Chemical Education Today
Reports from Other Journals
Research Advances by Angela G. King
Using Geography To Increase Hemoglobin Levels As they test their bodies, endurance athletes struggle with the challenge of getting enough oxygen to cells. While some have turned to drugs such as erythropoietin (EPO) to address the problem and increase endurance, others have done so by altering training methods. In recent years the “live high–train low” (LHTL) approach has been a popular concept among elite endurance athletes. The approach is based on the expectation that living at higher altitudes where the air is thinner will, in turn, improve sea-level performances. Scientists have suggested that the LHTL strategy increases hemoglobin mass (Hbmass) and red cell volume (RCV) with the absolute training intensity afforded at sea level. But there has not been consensus concerning this result due to experimental design. In 2006, Jon Wehrlin and colleagues from Switzerland and Norway undertook a study of athletes to investigate the effects living at an altitude of ~2,500 m and training at lower levels for 24 days had on erythropoiesis, the formation or production of red blood cells, by using direct measurement of Hbmass (Figure 1). Seventeen elite athletes, a mix of men and women from national orienteering and cross-country teams, participated in the study and were assigned to either the altitude group (AG) or
the control group (CG). AG athletes lived 18 hours per day at 2,456 m and trained at 1,000 or 1,800 m above sea level (in the Swiss Alps) for a period of 24 days, termed the LHTL phase of the study. CG athletes trained for 24 days in a normal fashion, living and training at 500–1,600 m. About a month before the study began, all athletes were assessed for bone marrow iron stores and one day before the study began, they completed a pre-test that included submitting a blood sample, performing a maximal oxygen uptake (VO2max) test and running a 5,000 m time trial, with heart rate, rating of perceived exertion, and blood lactate levels monitored during and after the event. During the study timeframe, athletes submitted blood samples on day 1, 12, and 24, and 8 days after the 24 day training period, both AG and CG participants completed a post-test with measurements mirroring the pre-test. From the blood samples, scientists measured Hbmass, red cell volume (RCV), and blood volume according to previously established methodology. The results showed that the LHTL approach increased the athletes’ Hbmass and RCV by approximately 5% over a 24-day period while there was no change in these levels for the CG. The increases in Hbmass and RCV are credited with an observed decrease in times for the 5,000 time trial run and other changes, such as increase in VO2max.
Figure 1. Study design for LHTL approach. A–F refer to different segments of the study as described in the original paper. *Measurements in the altitude group only. (Figure 1 from Wehrlin, J. P.; Zuest, P.; Hallén, J.; Marti, B. Live High-Train Low for 24 Days Increases Hemoglobin Mass and Red Cell Volume in Elite Endurance Athletes. J. Appl. Physiol. 2006, 100, 1938–1945, used with permission.)
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Chemical Education Today
This study clearly indicates that athletes have alternatives to performance-enhancing drugs, and that there is a molecular basis of support for the LHTL approach to training elite athletes. More Information 1. Wehrlin, Jon Peter; Zuest, Peter; Hallen, Jostein; Marti, Bernard. Live High–Train Low for 24 Days Increases Hemoglobin Mass and Red Cell Volume in Elite Endurance Athletes. J. Appl. Physiol. 2006, 100, 1938–1945. 2. This Journal has published an excellent article that uses hemoglobin to bridge biochemistry and physiology. See J. Chem. Educ. 2001, 78, 757. An earlier article (J. Chem. Educ. 1985, 62, 1122) details an experiment that allows students to construct plots of saturation rate as a function of oxygen’s partial pressure.
NO Levels and Exercise Lower-level chemistry classes often introduce nitric oxide’s role in air pollution, but when discussing the free radical with students, teachers sometimes forget to mention its biological role, for which it was named Molecule of the Year in 1992 by Science. NO is produced from l-arginine upon catalysis by the three distinct forms of the enzyme nitric oxide synthase (NOS). Once generated in the body, nitric oxide activates guanylate cyclase, which catalyzes the production of guanosine 3′,5′-monophos-
phate, which in turn can lead to physiological responses such as cell adhesion, vasodilation, and neurotransmission. Through other avenues, nitric oxide is involved in long-term memory and immune system regulation. One form of NOS, calcium-independent inducible NOS (iNOS), is induced by pro-inflammatory cytokines, and thus its levels may greatly vary but do not change rapidly. Inflammation is associated with increased levels of NO from iNOS stimulated by the presence of these cytokines. Increases in intracellular calcium concentrations stimulate the activity of the remaining two forms of NOS, from either endothelial cells (eNOS) or neurons (nNOS). Both eNOS and nNOS are constitutively expressed enzymes, meaning there are small amounts constantly produced in the body. In the lung, these enzymes generate NO, which is involved in the regulation of vascular tone and airways through many mechanisms. Nitric oxide mediates vasodilation, bronchodilation, and lung inflammation. Since it can affect both pulmonary circulation (through vasodilation) and airway function (via bronchodilation), NO levels could be important during muscular exercise when both blood flow and ventilation levels are high. Hypoxemia is a medical condition where [O2] in the blood is very low, and it can be caused by a variety of physiological situations. Intense exercise can lead to a condition called exercise-induced hypoxemia (EIH).
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Chemical Education Today
Reports from Other Journals A research team led by Pascale Kippelen hypothesized that exhaled NO levels may be modified in endurance-trained athletes during and after intense exercise. They studied untrained participants and athletes with and without EIH, and measured their exhaled NO before, during, and after exercise. Exhaled NO concentration (CNO) and exhaled NO output (VNO) were measured by an NO analyzer. The scientists found that CNO decreased in all participants but returned to basal values and remained steady after one hour of recovery. VNO rose significantly during exercise, for all subject groups, rapidly dropped after exercise, and was similar to basal values after one hour of recovery. These results also suggest that iNOS is probably not the cause of increased NO output associated with heavy exercise due to the transient nature of changes in NO levels. Thus scientists have concluded that it is improbable that pulmonary inflammation, which would induce iNOS, occurs in the athletes who develop a hypoxemia. Both VNO and CNO were lower in athletes with EIH than in untrained participants during basal conditions, while exercising, and during recovery. Scientists are still investigating the possibility of NO downregulation in endurance-trained athletes to explain observed NO exhalation levels. More Information 1. Kippelen, Pascale; Caillaud, Corinne; Robert, Emmanuelle; Masmoudi, Kaouthar; Prefaut, Christian. Exhaled Nitric Oxide Level during and after Heavy Exercise in Athletes with Exercise-Induced Hypoxaemia. Pflugers Archiv.: Eur. J. Physiol. 2002, 444, 397–404. 2. J. Chem. Educ. 1995, 72, 686 unites the history, environmental impact, and physiology of NO and will be of interest to readers at many levels of understanding. 3. Due to nitric oxide’s physiological properties, drugs that release it are the subject of much research. See J. Chem. Educ. 2002, 79, 1427. 4. Kipperlen’s research, which is centered on the pathophysiology of respiratory disorders in the athletic population, is described online at http://www.abdn.ac.uk/ims/staff/details.php?id=p.kippelen (accessed Jul 2008). 5. Anthropologists have also looked at NO generation as a response to high altitude living. See Erzurum, S. C.; Ghosh, S.; Janocha, A. J.; Xu, W.; Bauer, S.; Bryan, N. S.; Tejero, J.; Hemann, C.; Hille, R.; Stuehr, D. J.; Feelisch, M.; Beall, C. M. Higher Blood Flow and Circulating NO Products Offset High-Altitude Hypoxia among Tibetans. Proc. Natl. Acad. Sci. 2007, 104, 17593.
Better Method for Steroid Detection Amid growing concerns about sports “doping”, researchers in Indiana and China report development of a faster and more efficient method for detecting the presence of illegal anabolic steroids in urine. Their new method takes only a few seconds and involves no time-consuming sample preparation. Their recent study notes that use of banned substances by professional athletes to build muscle and gain a competitive advantage is a growing problem in sports such as track and field, baseball, football, and cycling. Although effective methods exist for detecting the presence of illegal steroids in urine, such as GC/MS and LC/MS, they are time-consuming and involve 1312
Figure 2. This instrument provides a faster, more efficient method for detecting illegal steroids in urine. Photo courtesy of Zheng Ouyang, Purdue University.
cumbersome preparation steps including extraction, hydrolysis, and derivatization. Zheng Ouyang, R. Graham Cooks, and colleagues developed a new steroid-testing method that combines two state-ofthe-art testing techniques called reactive desorption electrospray ionization (reactive DESI) and tandem mass spectrometry (Figure 2). Reactive DESI affords the advantage of derivatizing compounds in the course of ionization, which is performed on the raw sample. The resulting steroid test uses spray solutions of NH2OH to react with the carbonyl groups of steroids during the ionization process. The ion/molecule reaction adduct and the oxime formed in the reaction were observed in reactive DESI profiles for seven different anabolic steroids (Figure 3). Low mass cutoffs in this study were set at m/z 200 to eliminate the detection of other compounds found in urine that contain carbonyls but have smaller molar masses than steroids. Limits of detection for the pure steroid compounds were < 1 ng. In additional laboratory studies, the researchers used this new method to analyze fresh urine samples for the presence of tiny amounts of these same anabolic steroids. Solid-phase microextraction (SPME) easily and quickly concentrated the steroids to the detection level of ~20 ng/mL, which indicates the method would work at sports doping anabolic steroid levels. Researchers say the new method accurately identified the steroids in only a few seconds. A single drop of urine was placed on a substrate and the mass spectrum was recorded while the sample was sprayed with the selected reagent, in this case hydroxylamine. The DESI mass spectrum indicated which steroids were present, and samples could be processed at a rate approaching 1 sample/s. It is worth noting that the DESI high-thoughput instruments were developed through Purdue University’s Center for Analytical Instrumentation Development engineering prototyping program. This on-campus program produces a limited number of engineering prototypes, sold by Purdue at cost; this generates funding needed for subsequent prototyping endeavors. Prototype users return data to designers to both benefit the community of users and advance the technology. The expected result is that the experience with the systems will result in improvements to systems faster than conventional approaches.
Journal of Chemical Education • Vol. 85 No. 10 October 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
Chemical Education Today
More Information 1. Huang, Guangming; Chen, Hao; Zhang, Xinrong; Cooks, R. Graham; Ouyang, Zheng. Rapid Screening of Anabolic Steroids in Urine by Reactive Desorption Electrospray Ionization. Anal. Chem. 2007, 79, 8327–8332. 2. This Journal has published an article on drug testing in the chemistry lab. See J. Chem. Educ. 2005, 82, 1809. 3. This Journal has also published an article on the chemistry sports fanatics should know that includes performance-enhancing steroids. See J. Chem. Educ. 2002, 79, 813–819.
Figure 3. (A) Reactive DESI analysis of epitestosterone on ground glass, mass spectrum and (B) product ion MS2 spectra of the fragments of the ion/molecule reaction product m/z 322 and (C) the oxime ion m/z 304. Reprinted with permission from Anal. Chem. 2007, 79, 8327–8332. Copyright 2007 American Chemical Society.
4. More information on Purdue’s Engineering Prototyping Program is at http://www.purdue.edu/dp/caid/eep.php (accessed Jul 2008). 5. Another recent breakthrough in steroid detection involves hydropyrolysis. More information can be found at http://www.sciencedaily.com/releases/2008/03/080305104900.htm (accessed Jul 2008).
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/Oct/abs1310.html Abstract and keywords Full text (PDF) with links to cited URLs and JCE articles
Angela G. King is Senior Lecturer in Chemistry at Wake Forest University, P.O. Box 7486, Winston-Salem, NC 27109;
[email protected].
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