Method for evaluating the potential of 14C labeled plant polyphenols to cross the blood–brain barrier using accelerator mass spectrometry

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Abstract

Bioactive compounds in botanicals may be beneficial in preventing age-related neurodegenerative diseases, but for many compounds conventional methods may be inadequate to detect if these compounds cross the blood–brain barrier or to track the pharmacokinetics in the brain. By combining a number of unique technologies it has been possible to utilize the power of AMS to study the pharmacokinetics of bioactive compounds in the brain at very low concentrations. 14C labeled compounds can be biosynthesized by plant cell suspension cultures co-incubated with radioisotopically-labeled sucrose and isolated and separated into a series of bioactive fractions.

To study the pharmacokinetics and tissue distribution of 14C labeled plant polyphenols, rats were implanted with jugular catheters, subcutaneous ultrafiltration probes and brain microdialysis probes. Labeled fractions were dosed orally. Interstitial fluid (ISF) and brain microdialysate samples were taken in tandem with blood samples. It was often possible to determine 14C in blood and ISF with a β-counter. However, brain microdialysate samples 14C levels on the order of 107 atoms/sample required AMS technology. The Brain MicrodialysateAUC/SerumAUC ranged from .021- to .029, with the higher values for the glycoside fractions. By using AMS in combination with traditional methods, it is possible to study uptake by blood, distribution to ISF and determine the amount of a dose which can reach the brain and follow the pharmacokinetics in the brain.

Introduction

With the aging population, neurodegenerative diseases such as Alzheimer’s disease (AD) have become significant medical problems [1]. Currently there are no cures for these diseases and medications have only limited efficacy [2]. Supplements of bioactive plant polyphenols may be beneficial in protecting against the oxidative stress that is thought to be a major contributing factor in the development of neurodegenerative diseases [3].

One of the major issues in the efficacy of polyphenol rich supplements is bioavailability. Bioavailability can be extremely complicated to determine. Many plant polyphenols are destroyed in the digestive process. It is important to understand the pharmacokinetics and tissue distribution of the various polyphenol constituents of plant based supplements. It is not sufficient just to determine concentrations in blood because the blood–brain barrier (BBB) blocks many compounds from entering the brain, and the biological half life of compounds in the brain may be different from blood. Therefore to understand the mechanisms of potentially beneficial compounds, one must understand their kinetics in brain. Previous studies have used 14C labeled compounds to study their ability to cross the blood brain barrier by harvesting brain, and determining the 14C by AMS [4]. In these studies we used microdialysis (MD) to measure changes in 14C labeled chemicals over time in the brain extracellular fluid of awake, freely moving animals.

It has been shown in this study that 14C from labeled plant polyphenols, obtained by growing plant tissue in the presence of 14C labeled starting materials and chemical fractionation, can be tracked into tissues even in extremely small amounts because of the sensitivity of AMS. Fractionation of plant extract, bioavailability and tissue distribution testing of the plant polyphenols can provide insights into which compounds actually reach the target tissues and may be the effective ingredients. In this study total 14C label from both dosed compounds and metabolites was tracked into blood, ISF and brain.

Section snippets

Preparation of 14C labeled plant polyphenols

14C labeled polyphenols from different plant sources can be produced as described previously [5], [6]. Cell suspension cultures of plant material were co-cultured with 14C labeled sucrose in a specially designed chamber (Fig. 1). Cells were harvested, lysed and polyphenols were extracted with 70% aqueous acetone. Extracts were lyophilized and fractionated by vacuum chromatography on a Toyopearl resin polymer (TOSOH Bioseparation Specialists LLC, Montgomeryville, PA). Polyphenols were prepared

Results and discussion

Grape polyphenol fractions I–III which contained mostly proanthocyanidin monomers, dimers and trimers were poorly absorbed compared to fractions IV and V which contained the anthocyanin glycosides. Fig. 3 shows the relationship between the plasma, ISF and brain MD for two of the fractions. Probe recoveries were 93% for UF probes and 11.2% and 6.3% for fractions IV and V for MD probes. Concentration in target tissues is not necessarily proportional to plasma concentrations. The maximum plasma

Conclusion

Determination of the potential of bioactive compounds to reach the brain is difficult because the BBB restricts access of many chemicals making concentrations which reach the brain low. Use of 14C labeled plant polyphenols for in vivo studies and AMS to analyze the samples make it possible to track even the small amounts of material which cross the BBB and enters the brain. Use of AMS tracking of plant polyphenols into the brain may be a useful technique of identifying components of plant

Acknowledgements

The authors gratefully acknowledge technical assistance of Pamela Lachcik, Jane Einstein and Tom Kubley.

This project was supported by the NIH Office of Dietary Supplements and NCCAM Grant P50 AT 00477.

References (18)

  • R. Brookmeyer et al.

    Alzheimer’s Dementia

    (2007)
  • Z. Khachaturian et al.

    Alzheimer’s Dementia

    (2009)
  • E.M. Janle et al.

    Microdialysis and ultrafiltration

  • G. Leegsma-Vogt et al.

    Life Sci.

    (2003)
  • G.S. Jackson et al.

    Nucl. Instrum. Methods Phys. Res. Section B: Beam Interactions Mater. Atoms

    (2004)
  • Y.T. Wu et al.

    J. Chromatogr. A

    (2009)
  • Y. Kou et al.

    Int. J. Pharm.

    (2008)
  • L.C. Lin et al.

    Anal. Chim. Acta

    (2007)
  • L. Rossi et al.

    Neurochem. Res.

    (2008)
There are more references available in the full text version of this article.

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