Meet a Member of NACCAM
This article is an extended version of the interview with Dr. Barnes in printed copies of this newsletter.
Stephen Barnes, Ph.D.
Stephen Barnes, Ph.D., is a member of NCCAM's National Advisory Council for Complementary and Alternative Medicine (NACCAM). He is associate director of NCCAM's Botanicals Center for Age-Related Diseases, a collaboration between Purdue University and the University of Alabama at Birmingham (UAB). The center's projects include the effects of isoflavones (estrogen-like substances found in some plants, including soy and kudzu root) on bone and vascular health in postmenopausal women, and their effects on the onset of cataracts in aging. Isoflavones are members of a broader class of a group of chemical components called polyphenols that are widely found in plants, including in the skins and seeds of grapes and in many other fruits and berries.
Dr. Barnes is an expert in high-technology research techniques, especially mass spectrometry1. He is professor of pharmacology and toxicology, biochemistry and molecular genetics, environmental health sciences, genetics, and vision sciences at UAB, where he is also director of the Comprehensive Cancer Center Mass Spectrometry and Proteomics Shared Facility, and senior scientist at the Comprehensive Cancer Center and the Center for Aging. Dr. Barnes received his Ph.D. in biochemistry from the University of London, United Kingdom. He is also carrying out other NIH-supported research on cancer prevention, skin diseases, and bile acids.
Why use mass spectrometry to study botanicals in CAM?
We have important questions about botanicals used as CAM—such as what is in them, at what concentrations, and what the structures of their constituents (ingredients) look like. Mass spectrometry and its various instruments are vital tools in CAM research for answering them, because of their extreme precision in measuring any compound, large or small. [In chemistry, a compound is a combination of two or more different elements or atoms.]
A great challenge that lies before us is the variation in substances not only among plants, but also within them. Plants are incredible chemical factories. They don't make their chemicals (called phytochemicals) just to suit us, but for their own survival. For example, certain compounds in the petal of a flower give the "come hither" sign to bees, aiding pollination, while others fight off attacking insects or microbes. A compound in a plant always leaves the same "fingerprint" of its constituents when analyzed by mass spectrometry.
How does your team approach analyzing a botanical?
First, we make sure that the botanical has been correctly identified by a plant expert such as our colleague Dr. James E. Simon at Rutgers University. Then we remove the phytochemicals from the botanical by soaking it in a solvent such as alcohol, often with a little added water. We inject a small volume of this extract into a tube that contains a fine powder, and pump a solution through the tube under high pressure. Some of the compounds pass through quickly since they don't "like" the powder, and others move more slowly. By changing the composition of the solution, we can speed up the movement of those compounds that linger on the powder.
As the compounds emerge from the tube, they go through a process called electrospray ionization, in which they are sprayed out through a hollow needle that carries a large positive or negative charge (3000 volts, plus or minus). As you would also find with an inkjet printer, this spray rapidly evaporates, forcing all the charged molecules in the droplets closer together. The droplets and everything in them explode, which transfers the compounds into the gas phase, and they enter the mass spectrometer through a pinhole. This process is known as liquid chromatography-mass spectrometry (LC-MS).
Inside the spectrometer, we find a vacuum similar to that of outer space. This allows the ions to move around freely without having to bump into air molecules, including when we apply the effects of voltage gradients and magnetic fields. The motion of the ions is thus "pure" and, more importantly, analyzable.
How does a mass spectrometer recognize particular compounds?
All compounds have a defined weight or mass. If we know a compound's chemical formula, we can determine its molecular weight exactly and hence the mass-to-charge (m/z) ratio of its molecular ion. In the opposite way, if we observe a molecular ion's m/z ratio, then we can find out its molecular weight. This helps us identify the compound.
This task is not always so straightforward, however. For example, if you have traveled through airport security recently, you may have seen the security staff wipe a clean lint cloth over your bags and then put the cloth in an instrument. That instrument is a mass spectrometer. It looks for evidence of known explosives, each of which has a characteristic m/z ratio. Of course, you could have a harmless substance in your bag that has an m/z ratio similar to that of an explosive. Similar uncertainty can arise when we are analyzing botanical compounds. Thus, to ensure that we identify them correctly, we combine knowledge of m/z ratios with how quickly the compounds move through the tube.
Are there any techniques in mass spectrometry that "take it up a notch"?
Yes. One is tandem mass spectrometry (TMS), which gives us two analyses of the ions. First the molecular ion that was observed before is selectively isolated; then it is sped up into a zone where it suddenly encounters a moderate pressure of gas molecules (helium, nitrogen, or argon). This shakes the molecule so much that it breaks apart. We can then analyze the resulting ion fragments to find out their m/z ratios and confirm—with more confidence—the compound's identity or suggest its likely structure.
Can you give us an example of a research challenge that mass spectrometry helped you address?
Kudzu
© Peggy Greb
Dr. Jeevan Prasain of UAB and I have used LC-MS to analyze isoflavones from soy dietary supplements and from bodily fluids of study volunteers. Some, but not all, of the supplements are made only from soybean sources. These typically contain an isoflavone, daidzein, connected to a glucose molecule; this forms a combination called daidzin. Daidzin produces a molecular ion with a particular fingerprint.
Other isoflavone supplements contain a mixture of isoflavones from soy and from kudzu root. Kudzu root also contains daidzein connected to glucose, but with some important differences from daidzin: a different name, puerarin; a different chemical "bridge" linking the isoflavone and glucose; and a different way of disintegrating when the compound encounters neutral gas during TMS.
In some isoflavone supplements, puerarin may be more than 80 percent of the total isoflavones. Our data have shown that the body takes up puerarin in different ways than it takes up daidzin [the soy counterpart]. This may account for puerarin's ability to improve glucose tolerance.
Is there any new technology of this type that you are particularly excited about—especially one that could be used to research CAM?
Yes, I can give several examples:
- We can use mass spectrometry to identify the proteins and peptide biomarkers—in blood, urine, or extracts of plants or clinical tissues—that are affected by CAM modalities used for preventing or treating disease. This research approach can yield wonderful insights as to how botanicals work.
- Researchers can use a laser to explore the composition of proteins or phytochemicals in a tissue slice. To do this, we first spread a thin layer of crystals (a crystalline matrix) over the tissue slice. This absorbs the energy of the laser. With each shot of the laser, we generate a cloud of ions that can be analyzed by a type of mass spectrometry called MALDI-TOF, short for matrix-assisted laser desorption ionization time-of-flight analysis.
- We give the ions made by the laser a kick by applying a short, nanosecond-accelerating (20 kV) pulse, and then we fire the ions up a tube. A detector times their arrival at the end of the tube. Small ions arrive first, and heavy ones later. Their time-of-flight allows us to accurately measure the mass (or weight) of each ion. After such analysis, we use computers to create maps of the compounds. In collaboration with Dr. Jim Mobley of UAB, we are using time-of-flight analysis to study how the proteins in the lens of the eye are distributed and how selected botanicals affect them over long periods of time.
- Mass spectrometers in which the ion detector is located inside superconducting magnets can be used to identify compounds of interest. In this approach, the magnets cause the ions to circulate around the detector, like the moons around Jupiter and Saturn. The speed at which the ions circulate depends on their mass, which can be measured very accurately.
- We are going to see more applications in CAM of accelerator mass spectrometry (AMS), which can measure long-lived radioisotopes with unprecedented sensitivity. It allows us to use tiny amounts—less than the amount of radioactivity in your wristwatch—of a botanical labeled with carbon 14 (a radioactive isotope of carbon) to be introduced into a system and followed for extended periods with tremendous accuracy. Our colleague Dr. Mary Ann Lila of the University of Illinois-Urbana-Champaign is an expert in using plant cells to produce botanicals labeled with carbon-14 for our studies.
What are a few of the major results so far from your center?
We have a great interest in isoflavones from certain plants, including soy, and especially in several types of substances called daidzein glucosides2 that they contain. These appear to have differences in where they go in the body and how they are metabolized. We have analyzed isoflavones both from soy dietary supplements and from the bodily fluids of study volunteers, and have found a kudzu root isoflavone glucoside that lowers blood glucose levels. It is absorbed without being changed and may compete with glucose for uptake.
Another finding is that grape seed extract fed to young rats caused changes in brain proteins that appear to be opposite of the changes caused by Alzheimer's disease. We are now studying in more detail what happens when one of those proteins, creatine kinase, is attacked via oxidative stress during the process of cognitive degeneration, as in Alzheimer's.
Also, Connie Weaver, Ph.D., director of our botanicals research center and distinguished professor of foods and nutrition at Purdue University, has been using accelerator mass spectrometry (AMS)3 to study the preservation of bone in women as they go through menopause. AMS allows measurements of the turnover of calcium-41 from bone with great sensitivity and rapidity. Using this technique, Dr. Weaver has examined the effects of soy protein isolate enriched with isoflavones on calcium turnover in the bones of the same patients for 5 years.
What are some things you have learned about CAM from your time on NACCAM?
Most scientific studies in CAM today are being carried out with the same rigor one expects from all NIH-funded investigators. CAM is an important area of study since it is being used widely by the public. It is necessary that NCCAM-funded research sort out which of the CAM modalities work. If the experiments are done well, the true benefit (or lack thereof) of the CAM treatment can be established.
Selected References
- Barnes S, Birt DF, Cassileth BR, et al. Technologies and experimental approaches at the National Institutes of Health Botanical Research Centers. American Journal of Clinical Nutrition. 2008;87(2):476S–480S.
- Cheong JM, Martin BR, Jackson GS, et al. Soy isoflavones do not affect bone resorption in postmenopausal women: a dose response study using a novel approach with 41Ca. Journal of Clinical Endocrinology and Metabolism. 2007;92(2):577–582.
- Deshane J, Chaves L, Sarikonda KV, et al. Proteomics analysis of rat brain protein modulations by grape seed extract. Journal of Agricultural and Food Chemistry. 2004; 52(26):7872–7883.
- Eliuk SM, Renfrew MB, Shonsey EM, et al. Active site modifications of the brain isoform of creatine kinase by 4-hydroxy-2-nonenal correlate with reduced enzyme activity: mapping of modified sites by Fourier transform-ion cyclotron resonance mass spectrometry. Chemical Research in Toxicology. 2007;20(9):1260–1268.
- Prasain JK, Jones K, Kirk M, et al. Profiling and quantification of isoflavonoids in kudzu dietary supplements by high-performance liquid chromatography and electrospray ionization tandem mass spectrometry. Journal of Agricultural and Food Chemistry. 2003; 51(15):4213–4218.
- Weaver CM, Barnes S, Wyss JM, et al. Botanicals for age-related diseases: from field to practice. American Journal of Clinical Nutrition. 2008;87(2):493S–497S.
Notes:
- Mass spectrometry is a method used with a variety of specialized instruments (mass spectrometers) for analytical purposes in chemistry. Each mass spectrometer separates out and identifies all the chemicals in a substance according to their mass and electrical charge.
- Daidzein is a component of the isoflavones in soy; glucosides are chemical compounds that yield glucose (sugar) when broken down by hydrolysis, a chemical reaction that involves water.
- Accelerator mass spectrometry uses an ultrasensitive technique that takes mass spectrometry analysis to a high level through an advanced device called a particle accelerator.
