin) than in umbilical blood. R 16.0 c   L- Co < 8.0 6) La 4.0 U 0.0 b V V V   00 AA V  VAA, 0 300 600 900 1200 FFA concentration  ItEq/1) 1500 Figure I relationship between free VPA fraction and:  a) albumin concentration;  b) FFA concentration in mixed cord serum (0); maternal serum, before oxytocin (A) and after delivery (V). The relationship between free drug fraction and serum albumin was relatively weak  r = 0.55, P < 0.05). As clearly shown in Figure la, at equivalent valuesof serum albumin the free frac- tion was much smaller in cord than in matemal blood. By contrast, a strong positive correlation could be found between free VPA fraction and FFA  r = 0.86, P < 0.001, Figure lb). Table I Free VPA fraction, albumin and FFA concentration (means + s.d.)in the samplesexamined  n = 5 for each set). Albumin FFA  N)  ,umolll)  IiEqll) Maternalserum  pre-oxytocin) 12.2 ± 2.7 505 + 51 1099 + 199 Maternal serum  after delivery) 9.9 ± 2.3* 484 + 30 746 + 338* Cord serum 6.0 + 0.8* 545 + 42 398 ± 238* * significantly different from thevalues observed in maternalserum pre- oxytocin (P < 0.05).   significantly different from the value observed in maternal serum after delivery (P < 0.05).    Short report 761 Discussion These experiments clearly demonstrate that VPA binds to cord serum much more extensively than to maternal serum. These dataconfirm pre- liminary in vivo observations made by Nau et al.  1981), who studied four newborns of epileptic mothers and quoted a mean neonatal/maternal free VP fraction ratio of 0.72. The differential binding between motherand foetus (newborn) is consistent with and explains, at leastin part, clinical observations that total VPA levels at delivery are higher in neonatalthan in maternal blood (Dickinson et al., 1979; Froscher et al., 1981; Ishizaki et al., 1981; Nau et al., 1981, Kaneko etal., 1983). The presence of additional mechanisms, however, cannot be excluded since the 3-keto-metabolite of VPA, which is not highly protein-bound, has also been found in higherconcentrations in cordthan in maternal serum (Nau et al., 1981). In serum, VPA is bound virtually entirely to albumin (Monks   Richens, 1979) and the impairment in binding observed in pregnancy is known to correlate with a decrease in albuminconcentration (Perucca et al., 1981; Riva et al., 1984). Albumin, however, did not appear to be important in determining the transplacental gradient in VPA binding. In fact the level of this protein in cord blood was onlymarginally greater than that in the maternal blood. Further- more, the free drug fraction at equivalent values of albumin concentration was clearly much higher in the maternal samples indicating that other factors weremainly responsible for the binding difference. The striking elevation of FFA in maternal samples and their correlation with free VPA strongly suggest that these factors can be identi- fied at least in part as FFA. FFA have previously been shown to displace VPA from binding sites in a concentration-dependent manner (Monks   Richens, 1979; Riva et al., 1982,1984; Albani et al., 1983, and to be a dominant factor in govern- ingthe maternal swings infreefraction and the transplacental binding gradient ofanother highly albumin-bound drug diazepam,which is also found in greater concentration in cord as com- pared withmaternal serum (Ridd et al., 1982,1983). Since the dramatic maternal elevation of FFA is confined to the stress of the perinatal period, it is unlikely that thetransplacental bind ing gradientof these drugs is maintained throughout pregnancy. A final comment is required on the decrease in free VPA fraction observed in the maternal serum afterdelivery. In studies with diazepam and salicylic acid, Hamar   Levy  1980) repor- ted that administration of oxytocin in vivo  but not, at least for salicylate, its addition in vitro) caused a decrease in serum protein binding. In our own studies we observedan increase in VPA binding after administration of oxytocin and sub- sequent delivery. The effect, however, was asso- ciated with a 30 fall in serum FFA which could itself easily account for the observedchange in binding capacity. The assessmentof any effect of oxytocinper se will require more detailed investi- gations, possibly including comparison witha group of women not treated with this drug. This work has been executed as a part of the  Progetto finalizzato Medicina preventiva e Riabilitativa of the National Research Council of Italy, grant no. 83.02528.56 References Albani, F., Riva, R., Procaccianti, G., Baruzzi, A.   Perucca, E.  1983). Free fraction of valproic acid: in vitro time-dependent increase and correlation with free fatty acid concentration in human plasma and serum. Epilepsia, 24, 65-73. Dickinson, R.G.,Harland,R. C., Lynn, R. K., Smith, W. B.   Gerber,N.  1979). Transmissionof valproic acid (Depakene) across theplacenta: half-life of the drug in motherand baby. J. Pediatr., 94, 832-835. Duncombe, W. G.  1964). The colorimetric micro- determination of non-esterified fatty acids in plasma. Clin. Chim. Acta, 9, 122-125. Froscher, W., Eichelbaum,M., Niesen, M., Altmann,D.   von Unruh, G. E.  1981). Antiepileptic therapy with carbamazepine and valproic acid during pregnancy and lactation period. In Advances in epileptology: XII Epilepsy International Sym-posium. Eds Dam, M., Gram, L.   Penry, J. K. New York: Raven Press. Hamar, C.   Levy, G.  1980). Serum protein binding of drugs and bilirubin in newborn infants and their mothers. Clin. Pharmac. Ther., 28, 58-63. Ishizaki, T., Yokochi, K., Chiba, K., Tabuchi, T.   Wagatsuma, T.  1981). Placental transfer of anti- convulsants  phenobarbital, phenytoin, valproicacid) and the elimination from neonates. Pediatric Pharmac., 1, 291-303. Kaneko, S., Otani K., Fukushima, U., Sato, T., Nomura, Y.   Ogana, Y.  1983). Transplacental passage and half-life of sodium valproate in infants born to epileptic mothers. Br. J. clin. Pharmac., 15, 503-506. Laurell, C. B.  1966). Quantitative estimationof protein by electrophoresis in agarose gel contain- ing antibodies. Analyt. Biochem., 15,45-52. Monks, A.   Richens, A.  1979). Serum protein bind- ing of valproicacid and its displacent by palmitic acid in vitro. Br. J. clin. Pharmac., 8, 187-188. Nau, H., Rating, D., Koch, S., Hauzer, I.   Helge,H.  762 F. Albani et al.  1981). Valproic acid and its metabolites:placental transfer, neonatalpharmacokinetics, transfervia mother smilk and clinical status in neonates of epileptic mothers. J. Pharmac. exp. Ther.,219, 768-777. Perucca, E., Ruprah, M.   Richens, A.  1981). Altered drug binding to serum proteins in pregnant women: therapeutic relevance. J. Roy. Soc. Med., 74,422-426. Ridd, M. J., Brown, K. F., Moore, R. G., McBride, W. G.   Nation, R. L.  1982). Diazepam plasma binding in the perinatalperiod:influence of non- esterified fatty acids. Eur. J. clin. Pharmac., 22, 153-160. Ridd, M. J., Browne K. F., Nation, R. L.   Collier, C. B.  1983).Differential transplacental binding of diazepam: causes and implications.Eur. J. clin. Pharmac., 24, 595-601. Riva,R., Albani, F., Baruzzi, A., Galvani, I.   Perucca, E.  1982). Determination of unbound valproic acid concentration in plasmaby equili- brium dialysis and gas-liquid chromatography: methodological aspects and observations in epilep- tic patients. Ther. Drug. Monit., 4, 341 352. Riva, R., Albani, F., Contin, M., Baruzzi, A., Altomare, M., Merlini, G. P., Perucca, E.  1984). Mechanisms of altered drug binding to serum pro- teins in pregnant women: studies with valproic acid. Ther. Drug. Monit.,  in press).  Received October26, 1983, accepted January 22, 1984)
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