J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.M500185-JLR200 on August 1, 2005

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Journal of Lipid Research, Vol. 46, 2295-2298, October 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Separation of polyprenol and dolichol by monolithic silica capillary column chromatography

Takeshi Bamba*,{dagger}, Eiiciro Fukusaki1,*, Hiroshi Minakuchi§, Yoshihisa Nakazawa{dagger} and Akio Kobayashi*

* Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
{dagger} Hitachi Zosen Corporation, 2-2-11 Funamachi, Taisyou-ku, Osaka 551-0022, Japan
§ Kyoto Monotech, 13 Shimotsubayashi Shibanomiya-cho, Nishikyo-ku, Kyoto, Kyoto 615- 8035, Japan

Published, JLR Papers in Press, August 1, 2005. DOI 10.1194/jlr.M500185-JLR200

1 To whom correspondence should be addressed. e-mail: fukusaki{at}bio.eng.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
We attempted an analysis of naturally occurring polyprenol and dolichol using a monolithic silica capillary column in HPLC. First, the separation of the polyprenol mixture alone was performed using a 250 x 0.2 mm inner diameter (ID) octadecylsilyl (ODS)-monolithic silica capillary column. The resolution of the separation between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) exceeded by ≥2-fold the level recorded when using a conventional ODS-silica particle-packed column (250 x 4.6 mm ID) under the same elution conditions. Next, the mixture of the prenol type (polyprenol) and dolichol type (dihydropolyprenol) was subjected to this capillary HPLC system, and the separation of each homolog was successfully achieved. During the analysis of polyprenol fraction derived from Eucommia ulmoides leaves, dolichols were found as a single peak, including all-trans-polyprenol and cis-polyprenol previously identified.

This sensitive high-resolution system is very useful for the analysis of compounds that are structurally close to polyprenols and dolichols and that have a low content.

Abbreviations: ODS, octadecylsilyl; SFC, supercritical fluid chromatography

Supplementary key words high-performance liquid chromatography • profiling • metabolomics • metabolome


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Polyprenol is the generic name for linear 1,4-polyprenyl alcohols. Naturally occurring polyprenols can be classified into four categories (Fig. 1) : I) all trans form; only three such polyprenols are known, namely, solanesol (9-mer), spadicol (10-mer), and long-chain trans-polyprenol from Eucommia ulmoides Oliver; II) trans-trans-trans-polycis-prenols of the ficaprenol type; III) trans-trans-polycis-prenols, such as the bacteria prenol and beturaprenol types; IV) the dolichol type; the {alpha} terminal is saturated only in the dolichol type.



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  		Fig. 1. Chemical structures of natural polyprenols.

 
Since the first report of a polyprenol isolated from tobacco, named solanesol (1), further polyprenols have been isolated from animals, microbes, plants, etc. (2–4). The best-known dolichol plays an important role as a sugar-carrying intermediate in the biosynthesis of glycoproteins (5, 6). There are also many reports concerning the structure and chain-length distribution of polyprenols (4, 7–10).

For polyprenol analyses, reverse-phase HPLC using an octadecylsilyl (ODS)-silica particle-packed column has been widely used (8). This system of analysis is useful for separating polyprenol homologs based on the degree of polymerization. However, its resolution is insufficient for the baseline separation of geometric isomers and long-chain polyprenols, prompting us to develop recently a high-resolution analytical system for polyprenol, using supercritical fluid chromatography (SFC) (11, 12). In addition, the structure of polyprenols derived from E. ulmoides has been analyzed in detail using SFC, and the chain-length of cis and trans geometric isomers and their distribution in the harvest parts of E. ulmoides elucidated (13). We also succeeded in the separation of geometric isomers by connecting the monolithic silica column, which is the low backpressure in HPLC, as well as SFC (14).

With regard to the separation of the prenol type (polyprenol) and dolichol type (dihydropolyprenol), there has been no report concerning separation using a column chromatography such as HPLC until now; only the use of two-dimensional thin-layer chromatography (TLC) has been reported (15). Therefore, in this study, we tried to separate polyprenol and dolichol using a monolithic silica capillary column in HPLC. This monolithic silica column can be prepared in a fused-silica capillary using a sol-gel method developed by Nakanishi et al. (16, 17). This column facilitates increasing the resolution of separation (Rs) through extension of the column length because of the low backpressure (17, 18). Additionally, the capillary HPLC system using this column is useful for the analysis of the low-content natural product.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Materials
Polyprenol (prenol 16-21 from Gingko biloba leaves, which has the structure shown in Fig. 1III), and dolichol (dolichol 17-21 from bovine serum, which has the structure shown in Fig. 1IV), were purchased from Funakoshi Co. (Tokyo, Japan). Undecapolyprenol (C55) from Aianthus altissima, which has the structure shown in Fig. 1II, was obtained from Sigma-Aldrich Japan (Tokyo, Japan). For the HPLC analyses, HPLC-grade methanol, 2-propanol, and N-hexane were used (Merck; Darmstadt, Germany), and water was purified using a Millipore Milli-Q system (Bedford, MA).

Samples of E. ulmoides were collected at the Hitachi Zosen Corporation experimental station (Habu 2264-1 Innoshima, Hiroshima, Japan). Preparation of polyprenols from E. ulmoides leaf was performed according to the reported procedure of Bamba et al. (13).

Capillary HPLC
The HPLC of polyprenols was performed on an ODS-monolithic silica capillary column (MonoCap) [250 x 0.2 mm inner diameter (ID); macropore size, 2 µm; mesopore size, 13 nm; (GL Sciences Inc.; Tokyo, Japan) and 500 x 0.2 mm ID; macropore size, 2 µm; mesopore size, 13 nm (Kyoto Monotech Co., Kyoto, Japan)] using a dual pump apparatus (MP711; GL Sciences Inc.), and a UV-visible (UV-VIS) detector with a capillary optical fiber flow cell (set at 210 nm; GL Sciences Inc.). A carrier reservoir (CR791; GL Sciences Inc.), and a data station (SIC-480II data station; System Instruments Co., Hachioji, Japan) were also used. For elution purposes, a gradient was applied from an 80% methanol-2-propanol-water mixture (60:40:5; v/v/v) in pump A to 80% N-hexane-2-propanol (70:30; v/v) in pump B. The solvent flow rate was 4 µl/min. The end of the gradient was reached after 40 or 80 min, respectively, and then held at A-B (20:80; v/v) for 10 min. The column was at room temperature.

To investigate the sensitivity of this analysis system for polyprenol, 10 nl of 10 pg/nl chloroform solution of undecaprenol (100 pg) was injected into the capillary HPLC with 250 mm capillary column (N = 3).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
First, we tried to separate the polyprenol mixture (16–21 mer) derived from G. biloba leaves by using a 250 x 0.2 mm ID ODS-monolith capillary column. For elution purposes, a gradient using a mixed solvent system comprising MeOH, 2-propanol, N-hexane, and H2O was applied in this experiment. The Rs between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) showed 4.7 and a high value, which exceeded by ≥2-fold the level recorded when using a conventional ODS-packed column (250 x 4.6 mm ID, Rs = 2.1) under the same elution conditions (Fig. 2) . In addition, the detection sensitivity was markedly higher than that of a conventional system. In detail, the UV-VIS detector with the capillary optical fiber flow cell, with a light path length of 4 mm, was adopted for use within this analysis system. Accordingly, the injection amount of the sample was 50 ng (5 mg/ml, 10 nl), and the presence of even a few nanograms per individual component was sufficiently detectable. The sensitivity of this capillary HPLC system for polyprenol was investigated using undecaprenol (C55), and the average of signal to noise was 65 (N = 3) in the injection amount of 100 pg.



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  		Fig. 2. Chromatogram of authentic polyprenol mixtures separated using a 250 mm monolith capillary column. Conditions: mobile phase, pump A: methanol-2-propanol-water (60:40:5; v/v/v); pump B: N-hexane-2-propanol (70:30; v/v), A-B (80:20; v/v) to A-B (20:80; v/v) over 40 min; flow rate, 4.0 µl/min; UV detection at 210 nm. The numbers represent degrees of polymerization for polyprenol homologs.

 
Next, the mixture of polyprenols derived from G. biloba leaves (prenol 16-21) and dihydropolyprenols derived from bovine serum (dolichol 17-21) was subjected to this capillary HPLC system, and the separation of polyprenols and dolichols was successfully achieved (Fig. 3a) . It was proven that dolichol polymerized to the same degree was eluted behind the polyprenol under these conditions. There has been no report concerning the separation of polyprenol and dolichol within a liquid chromatography system until now, making this the first example. Additionally, the polyprenol fraction from E. ulmoides leaves, containing trans and cis isomers, was subjected to this analysis system. Consequently, although peaks thought to be dolichol were found (shown as circled peaks in Fig. 4a) , except for the trans and cis isomers confirmed previously (13), the separation of cis-polyprenols was insufficient.




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  		Fig. 3. Chromatogram of authentic polyprenol and dolichol mixtures separated using (A) 250 mm and (B) 500 mm monolith capillary column. Mobile phase, pump A to A-B (20:80; v/v) over (A) 40 min or (B) 80 min; flow rate, 4.0 µl/min; UV detection at 210 nm. The numbers represent degrees of polymerization for polyprenol and dolichol homologs. P, polyprenol; D, dolichol.

 



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  		Fig. 4. Chromatogram of polyprenol fraction from E. ulmoides leaves separated using (A) 250 mm and (B) 500 mm monolith capillary column. Mobile phase, pump A to A-B (20:80; v/v) over (A) 40 min or (B) 80 min and then held at A-B (20:80; v/v) for 10 min; flow rate, 4.0 µl/min; UV detection at 210 nm. The numbers represent degrees of polymerization for polyprenol and dolichol homologs. c, cis isomers; t, trans isomers; P, polyprenol; D, dolichol. The peaks presumed to be dolichol are circled (A). These peaks were identified in an earlier experiment (13).

 
Under the conditions used, the initial backpressure of this column was low at 5.3 Mpa, so improvement of the Rs was attempted by extending the column length. First, through the use of a 500 x 0.2 mm ID ODS-monolith capillary column, the Rs between the cis-polyprenol and dolichol in the polyprenols-and-dolichols mixture sample was checked. The Rs on prenol 18 increased to 1.8, compared with that of the 250 mm column (Rs = 0.99) (Fig. 3b). Subsequently, analysis of the polyprenol fraction derived from E. ulmoides leaves was similarly carried out, and effective separation of three components, i.e., the trans-polyprenols, cis-polyprenols, and dolichols, was found to have occurred (Fig. 4b).

Thus, this monolith capillary HPLC system was seen to allow the high-resolution analysis of a small sample, an analysis associated with increased and facilitated reproducibility, as compared with two-dimensional TLC. This sensitive high-resolution system is thus considered very useful for the analysis of structurally close and low-content polyprenol. The monolithic capillary column would be ideal as a powerful tool for metabolite analysis in various organisms, inasmuch as the column length can be freely adjusted in proportion to the necessary resolution and includes a widely variable separation mode, enabling various modifications, as well as the particle-packed column.


   ACKNOWLEDGMENTS
 
This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) through the Research Association for Biotechnology, the Kansai Bureau of Economy, Trade and Industry, and the Osaka Science and Technology Center. The authors are very grateful to Dr. K. Nakanishi (Kyoto University) and Messrs. M. Furuno and Y. Shintani (GL Sciences Inc.) for their helpful discussion and support for the experiments.

Manuscript received May 10, 2005 and in revised form July 17, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
  1. Rowland, R. L., P. H. Latimer, and J. A. Giles. 1956. Flue-cured tobacco. I. Isolation of solanesol, an unsaturated alcohol. J. Am. Chem. Soc. 78: 4680–4683.[CrossRef]

  2. Burgos, J., F. W. Hemming, J. F. Pennock, and R. A. Morton. 1963. Dolichol: a naturally-occurring C100 isoprenoid. Biochem. J. 88: 470–482.[Medline]

  3. Thorne, K. J. I., and E. Kodieck. 1966. The structure of bactoprenol, lipid formed by lactobacilli from mevalonic acid. Biochem. J. 99: 123–127.[Medline]

  4. Swiezewska, E., W. Sasak, T. Mankowski, W. Jankowski, T. Vogtman, I. Krajewska, J. Hertel, E. Skoczylas, and T. Chojnacki. 1994. The search for plant polyprenols. Acta Biochim. Pol. 41: 221–260.[Medline]

  5. Chojnacki, T., and G. Dallner. 1988. The biological role of dolichol. Biochem. J. 251: 1–9.[Medline]

  6. Burda, P., and M. Aebi. 1999. The dolichol pathway of N-linked glycosylation. Biochim. Biophys. Acta. 1426: 239–257.[Medline]

  7. Ibata, K., M. Mizuno, T. Takigawa, and Y. Tanaka. 1983. Long-chain betulaprenol-type polyprenols from the leaves of Ginkgo biloba. Biochem. J. 213: 305–311.[Medline]

  8. Chojnacki, T., and T. Vogtman. 1984. The occurrence and seasonal distribution of C50-C60-polyprenols and of C100-and similar long-chain polyprenols in leaves of plants. Acta Biochim. Pol. 31: 115–126.[Medline]

  9. Tangpakdee, J., and Y. Tanaka. 1998. Long-chain polyprenols and rubber in young leaves of Hevea brasiliensis. Phytochemistry. 48: 447–450.[CrossRef]

  10. Tateyama, S., R. Wititsuwannakul, D. Wititsuwannakul, H. Sagami, and K. Ogura. 1999. Dolichols of rubber plant, ginkgo and pine. Phytochemistry. 51: 11–15.[CrossRef]

  11. Bamba, T., E. Fukasaki, S. Kajiyama, K. Ute, T. Kitayama, and A. Kobayashi. 2001. High-resolution analysis of polyprenols by supercritical fluid chromatography. J. Chromatogr. A. 911: 113–117.[Medline]

  12. Bamba, T., E. Fukusaki, Y. Nakazawa, H. Sato, K. Ute, T. Kitayama, and A. Kobayashi. 2003. Analysis of long-chain polyprenols using supercritical fluid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. J. Chromatogr. A. 995: 203–207.[Medline]

  13. Bamba, T., E. Fukusaki, S. Kajiyama, K. Ute, T. Kitayama, and A. Kobayashi. 2001. The occurrence of geometric polyprenol isomers in the rubber-producing plant, Eucommia ulmoides Oliver. Lipids. 367: 727–732.

  14. Bamba, T., E. Fukusaki, Y. Nakazawa, and A. Kobayashi. 2004. Rapid and high-resolution analysis of geometric polyprenol homologues by connected octadecylsilylated monolithic silica columns in high-performance liquid chromatography. J. Sep. Sci. 27: 293–296.[Medline]

  15. Sagami, H., A. Kurisaki, K. Ogura, and T. Chojnacki. 1992. Separation of dolichol from dehydrodolichol by a simple two-plate thin-layer chromatography. J. Lipid Res. 33: 1857–1861.[Abstract]

  16. Tanaka, N., H. Kobayashi, K. Nakanishi, H. Minakuchi, and N. Ishizuka. 2001. Monolithic LC columns. Anal. Chem. 73: 420A–429A.[Medline]

  17. Ishizuka, N., H. Minakuchi, K. Nakanishi, N. Soga, H. Nagayama, K. Hosoya, and N. Tanaka. 2000. Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions. Anal. Chem. 72: 1275–1280.[Medline]

  18. Tanaka, N., H. Kobayashi, N. Ishizuka, H. Minakuchi, K. Nakanishi, K. Hosoya, and T. Ikegami. 2002. Monolithic silica columns for high-efficiency chromatographic separations. J. Chromatogr. A. 956: 35–49.[Medline]


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