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Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATB0,+, LAT1 and LAT2.

Wongthai P, Hagiwara K, Miyoshi Y, Wiriyasermkul P, Wei L, Ohgaki R, Kato I, Hamase K, Nagamori S, Kanai Y - Cancer Sci. (2015)

Bottom Line: Among aromatic amino acid transporters, ATB(0,+), LAT1 and LAT2 were found to transport BPA with Km values of 137.4 ± 11.7, 20.3 ± 0.8 and 88.3 ± 5.6 μM, respectively.Uptake experiments in cancer cell lines revealed that the LAT1 protein amount was the major determinant of BPA uptake at 100 μM, whereas the contribution of ATB(0,+) became significant at 1000 μM, accounting for 20-25% of the total BPA uptake in MCF-7 breast cancer cells.ATB(0,+), LAT1 and LAT2 transport BPA at affinities comparable with their endogenous substrates, suggesting that they could mediate effective BPA uptake in vivo.

View Article: PubMed Central - PubMed

Affiliation: Division of Bio-system Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Suita, Japan.

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Related in: MedlinePlus

Separation of p-boronophenylalanine (BPA) by HPLC. Chromatograms showing the separation of a BPA standard (0.2 pmol) (a), amino acid standards (0.2 pmol each) (b), a sample from the oocyte expressing LAT1 (c), and the control oocyte not expressing LAT1 (d). (a, b) The BPA peak was identified by retention time and spike study (not shown). Similarly, by comparison with amino acid standards and a spike study (not shown), the peaks neighboring BPA in oocyte samples were identified as alanine (Ala) and proline (Pro). (c, d) The LAT1-expressing oocyte and non-expressing control oocyte were incubated in the uptake buffer containing BPA. Samples from the oocytes were separated by HPLC. The increased BPA peak height in (c) showed that the uptake of BPA was mediated by LAT1. Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; His, histidine; Ser, serine.
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fig01: Separation of p-boronophenylalanine (BPA) by HPLC. Chromatograms showing the separation of a BPA standard (0.2 pmol) (a), amino acid standards (0.2 pmol each) (b), a sample from the oocyte expressing LAT1 (c), and the control oocyte not expressing LAT1 (d). (a, b) The BPA peak was identified by retention time and spike study (not shown). Similarly, by comparison with amino acid standards and a spike study (not shown), the peaks neighboring BPA in oocyte samples were identified as alanine (Ala) and proline (Pro). (c, d) The LAT1-expressing oocyte and non-expressing control oocyte were incubated in the uptake buffer containing BPA. Samples from the oocytes were separated by HPLC. The increased BPA peak height in (c) showed that the uptake of BPA was mediated by LAT1. Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; His, histidine; Ser, serine.

Mentions: Standard BPA and amino acid mixture were separated by HPLC after pre-column derivatization with NBD-F. NBD-BPA was eluted between NBD-alanine and NBD-proline (Fig.1a,b). NBD-BPA (0.1 pmol/injection) was detected with our HPLC specifications. BPA standards were linearly quantified from 0.1 to 12 pmol with a correlation coefficient r = 0.9995. To establish HPLC determination of BPA in biological sample, BPA transported into Xenopus oocytes by LAT1 was separated by HPLC (Fig.1c,d). The BPA peak of LAT1-expressing oocyte was higher than that of the control, indicating that BPA uptake by LAT1 was detected by this analytical procedure. Quantitative estimation of BPA in biological sample was carried out when the BPA peak height was more than two times higher than the background. To assess the recovery of BPA, samples prepared from oocytes were exogenously supplemented with 1 pmol of BPA before homogenization. The recovery was estimated to be 97 ± 1.25% (mean ± SD, n = 3).


Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATB0,+, LAT1 and LAT2.

Wongthai P, Hagiwara K, Miyoshi Y, Wiriyasermkul P, Wei L, Ohgaki R, Kato I, Hamase K, Nagamori S, Kanai Y - Cancer Sci. (2015)

Separation of p-boronophenylalanine (BPA) by HPLC. Chromatograms showing the separation of a BPA standard (0.2 pmol) (a), amino acid standards (0.2 pmol each) (b), a sample from the oocyte expressing LAT1 (c), and the control oocyte not expressing LAT1 (d). (a, b) The BPA peak was identified by retention time and spike study (not shown). Similarly, by comparison with amino acid standards and a spike study (not shown), the peaks neighboring BPA in oocyte samples were identified as alanine (Ala) and proline (Pro). (c, d) The LAT1-expressing oocyte and non-expressing control oocyte were incubated in the uptake buffer containing BPA. Samples from the oocytes were separated by HPLC. The increased BPA peak height in (c) showed that the uptake of BPA was mediated by LAT1. Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; His, histidine; Ser, serine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376436&req=5

fig01: Separation of p-boronophenylalanine (BPA) by HPLC. Chromatograms showing the separation of a BPA standard (0.2 pmol) (a), amino acid standards (0.2 pmol each) (b), a sample from the oocyte expressing LAT1 (c), and the control oocyte not expressing LAT1 (d). (a, b) The BPA peak was identified by retention time and spike study (not shown). Similarly, by comparison with amino acid standards and a spike study (not shown), the peaks neighboring BPA in oocyte samples were identified as alanine (Ala) and proline (Pro). (c, d) The LAT1-expressing oocyte and non-expressing control oocyte were incubated in the uptake buffer containing BPA. Samples from the oocytes were separated by HPLC. The increased BPA peak height in (c) showed that the uptake of BPA was mediated by LAT1. Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; His, histidine; Ser, serine.
Mentions: Standard BPA and amino acid mixture were separated by HPLC after pre-column derivatization with NBD-F. NBD-BPA was eluted between NBD-alanine and NBD-proline (Fig.1a,b). NBD-BPA (0.1 pmol/injection) was detected with our HPLC specifications. BPA standards were linearly quantified from 0.1 to 12 pmol with a correlation coefficient r = 0.9995. To establish HPLC determination of BPA in biological sample, BPA transported into Xenopus oocytes by LAT1 was separated by HPLC (Fig.1c,d). The BPA peak of LAT1-expressing oocyte was higher than that of the control, indicating that BPA uptake by LAT1 was detected by this analytical procedure. Quantitative estimation of BPA in biological sample was carried out when the BPA peak height was more than two times higher than the background. To assess the recovery of BPA, samples prepared from oocytes were exogenously supplemented with 1 pmol of BPA before homogenization. The recovery was estimated to be 97 ± 1.25% (mean ± SD, n = 3).

Bottom Line: Among aromatic amino acid transporters, ATB(0,+), LAT1 and LAT2 were found to transport BPA with Km values of 137.4 ± 11.7, 20.3 ± 0.8 and 88.3 ± 5.6 μM, respectively.Uptake experiments in cancer cell lines revealed that the LAT1 protein amount was the major determinant of BPA uptake at 100 μM, whereas the contribution of ATB(0,+) became significant at 1000 μM, accounting for 20-25% of the total BPA uptake in MCF-7 breast cancer cells.ATB(0,+), LAT1 and LAT2 transport BPA at affinities comparable with their endogenous substrates, suggesting that they could mediate effective BPA uptake in vivo.

View Article: PubMed Central - PubMed

Affiliation: Division of Bio-system Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Suita, Japan.

Show MeSH
Related in: MedlinePlus