Choline: An Important Micronutrient for Maximal Endurance-Exercise Performance?

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1 Scholarly Reviews International Journal of Sport Nutrition and Exercise Metabolism, 2008, 18, Human Kinetics, Inc. Choline: An Important Micronutrient for Maximal Endurance-Exercise Performance? Jason T. Penry and Melinda M. Manore Choline plays a central role in many physiological pathways, including neurotransmitter synthesis (acetylcholine), cell-membrane signaling (phospholipids), lipid transport (lipoproteins), and methyl-group metabolism (homocysteine reduction). Endurance exercise might stress several of these pathways, increasing the demand for choline as a metabolic substrate. This review examines the current literature linking endurance exercise and choline demand in the human body. Also reviewed are the mechanisms by which exercise might affect blood choline levels, and the links between methyl metabolism and the availability of free choline are highlighted. Finally, the ability of oral choline supplements to augment endurance performance is assessed. Most individuals consume adequate amounts of choline, although there is evidence that current recommendations might be insufficient for some adult men. Only strenuous and prolonged physical activity appears sufficient to significantly decrease circulating choline stores. Moreover, oral choline supplementation might only increase endurance performance in activities that reduce circulating choline levels below normal. Keywords: betaine, lecithin, homocysteine, folate, MTHFD1, review Choline is a micronutrient important for health in all people, from the sedentary individual to the elite athlete. The consequences of long-term choline deficiency have been well documented since the early 1900s, including steatosis (fatty liver) and death (Best & Huntsman, 1932). Moreover, recent work has shown that up to half the population might have a genetic allele related to an increased susceptibility to choline deficiency (Kohlmeier, da Costa, Fischer, & Zeisel, 2005). Thus, an applied understanding of the role of choline in human metabolism could be very important to a large segment of the population. Choline is found in a variety of foods (Cho et al., 2006; see Table 1), and limited quantities of choline can be synthesized from endogenous sources (Zeisel & Niculescu, 2005). As a result of this ubiquitous nature, it was previously thought that rapid changes in blood choline concentrations did not occur. Current research shows, however, that a strenuous exercise bout such as a marathon race can create a significant, short-term decrease in free (non-membrane-bound) choline in the The authors are with the Dept. of Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR

2 192 Penry and Manore Table 1 Choline and Betaine Content of Selected Foods and Supplements Food or supplement Total choline (mg) Total betaine (mg) Chicken liver (2 oz), pan fried Large egg (2.5 oz), hard boiled Pork chop (4 oz), pan broiled Chicken breast (4 oz), roasted 75 9 Beer, pint (16 fluid oz) Skim milk, one glass (8 fluid oz) 37 4 Medium white potato, baked Firm tofu (2 oz), nigari Spinach (2 oz), unprepared Whole-wheat bread (1 oz), slice 7 98 Twinlab Choline Cocktail (choline bitartrate), 8 oz prepared 1,500 0 Twinlab choline bitartrate, tablet Jarrow Lecithin Mega-PC 35, 1 gel Ultima Replenisher, 20 oz prepared 1 0 Twinlab betaine HCl, tablet Centrum, 1 multivitamin 0 0 Note. Adapted from USDA database for the choline content of common foods ( foodcomp/data/choline/choline.pdf, June 23, 2007) and product labels of included supplements. blood (Table 2). One hypothesized mechanism for this decrease in free choline is an increased demand for choline as a methyl-group donor during physiological stress (Kanter & Williams, 1995). This acute drop in free choline during strenuous exercise is subsequently thought to inhibit optimal muscle performance by decreasing the amount of choline available for acetylcholine synthesis, thereby inhibiting excitation contraction coupling at the neuromuscular junction (Conlay, Sabounjian, & Wurtman, 1992). If reductions in free choline do affect other physiological variables related to endurance performance (Conlay et al., 1992; da Costa, Badea, Fischer, & Zeisel, 2004), it is possible that supplementing this micronutrient during choline-depleting exercise could benefit individuals engaged in such activities. No peer-reviewed studies have explored this hypothesis. Answers to this question might have implications for any individual routinely placed under strenuous physical conditions, including endurance athletes, participants in extreme sports, and military personnel. This review examines the current research pertinent to the possible relationship between choline intake and endurance performance, beginning with an outline of the dietary sources of choline and normal intakes. Next, we explore the mechanisms by which exercise might affect blood choline concentrations and highlight the possible link between methyl metabolism and the availability of free choline in the blood. We summarize research examining the link between free choline and physical exercise in light of the increased demand for methyl groups with exercise. Finally, we assess the ability of oral choline supplements to augment endurance performance.

3 Table 2 Effects of Endurance Exercise on Acute Free Blood Choline Concentrations Study Sample gender* Activity Duration (min) Intensity (% VO 2max ) Baseline /Pre vs. post (nmol/ml) p Buchman et al., M + F marathon not given max effort 19.2 vs Buchman et al., M + F marathon max effort 9.6 vs Burns et al., M cycle % (105 min) + max effort (15 min) not given >.05 Conlay et al., a marathon not given max effort 10.1 vs. 6.2 <.001 Deuster et al., M load carriage ~110 70% 8.5 vs. 6.5 b >.05 Pierard et al., M combat course 7,200 ~35% 2.95% decrease <.01 Spector et al., M cycle 72 70% 8.5 vs b >.05 von Allwörden et al., M, 6 F cycle km/hr vs <.01 von Allwörden et al., M, 4 F cross-country run max effort vs >.05 Warber et al., M load carriage % 8.14 vs >.05 Note. M = male; F = female. a Gender of participants not disclosed in published paper. b Estimated from published figure. 193

4 194 Penry and Manore Dietary Sources of Choline The recommended adequate intake for choline is 550 mg/day in adult men and 425 mg/day in adult women (>19 years of age; Institute of Medicine [IOM], 1998). Pregnant and lactating women require additional choline, up to 450 and 550 mg/ day, respectively (IOM), because large amounts of this nutrient are lost across the placenta and in breast milk (Zeisel, Mar, Zhou, & da Costa, 1995). There is currently inadequate information to set a recommended dietary allowance (RDA) for choline (IOM), although it appears that some men might require more than is currently recommended. In a study by Fischer et al. (2007), 6 of 26 male participants developed symptoms of choline deficiency, including increased liver lipid content and possible muscle-cell membrane damage, when fed a diet containing the current adequate intake of 550 mg/day. Thus, it appears that additional research is needed to better understand the dietary requirements of free-living individuals. In the typical free-living diet, more than half the dietary choline demand is met by consuming red meat, poultry, milk, eggs, and fish (Cho et al., 2006). Coffee, beer, potatoes, and orange juice also contain significant amounts of choline (Cho et al.; see Table 1). Fischer et al. (2005) allowed 32 individuals (16 men and 16 women years of age) to consume food ad libitum from a metabolic kitchen and found that most met meet the daily recommended adequate intake for choline, with men consuming an average of 631 mg/day and women consuming 443 mg/day. The same researchers then used a 3-day food record to indirectly monitor choline intake in the same population and found values as low as 313 mg/day in both men and women. These low intakes of choline were attributed to the underreporting of total energy intake, which is common with food-recall records (Fischer et al., 2005). Most dietary choline is consumed in the form of phosphatidylcholine, a primary constituent of cell membranes (Cho et al., 2006). Choline can then be synthesized de novo from phosphatidylcholine in the liver by phosphatidylethanolamine N- methyltransferase (PEMT; Zeisel, 2006). PEMT activity is enhanced in individuals when estrogen is present (Fischer et al., 2007), allowing for additional synthesis of choline from physiological stores. Choline availability is paramount to a healthy fetus, and it is possible that this mechanism acts to buffer a developing fetus against dietary variability in choline consumption (Zeisel, 2006). Betaine, a metabolic derivative of choline, is also consumed in the diet. Because betaine plays a large role in regulating the osmotic balance in plants, it is found in large concentrations in plant foods (Craig, 2004; see Table 1). Most betaine in the diet comes from green leafy vegetables such as spinach (25% of the total choline consumed) and grain-derived foods including pasta (12%), white bread (9%), and cold cereal (8%; Cho et al., 2006). Although betaine cannot be converted directly into choline, it can be used as a methyl donor in the same metabolic pathways and, thus, reduces the amount of choline required by the body (Zeisel & Niculescu, 2005). As a result of this interrelated nature, researchers have begun to consider the two nutrients together when determining the total amount of choline intake per day (Cho et al.). Information concerning the dietary intakes of choline and betaine is limited, in part because of the previous lack of a valid database containing the choline and betaine content of common foods (Cho et al., 2006). With the recent advent of such

5 Choline and Endurance Performance 195 a database, however, it appears that choline and betaine intakes can be adequately measured by a semiquantitative food-frequency questionnaire (Cho et al.). In addition, a food-frequency questionnaire can accurately detect variance in consumed choline and betaine in a free-living population (Cho et al.), a key factor in better understanding choline and betaine intakes in humans. Individuals can complement their dietary intake of choline via a large range of supplements. Currently, most sport supplements contain choline in the form of choline bitartrate, a choline salt that has been shown to affect free blood choline concentrations within 45 min of ingestion (Spector et al., 1995). Another common supplemental form of choline is lecithin, a compound that is ~35% phospatidylcholine when purchased at health-food stores (Zeisel, 1994). Lecithin appears to have a larger (+265% vs. +86%) and more long-lasting (12 hr vs. 4 hr) impact on free blood choline concentrations than do choline salts (Wurtman, Hirsch, & Growden, 1977). Lecithin, however, has not been used in studies in which individuals receive supplementation during physical activity, which might be because of the small percentage of choline found in lecithin. In recent years, a number of choline-derived compounds such as cytidine 5 -diphosphocholine (CDP-choline) and alphaglyceryl-phosphorylcholine (alpha- GPC) have been used to successfully treat cognitive impairment in dementia disorders when phosphatidylcholine supplementation has been ineffective (Parnetti, Mignini, Tomassoni, Traini, & Amenta, 2007). Both CDP-choline and alpha-gpc are intermediary metabolites in the metabolic synthesis of phosphatidylcholine from choline (Deuster & Cooper, 2005), but it is currently unknown why CDP-choline and alpha-gpc are effective in treating such disorders but phosphatidylcholine is not (Parnetti et al.). It is possible that there is a similar form-specific effect when supplementing athletes, but no study to date has examined the effect of supplementing these metabolites in an athletic population. Additional research is necessary to determine which form of supplemental choline might have the greatest performance effect for strenuous physical activity. Choline Physiology The role of choline in the body is complex; it plays both a functional and a structural role in cells. Free choline can be found inside cells or circulating in the blood; choline in this form plays an important role in providing a substrate for cellular processes including acetylcholine synthesis and methyl-group metabolism (Deuster & Cooper, 2005). Normal concentrations of free choline are nmol/ml, although this value can be significantly higher after choline supplementation (Burns, Costill, & Fink, 1988). Alternatively, bound choline refers to choline in its structural role, incorporated into cell membranes (as phosphatidylcholine), lipoproteins, cell-signaling proteins, or other biological molecules (Deuster & Cooper). Blood baseline values for phospholipid-bound choline range from 2,000 to 2,500 nmol/ml (Buchman, Awal, Jenden, Roch, & Kang, 2000; Buchman, Jenden, & Roch, 1999). It is possible to exchange free and bound choline, because phosphatidylcholine is synthesized from free choline in the liver (Zeisel & Niculescu, 2005) and phosphatidylcholine is hydrolyzed to form choline in cholinergic neurons (Zhao, Frohman, & Blusztajn, 2001).

6 196 Penry and Manore Choline Utilization During Exercise Choline metabolism during exercise might be altered through a number of hypothesized mechanisms that are discussed as follows. Acetylcholine Synthesis Free choline concentrations are of particular interest to exercise physiologists, because reduced concentrations of free choline have been associated with weakened impulse transmission and impaired performance in skeletal muscle (Conlay et al., 1992). Because choline is an important building block of the neurotransmitter acetylcholine, it is not surprising that reductions in free choline result in an acute decline in acetylcholine synthesis (Bierkamper & Goldberg, 1980; Maire & Wurtman, 1985). This reduction in acetylcholine production can have a marked influence on muscle performance, because it inhibits the ability of the α-motor neuron to communicate with the motor end plate in muscle excitation contraction coupling (Conlay et al., 1992). Reduced acetylcholine availability might also impair neuron function in the brain, but the degree to which free choline concentrations influence cognitive performance in adults is not clear (Deuster, Singh, Coll, Hyde, & Becker, 2002; Pierard et al., 2004). Cell-Membrane Integrity Choline can also be incorporated as a phospholipid (phosphatidylcholine) in cell membranes. Prolonged choline deficiencies cause the body to mobilize the phosphatidylcholine located in its own cell membranes, compromising membrane integrity and allowing intramembranous materials to leak into the surrounding fluid (da Costa et al., 2004). In fact, the increased amount of serum creatine phosphokinase that leaks from porous muscle-cell membranes has been suggested as a diagnostic tool for choline deficiency (da Costa et al.). Stripping muscle-cell membranes of phospholipids also reduces the mechanical stress these cells can withstand (da Costa et al.), resulting in muscle damage that might be associated with muscle fatigue similar to that induced by reduced acetylcholine availability. Methyl-Group Metabolism Methionine, an essential amino acid, is used as a methyl-group donor in many metabolic reactions. After donating its methyl group, methionine is metabolized to homocysteine (Figure 1). Homocysteine is an extremely bioreactive molecule and has been identified as a major contributor to endothelial damage and cardiovascular disease (Refsum & Ueland, 1998). To recycle homocysteine into methionine, a methyl-group donor must be used. There are two pathways for the conversion of homocysteine to methionine (Olthof, Brink, Katan, & Verhoef, 2005). The first uses methyl tetrahydrofolate (methyl-thf) as the methyl donor and is catalyzed by the enzyme methionine synthase (MS). The alternative homocysteine methionine pathway uses betaine as the methyl donor and is catalyzed by the enzyme betaine-homocysteine S-methyltransferase (BHMT). Betaine is synthesized from choline in a nonreversible oxidation

7 Choline and Endurance Performance 197 Figure 1 A brief overview of methionine-homocysteine methyl metabolism. Note the two pathways used to recycle homocysteine to methionine: the choline-dependent BHMT pathway on the left and the folate-dependent MS pathway on the right. Adapted from Olthof et al. reaction (Olthof et al.). As stated previously, betaine can also be consumed in foods (Cho et al., 2006), and dietary betaine appears to be more efficient than dietary choline in its ability to convert homocysteine to methionine (Olthof et al.). Both the MS and BHMT pathways operate simultaneously, but certain conditions can cause the body to favor one pathway over the other. Folate plays an important role in the performance of the MS pathway, and cells with low folate availability will show reduced MS activity (Fiskerstrand, Ueland, & Refsum, 1997). It is not surprising that activity of the BHMT pathway must also be increased under reduced folate concentrations to maintain stable homocysteine concentrations (Jacob, Jenden, Allman-Farinelli, & Swendseid, 1999), thus placing a greater demand on physiological betaine stores (Trimble, Molloy, Scott, & Weir, 1993). Recent research has shown that up to half the population might carry an allele for a less effective methyl-thf dehydrogenase (MTHFD1 1958A; Kohlmeier et al., 2005), indicating that some individuals might require higher amounts of dietary folate to minimize BHMT activity and spare available choline (Kohlmeier et al., 2005). Unfortunately, there is no inexpensive and quick method to determine whether an individual carries the MTHFD1 1958A allele. Another factor that might influence the degree to which the body uses the BHMT pathway is the quantity of methyl metabolism that must be carried out in the liver. Blood homocysteine concentrations are attenuated after a methionine load when supplemented with betaine or phosphatidylcholine but not when supplemented with folate (Olthof et al., 2005). Thus, the body might use the BHMT pathway more when homocysteine production is high (and the demand for methyl metabolism is

8 198 Penry and Manore high), such as during strenuous endurance exercise (Herrmann et al., 2003; Konig et al., 2003). No research to date has examined this possible link between an increase in blood homocysteine and any change in free choline concentrations. Fluid Redistribution Transient shifts in fluid between the bloodstream and interstitial space might interfere with the ability to detect changes in free choline in the bloodstream during exercise (Kanter & Williams, 1995). Researchers must take this factor into account when designing experiments. Otherwise, choline utilization during exercise might appear greater than it actually is because of the redistribution of free choline stores dissolved in the interstitial fluid. Changes in Free Choline Concentrations With Exercise Generally, research has shown that free choline concentrations decrease during intense endurance exercise (Table 2). The impact of exercise on free choline concentrations was first described by Conlay et al. (1986), who observed a significant decrease in free choline concentrations (40% decrease, p <.001) from prerace to postrace in marathon runners. Later, von Allwörden, Horn, Kahl, and Feldheim (1993) observed significant decreases in free choline concentrations in triathletes cycling for 2 hr at 35 km/hr, and Buchman et al. (1999) measured significant decreases in a second cohort of marathon runners. Pierard et al. (2004) also noted significant reductions in free choline in military cadets after they completed a 5- day combat-training course. Not all types of exercise, however, have been shown to elicit a significant decrease in free choline concentrations. Burns, Costill, and Fink (1988) found that trained male cyclists who pedaled at 70% of their VO 2max for 105 min and then completed a 15-min time trial showed no change in free choline concentrations. A similar result was observed by Spector et al. (1995) in trained cyclists who exercised at 150% VO 2max for 2 min and 70% VO 2max for 70 min. Moreover, von Allwörden et al. (1993) found no significant decrease in free choline concentrations in adolescent cross-country runners after a local competition. Finally, neither a 4-hr treadmill load carriage test at 38% VO 2max (Warber et al., 2000) nor a 1.5-hr treadmill load carriage test at 70% VO 2max (Deuster et al., 2002) decreased free choline concentrations in male soldiers. These equivocal findings might result from differences in experimental design. Research protocols showing a significant postexercise decline in free choline concentrations are especially arduous in nature. Warber et al. (2000) suggest that free choline depletion does not depend on mode of exercise but, rather, on duration and intensity of the work bout. Moreover, results published by Spector et al. (1995) showed a mild negative relationship between time spent cycling and plasma choline concentrations (r =.60). Longer work bouts at lower intensities or shorter work bouts at higher intensities do not appear to be sufficient to deplete free choline concentrations below baseline concentrations. To significantly reduce the amount of free choline found in the blood, it appears that an individual must be exposed

9 Choline and Endurance Performance 199 to a relatively long work bout (>2 hr) at a relatively high work intensity (>70% VO 2max ). If this is true, the situations when free choline is significantly reduced below baseline concentrations might be limited to those participating in ultraendurance events (Warber et al.). Impact of Reductions in Free Choline on Endurance Performance Although research consistently shows a decrease in free choline concentrations with strenuous endurance activity, little research has examined the effect of such changes on endurance performance. The conclusions from current research have been limited because of small sample sizes or confounding study variables. Two hypothesized mechanisms through which decreases in free choline concentrations could influence endurance performance are reduced work capacity and impaired cognitive function. Buchman et al. (1999) showed a weak correlation (r =.47, p =.036) between actual and predicted time to finish in marathoners who best maintained their free choline concentrations. In other words, individuals whose free choline concentrations did not drop were better able to maintain their goal pace throughout the marathon. Concurrently, Pierard et al. (2004) found that performance on several cognitive tests was compromised after a 5-day combattraining course that significantly depleted free choline stores. The participants bodies were affected by the 5-day ordeal in many other respects, however, not the least of which were severe negative energy balance (5,000 8,000 kcal/day expended vs. 3,500 kcal/day consumed) and sleep deprivation (4 hr/night). Thus, additional research is needed to ascertain the relationship, if indeed there is one, between reduced free choline concentrations and endurance performance. Impact of Choline Supplementation on Endurance Performance Studies examining the effect of choline supplementation on endurance performance fall into two general groups. The first consists of studies in which supplementation elevates free choline concentrations above those that are normally found in the blood. The second group of studies uses a choline supplement to offset a drop in free choline concentrations that would normally occur with strenuous exercise. Supplementation Above Normal Physiological Concentrations Most exercise studies have examined the effect of increasing free choline concentrations above an individual s normal physiological concentrations before an exercise bout (Buchman et al., 2000; Burns et al., 1988; Deuster et al., 2002; Warber et al., 2000). These studies found no significant performance effect of this type of supplementation, nor did they find significant change in any cognitive or physical variable measured, including memory tasks, time to fatigue, and %VO 2max during an activity.

10 200 Penry and Manore In one noteworthy study, Spector et al. (1995) examined the effect of choline supplementation on cycling time to exhaustion in 10 trained cyclists. A doubleblind crossover design was employed, with the participants diets screened to ensure relative homogeneity of choline intake. Forty-five minutes before each test, participants consumed 200 ml of a beverage containing 6% glucose, 70 mg sodium, 25 mg potassium, and a mixture of B vitamins (specific quantities unnamed) or a similar beverage containing 2.43 g of choline bitartrate. The participants then began cycling at 70% of their VO 2max. Twenty-five minutes into the test, the participants received 200 ml of the same beverage they had consumed before the test began. No significant difference was observed between trials in cycle time to exhaustion or any physiological variable. It should be noted, however, that although the supplement significantly increased free choline concentrations above baseline values, free choline concentrations of both the control and treatment trials were not significantly different from baseline at the conclusion of the cycling test. Because research has shown that particularly strenuous exercise can significantly decrease free choline concentrations, additional research is needed to determine the effect of choline supplementation under the conditions of an acute exercise-induced free choline deficiency. Supplementation to Maintain Normal Physiological Concentrations Only one study (published as an abstract; Sandage, Sabounjian, White, & Wurtman, 1992) has examined the effect of choline supplementation on endurance performance in individuals who demonstrated a significant exercise-induced decrease in free choline from baseline concentrations. In that crossover study, Sandage et al. reported that ingestion of a 2.8 g choline citrate supplement 1 hr before and at the 10-mile mark of a 20-mile run maintained plasma choline concentrations, whereas plasma choline concentrations fell in individuals receiving a placebo. Participants who ingested the choline supplement also had faster finish times for the 20-mile run than those who consumed a placebo (p <.05, actual times not reported). It is unknown why this study was never published as a full paper, although it is important as the only choline-supplementation study in which the authors were able to elicit a significant drop in plasma choline concentrations in the control group. Although Sandage et al. s (1992) study is thought provoking, it is also quite interesting that no subsequent studies have examined the effect of choline supplementation under these specific circumstances. It is likely that this is because of a combination of factors, notably the arduous nature of the exercise protocol needed to cause a decline in free choline and the difficulty of finding a suitable performance measure. Moreover, it is also possible that trained individuals have some resistance to acute choline depletion (Conlay et al., 1992), although this has not been studied.

11 Choline and Endurance Performance 201 Summary and Recommendations Current research underscores the importance of choline in proper functioning of the human body. It appears that maintaining free blood choline concentrations is necessary for optimal cognitive and muscular performance; however, the effect of oral choline supplementation on endurance performance is equivocal. It seems certain that raising free blood choline concentrations above those normally found in the circulation has no beneficial effect on cognitive or physical-endurance performance. In physical endeavors of a particularly long and strenuous nature, free choline might drop below baseline concentrations. To date, only one abbreviated study has examined the effect of choline supplementation in these types of physical events. It is possible that choline supplementation in situations when free choline concentrations are acutely decreased improve performance by preventing a decline in free choline concentrations. Future research on this topic must select an exercise modality that will minimize the ancillary effects of strenuous exercise while retaining the ability to stimulate the types of metabolism that cause a significant decrease in an unsupplemented test population. The link between blood homocysteine concentrations and free choline concentrations also deserves attention. Blood homocysteine concentrations are increased after a bout of exercise, provided that the activity is both continuous and strenuous in nature (Joubert & Manore, 2006). Free choline concentrations have been shown to decrease under similar physiological conditions. Moreover, the central role that choline plays in converting homocysteine to methionine under exercise conditions supports a link between these findings. Choline supplementation during such exercise bouts might reduce the accumulation of blood homocysteine concentrations via increased BHMT activity, and adequate folate status might offset the depletion of choline during exercise by encouraging MS activity and lessening the demand on the BHMT pathway. Accordingly, it would seem that choline stores would be used more slowly and performance improved by limiting the amount of homocysteine produced during endurance exercise or by directly reducing the amount of choline used by the body for purposes other than acetylcholine synthesis. This latter point might be particularly important in athletes carrying the MTHFD1 1958A allele, who might need to consume extra folate in their diets to spare physiological choline stores. Additional research is needed to clarify the relationship among acute changes in blood homocysteine, free choline, genetics, and endurance performance. References Best, C.H., & Huntsman, M.E. (1932). The effect of the components of lecithine upon deposition of fat in the liver. The Journal of Physiology, 75, Bierkamper, G.G., & Goldberg, A.M. (1980). Release of acetylcholine from the vascular perfused rat phrenic nerve-hemidiaphragm. Brain Research, 202,

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