AJAC  Vol.3 No.6 , June 2012
The Development of S-Equol Diastereoisomer Specific ELISA
Abstract: Equol is a metabolite of soybean isoflavone, daidzein, and many health benefits are expected. Endogenous equol in urine is S-equol and mostly exists as glucuronate or sulfate conjugate. In this study we preliminary established the simple enzyme-linked immunosorbent assay (ELISA) without deconjugation, then developed the S-equol specific ELISA involves deconjugation showing high stereospecificity to S-equol without using stereospecific antibody. For the simple ELISA, we used a polyclonal antibody that targets the regions not influenced by inhibition by conjugation of glucuronate and sulfate and achieved the correlation coefficient; r = 0.975, but the value was 30 % lower than high performance liquid chromatography (HPLC). Developing upon this we invented the specific ELISA established from S-equol homogeneous combination for the standard and enzyme-labeled antigen to enhance stereospecificity. The correlation with HPLC was favorable: r = 0.986, y = 0.996x – 6. Compared to the previous method using (R,S)-equol combination, cross-reactivity with R-equol was reduced from 65 to 13 %, and that with daidzein from 0.31% to 0.08%, markedly increased in the specificity. This study is expected to be applied for both simple clinical researches, and stereospecific immunoassays in which specific antibody preparation is difficult.
Cite this paper: T. Minekawa, A. Kambegawa, K. Shindome, S. Takehara and H. Arakawa, "The Development of S-Equol Diastereoisomer Specific ELISA," American Journal of Analytical Chemistry, Vol. 3 No. 6, 2012, pp. 448-454. doi: 10.4236/ajac.2012.36059.

[1]   A. M. Duncan, B. E. Merz-Demlow, X. Xu, W. R. Phipps and M. S. Kurzer, “Premenopausal Equol Excretors Show Plasma Hormone Profiles Associated with Lowered Risk of Breast Cancer,” Cancer Epidemiology, Biomarkers and Prevention, Vol. 9, No. 6, 2000, pp. 581-586.

[2]   H. Akaza, N. Miyanaga, N. Takashima, S. Naito, Y. Hirao, T. Tsukamoto, T. Fujioka, M. Mori, W. J. Kim, J. M. Song and A. J. Pantuck, “Comparisons of Percent Equol Producers between Prostate Cancer Patients and Controls: Case-Controlled Studies of Isoflavones in Japanese, Korean and American Residents,” Japanese Journal of Clinical Oncology, Vol. 34, No. 2, 2004, pp. 86-89. doi:10.1093/jjco/hyh015

[3]   H. Akaza, N. Miyanaga, N. Takashima, S. Naito, Y. Hirao, T. Tsukamoto and M. Mori, “Is Daidzein Non-Metabolizer a High Risk for Prostate Cancer? A Case-Controlled Study of Serum Soybean Isoflavone Concentration,” Japanese Journal of Clinical Oncology, Vol. 32, No. 8, 2002, pp. 296-300. doi:10.1093/jjco/hyf064

[4]   K. Ozasa, M. Nakao, Y. Watanabe, K. Hayashi, T. Miki, K. Mikami, M. Mori, F. Sakauchi, M. Washio, Y. Ito, K. Suzuki, K. Wakai, A. Tamakoshi and JACC Study Group, “Serum Phytoestrogens and Prostate Cancer Risk in a Nested Case-Control Study among Japanese Men,” Cancer Science, Vol. 95, No. 1, 2004, pp. 65-71. doi:10.1111/j.1349-7006.2004.tb03172.x

[5]   K. D. Setchell, N. M. Brown and E. Lydeking-Olsen, “The Clinical Importance of the Metabolite Equol-A Clue to the Effectiveness of Soy and Its Isoflavones,” Journal of Nutrition, Vol. 132, No. 12, 2002, pp. 3577-3584.

[6]   T. Ueno, S. Uchiyama and N. Kikuchi, “The Role of Intestinal Bacteria on Biological Effects of Soy Isoflavones in Human,” Journal of Nutrition, Vol. 132, 2002, p. 594S.

[7]   E. Lydeking-Olsen, J. B. E. Jensen, K. D. R. Set-chell, M. Damhus and T. H. Jensen, “Isoflavone-Rich Soymilk Prevents Bone-Loss in the Lumbar Spine of Postmeno- pausal Women. A 2 Year Study,” Journal of Nutrition, Vol. 132, 2002, p. 581S.

[8]   T. Maruo, M. Sakamoto, C. Ito, T. Toda and Y. Benno, “Adlercreutzia Equolifaciens gen. nov., sp. nov., an Equol-Producing Bacterium Isolated from Human Faeces, and Emended Description of the Genus Eggerthella,” International Journal of Systematic and Evolutionary Microbiology, Vol. 58, Pt. 5, 2008, pp. 1221-1227.

[9]   S. Uchiyama, T. Ueno and T. Suzuki, “Identification of a Newly Isolated Equol-Producing Lactic Acid Bacterium from the Human Feces,” Journal of Intestinal Microbiology, Vol. 21, No. 3, 2007, pp. 217-220 [in Japanese].

[10]   K. Hirayama, “Metabolism of Soy Isoflavones by Intestinal Flora,” Journal of Intestestinal Microbiology, Vol. 19, No. 1, 2005, pp. 17-23 [in Japanese].

[11]   T. J. Lundh, H. Pettersson and K.H. Kiessling, “Liquid Chromatographic Determination of the Estrogens Daid- zein, Formononetin, Cou-mestrol, and Equol in Bovine Blood Plasma and Urine,” Journal of the Association of Official Analytical Chemists, Vol. 71, No. 5, 1988, pp. 938-941.

[12]   A. A. Franke and L. J. Custer, “High-Performance Liquid Chromatographic Assay of Isofla-vonoids and Coumestrol from Human Urine,” Journal of Chromatography B: Biomedical Sciences and Applications, Vol. 662, No. 1, 1994, pp. 47-60. doi:10.1016/0378-4347(94)00390-4

[13]   L. Coward, M. Kirk, N. Albin and S. Barnes, “Analysis of Plasma Isoflavones by Reversed-Phase HPLC-Multiple Reaction Ion Monitoring-Mass Spectrometry,” Clinica Chimica Acta, Vol. 247, No. 1-2, 1996, pp. 121-142. doi:10.1016/0009-8981(95)06242-4

[14]   E. Brouwers, R. L’homme, N. Al-Maharik, O. Lapcík, R. Hampl, K. W?h?l?, H. Mikola and H. Adlercreutz, “Time-Resolved Fluoroimmunoassay for Equol in Plasma and Urine,” Journal of Steroid Bio-chemistry and Molecular Biology, Vol. 84, No. 5, 2003, pp. 577-588. doi:10.1016/S0960-0760(03)00071-2

[15]   D. C. Talbot, R. M. Ogborne, T. Dadd, H. Adlercreutz, G. Barnard, S. Bugel, F. Kohen, S. Marlin, J. Piron, A. Cassidy and J. Powell, “Monoc-lonal Antibody-Based Time-Resolved Fluorescence Immunoassays for Daidzein, Genistein, and Equol in Blood and Urine: Application to the Isoheart Intervention Study,” Clinical Chemistry, Vol. 53, No. 4, 2007, pp. 748-756. doi:10.1373/clinchem.2006.075077

[16]   T. Minekawa, A. Kambegawa, K. Shindome, H. Ohkuma, K. Abe, H. Maekawa and H. Arakawa. “Development of Bioluminescent Enzyme Immunoassay for S-Equol Using Firefly Luciferase and Its Application to the Assessment of Equol-Producer Status,” Chemical and Pharmaceutical Bulletin, Vol. 59, No. 1, 2011, pp. 84-87. doi:10.1248/cpb.59.84

[17]   H. Adlercreutz, J. van der Wildt, J. Kinzel, H. Attalla, K. W?h?l?, T. M?kel?, T. Hase and T. Fotsis, “Lignan and Isoflavonoid Conjugates in Human Urine,” Journal of Steroid Biochemistry and Molecular Biology, Vol. 52, No. 1, 1995, pp. 97-103. doi:10.1016/0960-0760(94)00146-D

[18]   K. D. Setchell, C. Clerici, E. D. Lephart, S. J. Cole, C. Heenan, D. Castellani, B. E. Wolfe, L. Nechemias- Zimmer, N. M. Brown, T. D. Lund, R. J. Handa and J. E. Heubi, “S-Equol, a Potent Ligand for Estrogen Receptor Beta, Is the Exclusive Enantiomeric form of the Soy Isoflavone Metabolite Produced by Human Intestinal Bacterial Flora,” The American Journal of Clinical Nutrition, Vol. 81, No. 5, 2005, pp. 1072-1079.

[19]   K. D. Setchell, N. M. Brown, P. Desai, L. Zimmer- Nechemias, B. E. Wolfe, W. T. Brashear, A. S. Kirschner, A. Cassidy and J. E. Heubi, “Bioavailability of Pure Isoflavones in Healthy Humans and Analysis of Commercial Soy Isoflavone Supplements,” Journal of Nutrition, Vol. 131, No. 4-Suppl, 2001, pp. 1362S-1375.