1Division of Medical Genetics, Hôpital Sainte-Justine, Université de Montréal; 2Laboratoire de Physiologie materno-fætale, Département des Sciences Biologiques, Université du Québec à Montréal; and 3Département d'Obstétrique et de Gynécologie, Hôpital Sainte-Justine, Montreal, Quebec, Canada H3T 1C5
Submitted 1 August 2003 ; accepted in final form 20 February 2004
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ABSTRACT |
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membrane transport; valproate; maternofetal; xenobiotics; acylcarnitine
To date, few publications have described human placental carnitine transport (23, 27, 31). The first report (31), using an in vitro placental perfusion technique, concluded that carnitine is taken up by placental tissue in a stereospecific manner, indicating a mediated process. The second study (27) showed high-affinity, Na+-dependent binding of carnitine to human placental brush-border membranes but no transport. The third study (23), in JAR human placental choriocarcinoma cells, showed high-affinity Na+-dependent carnitine transport (Km = 12.3 ± 0.5 µM).
Recently, a high-affinity carnitine transporter, OCTN2, was cloned from human placenta (38, 45), but its localization in the placenta is unknown. OCTN2 is unique in that it transports carnitine with high affinity in a Na+-dependent manner and transports organic cations in a Na+-independent manner (4, 21, 44). Mutations in the human OCTN2 gene cause primary systemic carnitine deficiency (SCD; OMIM 212140 [OMIM] ), an autosomal recessive disease associated with cardiomyopathy, muscle weakness, fasting hypoglycemia, and sudden death (13). The deduced OCTN2 protein has 557 amino acids, a molecular mass of 63 kDa, and 12 putative transmembrane domains.
To date, three carnitine transporters have been identified: OCTN1, OCTN2, and OCTN3. They belong to the organic cation transporter (OCT) family and differ in their affinity and capacity for carnitine transport, energization of transport, and sensitivity to inhibitors. OCT transporters are expressed in several tissues, but in most cases their intracellular localization is unknown.
Our aim was to characterize the mechanism of carnitine transport in human placenta, specifically at the brush-border membrane (BBM). The BBM forms the interface between the fetus and the maternal circulation, and BBM transport is the first step of uptake from mother to fetus. The results in this article support the hypothesis that human placental BBM carnitine uptake is mediated by OCTN2.
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MATERIALS AND METHODS |
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Membrane vesicle preparation. BBM vesicles were isolated from normal human term placentas obtained within 1 h of delivery using the Mg2+ precipitation method of Schmitz et al. (32). Briefly, the placentas were placed in 0.9% NaCl at 4°C, and the cord, amniochorion, and decidua were removed. Tissue obtained from the central part of the placenta was cut into 2- to 5-mm fragments and homogenized at 4°C in (in mM) 50 mannitol, 5 EGTA, and 10 Tris-HEPES (pH 7.5) with a small Waring blender (3 times for 30 s each). MgCl2 was added to a final concentration of 10 mM, and the homogenate was stirred on ice for 20 min. The homogenate was centrifuged for 10 min at 2,000 g, and then the supernatant was centrifuged for 20 min at 20,000 g. The pellet was resuspended in intravesicular buffer (see below) using a Potter-Elvehjem apparatus and then centrifuged for 15 min at 1,900 g. The resultant supernatant was centrifuged at 30,900 g for 20 min. With a 25-gauge needle and syringe, the final BBM pellet was resuspended in intravesicular buffer (in mM: 50 Tris-HEPES, 250 KCl, and 125 mannitol, pH 7.5) at a protein concentration of 1520 mg/ml. All centrifugation steps were performed in a Beckman J2-21 rotor. A sample of each final BBM preparation was removed for protein determination according to the method of Lowry et al. (14) using bovine serum albumin (BSA) as a standard. BBM were stored in liquid nitrogen until uptake studies. The basal plasma membrane (BPM) fraction was prepared using a discontinuous Ficoll gradient as described by Lafond et al. (11). The protocol was approved by the ethics committees of St. Justine's Hospital. Placentas were used after informed consent was obtained from mothers after they gave birth.
Purity of BBM and BPM. Membrane purity was assessed by measuring the enrichment of marker enzymes alkaline phosphatase (8) for the BBM and Na+/K+-ATPase (15) for the BPM.
Uptake studies. Uptake of [3H]carnitine was measured by using a rapid filtration method with manifold with cellulose nitrate filters of 0.65-µm pore size from Sartorius (Göttingen, Germany). The human placental BBM were usually resuspended in a final concentration of 1520 mg/ml in (in mM) 50 Tris-HEPES, 250 KCl, and 125 mannitol, pH 7.5. Next, a 4-µl vesicle suspension was mixed in a final volume of 50 µl of incubation medium (in mM: 50 Tris-HEPES, 100 KCl, 150 NaCl, and 125 mannitol, pH 7.5). Briefly, the reaction was started by mixing the vesicles with the incubation medium, to which the required amount of [3H]carnitine (82 Ci/mmol) had been added. The reaction was stopped by adding 1 ml of ice-cold stop solution. The solution was then filtered on 0.65-µm (Micro Filtration System) nitrogen cellulose filters and washed three times with 1 ml of nonradioactive ice-cold stop solution. Filters were dissolved in minivials by 15-min incubation with 5 ml of Filter Count (United Technologies Packard) and continuous shaking. 3H radioactivity was determined by using a Minaxi Tri-Carb series 4000 model 4450 scintillation counter (United Technologies Packard). All vials were counted for 5 min. Values presented represent the mean of triplicate or quadruplicate determinations. Corrections were made for [3H]carnitine bound to the filters in the absence of vesicles, which was always <0.1% of added counts.
Assays with xenobiotic inhibitors. Membrane vesicles were prepared as described above. The incubation medium contained (final concentrations) 1 µM L-[3H]carnitine (4 µCi/assay), 50 mM Tris-HEPES, 0.1 mM MgSO4, 100 mM KCl, 150 mM NaCl, 125 mM mannitol, pH 7.5, and 500 µM of the xenobiotic. Transport was started by adding 4 µl of membrane suspension to 46 µl of preheated incubation medium (37°C). The reaction was stopped after 8 min, which was determined to be optimal in preliminary studies.
Assays with carnitine analogs. BBM vesicles were incubated as described above in the presence of 50 µM of different acylcarnitine (acetyl-D,L-, propionyl-L-, butyryl-L-, isovaleryl-L-, octanoyl-L-, and palmitoyl-L-carnitine). These analogs are known to significantly inhibit carnitine uptake via OCTN2 (21).
Membrane localization of OCTN2 by Western blot analysis. Rabbit polyclonal antibodies were raised (Research Genetics, Huntsville AL) against a synthetic polypeptide, QWQIQSQTRMQKDGEESPT, corresponding to amino acids 532550 of mouse OCTN2. Whole placentas, BBM, or BPM fractions were isolated and homogenized in 4 ml of buffer containing 10 mM Tris-HCl and 50 mM mannitol (pH 7.4) using a Polytron homogenizer. The solution was then dispersed ultrasonically. After mixing in 2x sample buffer (4% SDS, 20% glycerol, 200 mM dithiothreitol, 120 mM Tris, pH 6.8, and 0.002% bromphenol blue), samples were denatured in a boiling water bath for 5 min and then resolved on 7.5% SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore, Bedford, MA). The membrane was incubated for 30 min in PBS buffer (in mM: 150 NaCl, 2.5 KCl, 5 Na2HPO4, and 1.5 KH2PO4) containing 0.02% wt/vol sodium azide and 12% (vol/vol) skim milk and then was incubated overnight with polyclonal antipeptide antibody in PBS buffer containing 0.02% sodium azide and 1% BSA, washed in PBS with 0.1% Tween 20, and incubated with secondary antibody, goat anti-rabbit IgG, and horseradish peroxidase-linked whole antibody (Sigma, St. Louis, MO). The membrane was washed as described above, and the proteins were detected by chemiluminescence using BM chemiluminescence ELISA substrate (POD) (Boehringer Mannheim, Laval, QC, Canada). Molecular weights were estimated by using prestained SDS-PAGE standards, broad range (Bio-Rad Laboratories, Hercules, CA).
Statistical analysis. Results are reported as means ± SE of at least three samples. Statistical analyses were performed using analysis of variance (ANOVA) with GraphPad InStat (GraphPad Software, San Diego, CA). For the determination of uptake kinetic parameters, the GraphPad Prism version 3.00 software program was used. Inhibition studies using valproate were analyzed using linear regression of Lineweaver-Burk plots.
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RESULTS |
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Binding vs. transport of L-[3H]carnitine in placental BBM. Nonspecific binding of the substrate to the vesicle surface, which causes an overestimation of transport into the vesicle, was calculated as the uptake of L-carnitine by the vesicles at infinite osmolarity. At 50 min of incubation, increasing medium osmolality decreased L-carnitine uptake (Fig. 1), indicating that L-carnitine is taken up into an osmotically sensitive vesicular space. The relationship between uptake and the reciprocal of osmolality was linear. The intercept on the ordinate (zero intravesicular volume) is a measurement of nonspecific binding (2). The binding of L-carnitine measured in the presence of a Na+ gradient (50.4 pmol/mg protein) represents 36% of the L-carnitine uptake measured under standard conditions.
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Effect of structural analogs of carnitine on L-carnitine transport by human placental BBM vesicles. The uptake of L-[3H]carnitine by human placental BBM vesicles was characterized in the presence of various carnitine analogs known to inhibit carnitine transport in other systems (2, 21, 35, 44) (Table 1). D-, Acetyl-D,L-, propionyl-L-, butyryl-L-, isovaleryl-L-, octanoyl-L-, and palmitoyl-L-carnitine all significantly inhibited carnitine transport by the BBM vesicles (inhibition between 29 and 75%; P < 0.001). These results show that the transport is specific for carnitine and its acyl derivatives. Short-chain as well as long-chain acylcarnitines are effective substrates.
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Detection and localization of OCTN2. Extracts of human placental BBM and BPM were analyzed by Western blotting. The polyclonal OCTN2-specific antibody detected a BBM protein with an apparent molecular mass of 80 kDa (Fig. 7). No OCTN2 immunoreactivity was observed in BPM.
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DISCUSSION |
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Na+, temperature, pH, and osmolarity dependence of carnitine uptake. Our study demonstrates that the uptake of L-carnitine declined with increasing external osmolarity, suggesting that L-carnitine was not only adsorbed onto vesicles but also transported into the intravesicular space (Fig. 1). This contrasts with data reported by Roque et al. (27). We performed osmolarity studies after 10 min as described by Roque et al., and like those investigators, we found no osmolarity dependence under these conditions (data not shown). This suggests that the conditions used by Roque et al. may not have permitted the reaction to continue until completion and may have lacked the sensitivity necessary to detect concentration dependence. Our data suggest that these preparations are a suitable model in which to study the characteristics of carnitine uptake across the apical membrane of the human placental syncytiotrophoblast.
Our study shows Na+- and temperature-dependent transport in placental BBM uptake of carnitine (Figs. 2 and 3). The uptake was high at 37°C and low at 4°C. Moreover, carnitine uptake by placental BBM was not significantly affected by pH (Fig. 5).
Kinetics and inhibition of carnitine transport.
We demonstrated that L-carnitine uptake in placental BBM vesicles is a saturable process with high affinity (Km = 11.09 ± 1.32 µM) (Fig. 6A) similar to the known properties of OCTN2. Similar high-affinity transport of carnitine was observed in human kidney cells (38), in HLF cells (47), and in Caco-2 cells (3). The affinity constant obtained in our study, Km = 11.09 ± 1.32 µM, is also close to that reported for the Na+-dependent carnitine transport in human placental choriocarcinoma cells (Km 12.3 µM) (23).
Many of the properties observed for human placental L-carnitine transport resemble those reported for OCTN2, including sodium and temperature dependence (Figs. 2 and 3) and high affinity for carnitine. The Km for L-carnitine transport in human placental BBM, 11.09 ± 1.32 µM, is of the same order as that reported for human kidney OCTN2 (4.3 µM) (38).
An apparent discrepancy between our results and those of Roque et al. (27) is Roque et al.'s conclusion regarding carnitine binding but not transport in BBM. We think that the difference in our conclusions is due to technical reasons. Using 10-min incubation to study the effect of osmolarity on L-carnitine uptake and/or binding as described by Roque et al. (27), we reproduced their results, finding no change of carnitine binding and/or uptake with osmolarity (data not shown). In contrast, a 50-min incubation period (Fig. 1) allows transport to proceed sufficiently to reveal a clear effect of medium osmolarity on L-carnitine handling, confirming that L-carnitine is transported into BBM vesicles and is not simply bound to their exterior.
To better define L-carnitine transport via OCTN2, we examined some structural analogs of carnitine, such as short-chain fatty acid esters of carnitine and compounds that inhibit L-carnitine transport via OCTN2 (4, 21, 25, 35, 37, 44). Short-chain acyl esters of L-carnitine are used in the treatment of a wide range of disorders (34, 42) and also can accumulate abnormally in several inborn errors of metabolism (26). As shown in Table 1, the short-chain acyl esters of L-carnitine significantly inhibited carnitine uptake by human placental BBM. Our results also show that carnitine uptake was significantly inhibited by verapamil, valproate, pyrilamine, and tetraethylammonium (Table 1) as previously reported for OCTN2-mediated L-[3H]carnitine uptake in both humans and rats (21, 44). These drugs are sometimes used in pregnancy, such as verapamil for fetal tachyarrhythmia (7) and valproate for maternal epilepsy. Of note, valproate is a teratogen (9), and the therapeutic level of valproate, 300700 µM, coincides with that used in our study (500 µM). Valproate is known to interfere with carnitine-related metabolic processes and can induce carnitine deficiency in cells (21, 24, 40) and patients (1, 41).
Different modes of inhibition have been described for the effect of drugs on carnitine transport. For valproate, we demonstrated a competitive inhibition of L-carnitine uptake in human placental BBM, with decrease of the affinity for carnitine in the presence of valproate (Km = 32.30 ± 3.46 µM) (Fig. 6, A and B). The maximum velocity of L-carnitine transport was not affected (37.63 ± 1.17 pmol·mg protein1·min1). Previous studies using HEK-293 cells transfected by human OCTN2 (21) showed competitive inhibition of L-carnitine transport by valproate. Recently, it was reported (17) that valproate is transported by a proton-dependent transporter in human placental BBM vesicles with a Km of 1.04 mM. Of note, other studies suggest that the inhibition of OCTN2 by verapamil may involve both competitive and noncompetitive inhibition (19).
Fetal valproate syndrome is a characteristic cluster of malformations and intellectual disabilities reported in children exposed to valproate during fetal life (9). The relationships, if any, of OCTN2-mediated carnitine transport to fetal valproate syndrome are speculative but merit further study. Similarly, OCTN2-mediated placental transport may be pertinent to the therapeutic use of acylcarnitines or the accumulation of acylcarnitines in maternal or fetal inborn errors of organic acid metabolism.
Immunochemical identification and localization. To confirm that the uptake of L-carnitine in human placenta is mediated by OCTN2, a mouse OCTN2-specific antibody was produced and used against a total homogenate of human placenta and against human placental BBM and BPM vesicles (Fig. 7). Immunoblotting of total homogenate and of the BBM preparation showed an immunoreactivity to the antibodies against OCTN2 (Fig. 7). However, no reactivity was observed in BPM preparation. The molecular mass (80 kDa) of the reactive band differs from that deduced for OCTN2 (63 kDa), perhaps because of atypical proprieties of migration related to posttranslational modification such as glycosylation, or perhaps representing a previously unreported OCTN2 isoform. Similar differences in molecular mass between that predicted from primary structure and that observed on electrophoretic migration were reported in mice by Tamai et al. (37), who used antibodies against mouse OCTN2 and obtained bands of apparent molecular mass between 70 and 80 kDa in several mouse tissues.
Our Western blotting results are in accord with many studies showing that OCTN2 is localized in BBM of other epithelial tissues, including mouse and rat kidney (36, 12), chicken intestine (2), and human intestinal Caco-2 cells (3).
The absence of reactivity with the anti-OCTN2 antibody in our human placental BPM vesicles supports the notion that placental OCTN2 is confined to BBM (Fig. 7). In BPM, carnitine may diffuse or be transported by another molecule. To our knowledge, carnitine uptake has not been described in placental BPM of any species. It will be interesting to compare L-carnitine uptake in the BBM and the BPM. Transport mechanisms frequently differ between BBM and BPM, such as those for lactate (6) and L-tryptophan (10).
Our study is consistent with a major role for OCTN2 in human placental carnitine transport. We cannot exclude a role for other transporters. For instance, the OCTN transporter subfamily includes two other members: OCTN1 and OCTN3. Each can transport carnitine, although with characteristics different from those of OCTN2 (46, 37). Human OCTN1 is expressed in several tissues, including placenta, and transports carnitine in a Na+-dependent manner, although the affinity of human OCTN1 for carnitine has not been reported (39, 37, 46). Rat intestinal OCTN1 reportedly interacts with carnitine with low affinity and in a Na+-independent manner (43). In mice, OCTN1 mediates Na+-dependent carnitine uptake with low affinity, although its expression in placenta has not been studied (37). OCTN3 has been cloned only in mice (37) and mediates Na+-independent carnitine uptake. In mice, it is expressed predominantly in testis and weakly in kidney, but its expression in the placenta was not described. Because we found that carnitine uptake by human placental BBM vesicles was Na+ dependent, it is unlikely that OCTN3 plays a role in carnitine transport by placental BBM. We cannot exclude some role of OCTN1 or other transporters in carnitine uptake by human placental BBM vesicles.
We used L-proline to test whether L-carnitine is transported by the ATB0,+ system, which transports both proline and carnitine (18) and is principally expressed in intestine, lung, and mammary gland. ATB0,+ is a Na+-dependent transporter with a low affinity for carnitine (Km = 0.83 mM). Because proline did not inhibit carnitine uptake, and in view of the high affinity of placental carnitine transport (Fig. 6), there is no evidence in favor of ATB0,+-mediated carnitine uptake in human placental BBM.
In summary, the properties of carnitine transport in BBM vesicles and the demonstration herein of immunoreactive OCTN2 in human placental BBM points to a hypothesis that OCTN2 may mediate most and possibly all of maternofetal carnitine transport. Because OCTN2 is multifunctional and mediates the transport of many drugs, we speculate that this transporter is also involved in the transfer of these drugs from mother to fetus. These results thus have potential clinical implications for maternofetal nutrient transfer and for the pharmacology and transfer of cationic drugs in the fetus.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Durán JM, Peral MJ, Calonge ML, and Ilundáin AA. Functional characterization of intestinal L-carnitine transport. J Membr Biol 185: 6574, 2002.[CrossRef][ISI][Medline]
3. Elimrani I, Lahjouji K, Seidman E, Roy MJ, Mitchell GA, and Qureshi I. Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 284: G863G871, 2003.
4. Ganapathy ME, Huang W, Rajan DP, Carter AL, Sugawara M, Iseki K, Leibach FH, and Ganapathy V. -Lactam antibiotics as substrates for OCTN2, an organic cation/carnitine transporter. J Biol Chem 275: 16991707, 2000.
5. Hahn P. The development of carnitine synthesis from -butyrobetaine in the rat. Life Sci 28 : 10571060, 1981.[CrossRef][ISI][Medline]
6. Inuyama M, Ushigome F, Emoto A, Koyabu N, Satoh S, Tsukimori K, Nakano H, Ohtani H, and Sawada Y. Characteristics of L-lactic acid transport in basal membrane vesicles of human placental syncytiotrophoblast. Am J Physiol Cell Physiol 283: C822C830, 2002.
7. Ito S. Transplacental treatment of fetal tachycardia: implications of drug transporting proteins in placenta. Semin Perinatol 25: 196201, 2001.[ISI][Medline]
8. Kempson SA, Kim JK, Northrup TE, Knox FG, and Dousa TP. Alkaline phosphatase in adaptation to low dietary phosphate intake. Am J Physiol Endocrinol Metab Gastrointest Physiol 237: E465E473, 1979.
9. Kozma C. Valproic acid embryopathy: report of two siblings with further expansion of the phenotypic abnormalities and a review of the literature. Am J Med Genet 98: 168175, 2001.[CrossRef][ISI][Medline]
10. Kudo Y and Boyd CAR. Characterisation of L-tryptophan transporters in human placenta: a comparison of brush border and basal membrane vesicles. J Physiol 531: 405416, 2001.
11. Lafond J, Moukdar F, Rioux A, Ech-Chadli H, Brissette L, Robidoux J, Masse A, and Simoneau L. Implication of ATP and sodium in arachidonic acid incorporation by placental syncytiotrophoblast brush border and basal plasma membranes in the human. Placenta 21: 661669, 2000.[CrossRef][ISI][Medline]
12. Lahjouji K, Malo C, Mitchell GA, and Qureshi IA. L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles. Biochim Biophys Acta 1558: 8293, 2002.[ISI][Medline]
13. Lahjouji K, Mitchell GA, and Qureshi IA. Carnitine transport by organic cation transporters and systemic carnitine deficiency. Mol Genet Metab 73: 287297, 2001.[CrossRef][ISI][Medline]
14. Lowry FH, Rosebrough NJ, Farr AL, and Randall RF. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 269275, 1951.
15. Mircheff AK and Wright EM. Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na+,K+-ATPase rich membranes and the distribution of enzyme activities. J Membr Biol 28: 309333, 1976.[ISI][Medline]
16. Montgomery JA and Mamer OA. Measurement of urinary free and acylcarnitines: quantitative acylcarnitine profiling in normal humans and in several patients with metabolic errors. Anal Biochem 176: 8595, 1989.[ISI][Medline]
17. Nakamura H, Ushigome F, Koyabu N, Satoh S, Tsukimori K, Nakano H, Ohtani H, and Sawada Y. Proton gradient-dependent transport of valproic acid in human placental brush-border membrane vesicles. Pharm Res 19: 154161, 2002.[CrossRef][ISI][Medline]
18. Nakanishi T, Hatanaka T, Huang W, Prasad PD, Leibach FH, Ganapathy ME, and Ganapathy V. Na+- and Cl-coupled active transport of carnitine by the amino acid transporter ATB0,+ from mouse colon expressed in HRPE cells and Xenopus oocytes. J Physiol 532: 297304, 2001.
19. Ohashi R, Tamai I, Inano A, Katsura M, Sai Y, Nezu J, and Tsuji A. Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein. J Pharmacol Exp Ther 302: 12861294, 2002.
20. Ohashi R, Tamai I, Nezu JI, Nikaido H, Hashimoto N, Oku A, Sai Y, Shimane M, and Tsuji A. Molecular and physiological evidence for multifunctionality of carnitine/organic cation transporter OCTN2. Mol Pharmacol 59: 358366, 2001.
21. Ohashi R, Tamai I, Yabuuchi H, Nezu J, Oku A, Sai Y, Shimane M, and Tsuji A. Na+-dependent carnitine transport by organic cation transporter (OCTN2): its pharmacological and toxicological relevance. J Pharmacol Exp Ther 291: 778784, 1999.
22. Penn D, Schmidt-Sommerfeld E, and Wolf H. Carnitine deficiency in premature infants receiving total parenteral nutrition. Early Hum Dev 4: 2334, 1980.[ISI][Medline]
23. Prasad PD, Huang W, Ramamoorthy S, Carter AL, Leibach FH, and Ganapathy V. Sodium-dependent carnitine transport in human placental choriocarcinoma cells. Biochim Biophys Acta 1284: 109117, 1996.[ISI][Medline]
24. Raskind JY and El-Chaar GM. The role of carnitine supplementation during valproic acid therapy. Ann Pharmacother 34: 630638, 2000.[Abstract]
25. Rebouche CJ and Mack DL. Sodium gradient-stimulated transport of L-carnitine into renal brush border membrane vesicles: kinetics, specificity, and regulation by dietary carnitine. Arch Biochem Biophys 235: 393402, 1984.[ISI][Medline]
26. Roe CR and Coates PM. Acyl-CoA dehydrogenase deficiencies. In: The Metabolic Basis of Inherited Disease, edited by Scriver CR, Beaudet AL, Sly WS, and Valle D. New York: McGraw-Hill, 1989, vol. I, p. 889914.
27. Roque AS, Prasad PD, Bhatia JS, Leibach FH, and Ganapathy V. Sodium-dependent high-affinity binding of carnitine to human placental brush border membranes. Biochim Biophys Acta 1282: 274282, 1996.[ISI][Medline]
28. Scaglia F, Wang Y, and Longo N. Functional characterization of the carnitine transporter defective in primary carnitine deficiency. Arch Biochem Biophys 364: 99106, 1999.[CrossRef][ISI][Medline]
29. Schiff D, Chan G, Seccombe D, and Hahn P. Plasma carnitine levels during intravenous feeding of the neonate. J Pediatr 95: 10431046, 1979.[ISI][Medline]
30. Schmidt-Sommerfeld E, Novak M, Penn D, Wieser PB, Buch M, and Hahn P. Carnitine and the development of newborn adipose tissue. Pediatr Res 12: 660664, 1978.[ISI][Medline]
31. Schmidt-Sommerfeld E, Penn D, Sodha RJ, Progler M, and Schneider H. Transfer and metabolism of carnitine and carnitine esters in the in vitro perfused human placenta. Pediatr Res 19: 700706, 1985.[Abstract]
32. Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, and Crane RK. Purification of the human intestinal brush border membrane. Biochim Biophys Acta 323: 98112, 1973.[ISI][Medline]
33. Shenai JP and Borum PR. Tissue carnitine reserves of newborn infants. Pediatr Res 18 : 679682, 1984.[Abstract]
34. Spagnoli A, Lucca U, Menasce G, Bandera L, Cizza G, Forloni G, Tettamanti M, Frattura L, Tiraboschi P, and Comelli M. Long-term acetyl-L-carnitine treatment in Alzheimer's disease. Neurology 41: 17261732, 1991.[Abstract]
35. Stieger B, O'Neill B, and Krähenbühl S. Characterization of L-carnitine transport by rat kidney brush-border-membrane vesicles. Biochem J 309: 643647, 1995.[ISI][Medline]
36. Tamai I, China K, Sai Y, Kobayashi D, Nezu J, Kawahara E, and Tsuji A. Na+-coupled transport of L-carnitine via high-affinity carnitine transporter OCTN2 and its subcellular localization in kidney. Biochim Biophys Acta 1512: 273284, 2001.[ISI][Medline]
37. Tamai I, Ohashi R, Nezu J, Sai Y, Kobayashi D, Oku A, Shimane M, and Tsuji A. Molecular and functional characterization of organic cation/carnitine transporter family in mice. J Biol Chem 275: 4006440072, 2000.
38. Tamai I, Ohashi R, Nezu J, Yabuuchi H, Oku A, Shimane M, Sai Y, and Tsuji A. Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2. J Biol Chem 273: 2037820382, 1998.
39. Tamai I, Yabuuchi H, Nezu J, Sai Y, Oku A, Shimane M, and Tsuji A. Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1. FEBS Lett 419: 107111, 1997.[CrossRef][ISI][Medline]
40. Tein I, DiMauro S, Xie ZW, and De Vivo DC. Valproic acid impairs carnitine uptake in cultured human skin fibroblasts. An in vitro model for the pathogenesis of valproic acid-associated carnitine deficiency. Pediatr Res 34: 281287, 1993.[Abstract]
41. Verrotti A, Greco R, Morgese G, and Chiarelli F. Carnitine deficiency and hyperammonemia in children receiving valproic acid with and without other anticonvulsant drugs. Int J Clin Lab Res 29: 3640, 1999.[CrossRef][ISI][Medline]
42. Wiseman LR and Brogden RN. Propionyl L-carnitine. Drugs Aging 12: 243250, 1998.[ISI][Medline]
43. Wu X, George RL, Huang W, Wang H, Conway SJ, Leibach FH, and Ganapathy V. Structural and functional characteristics and tissue distribution pattern of rat OCTN1, an organic cation transporter, cloned from placenta. Biochim Biophys Acta 1466: 315327, 2000.[ISI][Medline]
44. Wu X, Huang W, Prasad PD, Seth P, Rajan DP, Leibach FH, Chen J, Conway SJ, and Ganapathy V. Functional characteristics and tissue distribution pattern of organic cation transporter 2 (OCTN2), an organic cation/carnitine transporter. J Pharmacol Exp Ther 290: 14821492, 1999.
45. Wu X, Prasad PD, Leibach FH, and Ganapathy V. cDNA sequence, transport function, and genomic organization of human OCTN2, a new member of the organic cation transporter family. Biochem Biophys Res Commun 246: 589595, 1998.[CrossRef][ISI][Medline]
46. Yabuuchi H, Tamai I, Nezu J, Sakamoto K, Oku A, Shimane M, Sai Y, and Tsuji A. Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent transport of organic cations. J Pharmacol Exp Ther 289: 768773, 1999.
47. Yokogawa K, Miya K, Tamai I, Higashi Y, Nomura M, Miyamoto KI, and Tsuji A. Characteristics of L-carnitine transport in cultured human hepatoma HLF cells. J Pharm Pharmacol 51: 935940, 1999.[ISI][Medline]