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1 From the US Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND
2 Mention of a trademark or proprietary product does not constitute a guarantee of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. The US Department of Agriculture, Agricultural Research, Northern Plains Area is an equal opportunity, affirmative action employer and all agency services are available without discrimination. 3 Supported by the US Department of Agriculture. 4 Address reprint requests to HC Lukaski, USDA, ARS GFHNRC, Box 9034, Grand Forks, ND 58202-9034. E-mail: hlukaski{at}gfhnrc.ars.usda.gov.
ABSTRACT
Background: The role of zinc in promoting physiologic function during exercise is not well understood. Although some zinc-containing enzymes are postulated to regulate energy expenditure, data are limited on the effect of restricted dietary zinc on metabolic responses during exercise.
Objective: This study determined the effects of low zinc intake on carbonic anhydrase activity in red blood cells (RBCs) and cardiorespiratory function during exercise.
Design: In this double-blind, randomized crossover study, 14 men aged 20–31 y were fed low-zinc and supplemented (3.8 and 18.7 mg/d) diets made up of Western foods for 9-wk periods with a 6-wk washout. Peak work capacity, determined by using a cycle ergometer and a graded, progressive protocol, and a prolonged submaximal test (70% peak intensity for 45 min) were administered during the second and ninth weeks of each diet period.
Results: Dietary zinc did not affect hemoglobin or hematocrit. Low dietary zinc resulted in lower (P < 0.05) serum and erythrocyte zinc concentrations, zinc retention, and total carbonic anhydrase and isoform activities in RBCs. Peak oxygen uptake, carbon dioxide output, and respiratory exchange ratio were lower (P < 0.05), and ventilatory equivalents for metabolic responses during exercise were greater (P < 0.05), with low than with supplemental zinc intake. Similar functional responses were observed during prolonged, submaximal exercise.
Conclusion: These findings indicate that low dietary zinc is associated with significant reductions in zinc status, including RBC carbonic anhydrase activities, and impaired metabolic responses during exercise.
Key Words: Zinc depletion • erythrocyte carbonic anhydrase • cardiorespiratory function • humans
INTRODUCTION
Public health organizations encourage increased physical activity and consumption of healthful diets as preventive measures to reduce obesity and other chronic diseases among children and adults (1, 2). These recommendations underscore concerns about the interactions between nutrient intakes and physiologic function during exercise. Although considerable information exists about the roles of macronutrients in facilitation of adaptation to physical activity and promotion of health (3), there is a paucity of research data on which to develop recommendations for intakes of minerals, except iron (4), in the development of physical fitness and health. One mineral that is attracting interest among the public is zinc.
Epidemiologic studies show that many adults and children may not consume adequate dietary zinc. Estimates of the proportion of adults aged 19–50 y with inadequate zinc intakes range from 20% in men to nearly 40% in women (5). Surveys of physically active persons also indicate that low dietary zinc is common, particularly among individuals who participate in aerobic activities (6, 7), such as those recommended to promote health and well-being (1, 2).
Low zinc status hampers the physiologic functions required for optimal work performance. Low zinc intakes and reduced serum zinc concentrations have been associated with impaired muscle function, including reduced strength and increased propensity to fatigue (8, 9), and decreased power output during peak work capacity testing (10). Thus, low zinc status may lead to reduced physical function and performance.
Because zinc-containing enzymes number >200 in mammalian systems (11, 12), any effect of dietary zinc may be translated into metabolic and functional defects by zinc metalloenzymes. Carbonic anhydrase (EC 4.2.1.1), a zinc metalloenzyme, catalyzes the reversible hydration and dehydration of carbon dioxide, a product of cellular aerobic energy production (12). Removal of zinc from this metalloenzyme inactivates the enzyme (13). Studies in various species, including rodents, domestic fowl, calves, and lambs, have found that dietary zinc deficiency significantly reduces red blood cell (RBC) carbonic anhydrase activity (14–19) and, in a few cases, impairs respiratory function (14, 15). Sickle-cell anemia patients with biochemically determined zinc and other nutritional deficiencies had significantly decreased carbonic anhydrase protein in RBCs that increased significantly with zinc supplementation (20). Thus, reduced carbonic anhydrase activity in RBCs may be an indicator of zinc deficiency, particularly in humans.
The present study tested the hypothesis that restricted, compared with increased, dietary zinc in amounts consumed by physically active men would decrease zinc status and retention and RBC carbonic anhydrase activity and impair physiologic responses during controlled exercise.
SUBJECTS AND METHODS
Experimental design
Men engaged in regular physical activities, recreational or employment related, participated in a double-blind, crossover feeding study. On admission to the study, the men were matched by body mass index and assigned to receive either a basal low-zinc diet [3.5 mg/10.5 MJ (2500 kcal)] or the basal diet supplemented with 15 mg Zn as zinc sulfate (ZnSO4 · 7H2O) added to juice. These amounts of dietary zinc are consistent with reported zinc intakes of young men involved in various regular physical activities but not intense physical training (7). The volunteers consumed each diet for 9 wk, followed by a 6-wk washout period, and then received the other diet for 9 wk. The men lived in their usual residences and maintained their usual lifestyles with the exception of consuming the food and beverages provided and participating in scheduled testing. Phlebotomy, collection of excreta for determinations of zinc nutritional status and balance, and physiologic testing were performed at specified times during each dietary period.
Subjects
Fourteen men aged (
Registered dietitians planned the basal low-zinc diet that was composed of Western foods presented in a 3-d rotating menu cycle (Table 1). The energy distribution of the diet was 12% protein, 30% fat, and 58% carbohydrate. The basal, low-zinc diet was calculated (21) to supply recommended amounts of all essential nutrients except zinc. Limited amounts of salt, pepper, and selected low-energy carbonated beverages were individualized to the preference of each volunteer and then served consistently throughout the study. The range of daily energy intakes was 7.5–16.0 MJ (1750–3750 kcal).
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TABLE 1. Food items in the 3-d rotating menu for the basal low-zinc diet
All diet ingredients, except water, were weighed, prepared, and provided to the volunteers by the dietary staff. Volunteers ate one meal at the US Department of Agriculture Agricultural Research Service Grand Forks Human Nutrition Research Center on weekdays and consumed the remaining foods, after minimal reheating, away from the center. Foods were weighed to 0.1 g accuracy and were consumed quantitatively. The volunteers agreed to consume only the food and beverages provided by the dietary staff. On the basis of self-reports, the men did not consume other food or beverages.
Chemical analyses
Blood collection
Fasting venous blood samples were taken by phlebotomy during the second and ninth week of each dietary period and were limited to 50 mL/mo for routine health assessment and determination of zinc status. Blood was drawn into plastic syringes from an antecubital vein, which had been made visible by temporary use of a tourniquet, after the volunteers had fasted for 10 h. Samples were collected and prepared with care to avoid hemolysis. Hemoglobin and hematocrit were determined by using an automated clinical analyzer (Cell Dyn-3500; Abbott Instruments, Chicago, IL). Serum was processed within 90 min of the time the blood was obtained.
Zinc in serum and red blood cells
Serum for zinc determination was diluted 1:5 with distilled-deionized water; serum zinc was measured by using flame atomic absorption spectrometry with standards in a 5% glycerol matrix (22). Aliquots of hemolyzed RBCs were diluted and deproteinized with trichloroacetic acid were then analyzed for zinc by using atomic absorption spectrometry (23). Concurrent analysis of SeraChem I controls (Fisher Scientific, Orangeberg, NY) yielded values of 146.8 ± 0.6 (
Another venous blood sample was collected in a syringe containing 20 U heparin/mL; this amount of heparin, which contains negligible zinc, has been shown to not be a source of contamination in determinations of zinc in plasma and RBCs (24). After centrifugation (2800 x g at 4 °C for 15 min), plasma and white blood cells were removed, and packed RBCs were washed 3 times with normal saline followed by centrifugation. After the last wash, the supernatant fluid was removed and the packed RBCs were lysed with an equal volume of distilled-deionized water. Samples were centrifuged to remove cellular debris.
To determine carbonic anhydrase activity, aliquots of hemolyzed RBCs were diluted 1:50 and 1:100 with distilled-deionized water (25). An experienced analyst bubbled carbon dioxide through 5 mL cold distilled-deionized water (either with or without sample) for 1 min. Two milliliters of 50-mmol/L barbitol buffer (pH = 7.9) was added, and the time to attain a pH of 6.7 was recorded. A calibrated pH meter (Advanced pH Meter, model #840035; Technika, Phoenix, AZ) and fast-reacting electrode (#840016; Technika) were used to monitor pH in the reaction mixture (26). The 1:100 diluted sample gave the total carbonic anhydrase activity. The procedure was repeated with the 1:50 diluted sample, which was diluted again 1:1 with a solution containing 40 mmol bromopyruvate/L and 47 mmol Na2HPO4/L (pH = 7.5). The pH change was monitored as described above, which yields an estimate of carbonic anhydrase II activity (25, 26). Carbonic anhydrase isozyme I activity was estimated as the difference between total and isozyme II activities. The hemoglobin concentration of each hemolysate (mg hemoglobin/mL hemolysate) was determined with an automated cell counter. One enzyme unit is defined as the quantity of sample needed to result in a doubling of the reaction time without sample (26). Variability in carbonic anhydrase activity determinations was 2–3% within and 4–5% between days.
Zinc in food and excreta
All food was weighed with an accuracy of 0.1 g during preparation in the metabolic kitchen. Urine and feces were collected carefully to avoid trace mineral contamination. Duplicate diets at the 10.6-MJ (2500-kcal) intake were prepared daily for analysis and were blended in a plastic blender with stainless steel blades. Adjustments for differences in individual energy intakes were calculated proportionately.
The zinc content of 4 consecutive 3-d composites of diets and feces, obtained during the last 12 d of each dietary period, was determined by inductively coupled argon-plasma emission spectrophotometry (ICAP; Jarrell-Ash, Waltham, MA) after wet digestion of aliquots of freeze-dried, blended material with nitric and perchloric acids (23, 27). Urinary zinc, collected during the same 12-d periods, was determined by ICAP analysis of a diluted aliquot (23).
Concurrent replicate analysis of zinc in bovine liver samples (standard reference material 1577b; US National Institute of Standards and Technology, Gaithersburg, MD) yielded a value of 1.807 ± 0.037 (
Metabolic responses at rest and during exercise
Two types of exercise tests were administered: a graded, progressive, peak performance test and a prolonged submaximal test. Peak work capacity was determined by using a cycle ergometer (Monark; Varberg, Sweden) and a continuous, progressive exercise protocol that was terminated when the volunteer reached voluntary exhaustion. The submaximal test included a 5-min warmup at a work load equal to 50% peak oxygen uptake followed by 45 min at a work load of 70% peak oxygen consumption.
Exercise tests were performed during the second and ninth week of each dietary period. Two work capacity tests were performed on nonconsecutive days at each designated time period. The first test was used to acclimate or reacquaint the volunteer with the test environment and equipment; data from the second test were used to determine dietary effects on metabolic responses. Only one prolonged submaximal test was administered at the second and ninth week of each dietary treatment; it occurred within 7 d of completion of the second peak work capacity test. After an overnight fast, each volunteer rested for 5 min while seated on the ergocycle and then pedaled at 70 revolutions/min starting with an initial resistance of 0 kilopond (kP) and increasing by 0.5 kP every 3 min until he could not maintain the pedaling cadence or stopped the test. Heart rate was monitored continuously during the preexercise and exercise periods by using standard electrocardiographic leads (II, aVF, and CM5) and was recorded during the last 10 s of each min with a multichannel recorder. Oxygen uptake and carbon dioxide output were determined at rest and during exercise by indirect calorimetry every 2 s with an automated system (Q Plex I; Quinton Instruments, Seattle, WA). The oxygen and carbon dioxide analyzers were calibrated before each test with reference gas mixtures, the composition of which was previously determined by standard chemical procedures. The oxygen consumption and carbon dioxide data are presented as an average value per minute.
During the prolonged submaximal test, metabolic responses were assessed at rest and during the last 5 min of the exercise test. Heart rate was monitored throughout the test and was recorded as described above. The test-retest reproducibility of the physiologic responses during the peak work capacity and prolonged submaximal tests was ±2% for heart rate, oxygen uptake, and carbon dioxide output.
Statistics
Repeated-measures analysis of variance (ANOVA) with individual volunteers used as their own controls (28) was used to determine dietary treatment and time effects, with Tukey-Kramer contrasts, and to determine whether the order of presentation of the diets had an effect. There were no order effects for any dependent variables. Variance in the data is expressed as a pooled SD, which was calculated as the square root of the mean square error from the ANOVA. Because diet analyses used fewer independent samples (n = 5), only means and SEMs are presented for these data. t Tests were done to determine whether zinc balance data were different from 0 (28).
RESULTS
Body weight was maintained within ±2% of admission weight, and body composition was unchanged during the experiment (data not shown).
Zinc intake, excretion, and balance
Zinc excretion corresponded with zinc intake (Table 2). Urinary and fecal zinc losses were significantly lower with dietary zinc restriction. Zinc balance was significantly lower with the low than with the supplemental zinc intake. Zinc balance (intake – excretion) was significantly different (P < 0.05) from 0 during consumption of the zinc-supplemented diet; zinc balance was not significantly different from 0 during the low-zinc diet.
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TABLE 2. Zinc intake, excretion, and retention determined in 14 men1
Blood biochemical measures and indexes of zinc status
Zinc intake affected neither hemoglobin concentration nor total RBC count (Table 3). Serum zinc and RBC zinc concentrations, total carbonic anhydrase activity, and the activities of the carbonic anhydrase I and II isoforms in RBCs decreased significantly over time when the men consumed the low-zinc diet but did not change significantly when they ate the supplemental-zinc diet. Total carbonic anhydrase activity was significantly correlated (r = 0.92, P < 0.01) with erythrocyte zinc concentration.
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TABLE 3. Hematology and blood biochemical indicators of zinc status of 14 men consuming diets with 2 zinc intakes1
Functional responses during exercise
Peak oxygen uptake values ranged from 2.9 to 4.1 L/min or 43–50 mL · kg–1 · min–1 determined with high dietary zinc. Zinc intake affected exercise performance as well as functional responses during graded, peak exercise. The duration of exercise time, and hence the total work, to attain peak work performance tended to be less (6%) when dietary zinc was low (Table 4). Peak ventilatory volume and ventilatory equivalents for oxygen and carbon dioxide increased significantly, and peak oxygen consumption, carbon dioxide output, and respiratory exchange ratio decreased significantly over time when the volunteers received the low-zinc diet with no change when the men consumed the supplemental-zinc diet.
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TABLE 4. Functional and metabolic responses to peak exercise of 14 men consuming diets with 2 zinc intakes1
Low zinc intake tended to impair performance and alter physiologic function during the submaximal exercise test. When dietary zinc was low, 4 of the volunteers did not complete the 45-min test, which resulted in a decrease (11%; P = 0.10) in the average duration of submaximal exercise (Table 5) and total work performed. Heart rate, ventilatory volume, and ventilatory equivalents for oxygen and carbon dioxide were significantly increased, whereas oxygen consumption, carbon dioxide output, and respiratory exchange ratio were significantly decreased, over the time the men consumed the low-zinc diet but did not change significantly when they ate the supplemental-zinc diet.
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TABLE 5. Physiologic responses during prolonged, submaximal exercise of 14 men consuming diets with 2 zinc intakes1
DISCUSSION
The consequences of restricted dietary zinc depend on the physiologic state of the individual, the amount and bioavailability of the ingested zinc, and the duration of the low zinc intake (29). The present findings that low, compared with supplemental, dietary zinc result in significantly lower zinc retention, serum and RBC zinc concentrations, and RBC carbonic anhydrase activities indicate marginal zinc deficiency. Although zinc balance was similar to 0 (0.23 mg/d) when zinc intake was 3.7 mg/d, inclusion of an estimated surface loss of zinc (0.30 mg/d; 36) at a dietary zinc level of 3.8 mg/d suggests net zinc loss. Similarly, use of 0.31 mg of surface zinc loss for a zinc intake of 18.8 mg/d (30) reduces zinc balance from 1.46 to 1.15 mg/d. Importantly, zinc loss was associated with significantly lower RBC carbonic anhydrase activities with low compared with supplemental dietary zinc.
Previous studies found decreased RBC total carbonic anhydrase activity in animals and fowl fed low-zinc diets. Early studies of zinc-deficient rats (14) and chicks (15) reported gasping respiration and increased rates of respiration with significantly decreased RBC total carbonic anhydrase activity. Zinc-deficient calves (16), lambs (17), and rats (18, 19) also had decreased RBC total carbonic anhydrase activities. The reduction of RBC carbonic anhydrase activity depended on the amount and duration of zinc restriction; it ranged from 35% to 60% of that in controls fed zinc-adequate diets (31).
Reports of the responsiveness of RBC carbonic anhydrase to zinc intake in humans are limited. Prasad et al (20) studied patients with sickle cell anemia and found zinc deficiency and significantly lower plasma and RBC zinc concentrations than in healthy adults. The RBC carbonic anhydrase isoform I and II proteins were 40% less than values in zinc-adequate controls; after oral zinc therapy (150 mg/d for 4–60 wk), isozyme proteins increased by >20% in the zinc-deficient patients. Zinc supplementation significantly increased RBC and plasma zinc. Prasad et al (20) concluded that synthesis of the carbonic anhydrase apoenzyme requires zinc and that the apoenzyme does not accumulate if zinc is lacking. Canfield and Johnson (32) reported a significant correlation (r = 0.89) between RBC total carbonic anhydrase activity and dietary zinc ranging from 3 to 17 mg/d in men. Men fed graded amounts of dietary zinc (1, 2, 3, 4, and 10 mg/d) for 36-d periods had no changes in plasma zinc or RBC carbonic anhydrase activity (33). The brief duration of the treatment periods was probably insufficient to affect zinc status; no effects of dietary zinc were found on the activities of other zinc metalloenzymes.
Use of carbonic anhydrase activity as a putative mechanism linking low zinc status with physiologic functions supporting physical activity has a strong theoretical basis. Carbonic anhydrase was the first zinc-containing enzyme described (13), and isoforms are present in all organisms. At least 9 different isozymes are distributed in mammalian tissues, including RBCs, kidney, muscle, bone, and other organs (34). This broad distribution reflects varying capacities for carbon dioxide removal and acid-base regulation. Three isoforms of carbonic anhydrase are found in RBCs. Isoform I, which is present in the greatest concentration, has limited carbon dioxide hydration-dehydration capacity. Isoform II is the physiologically most important for carbon dioxide removal but is moderate in concentration. The third isoform has a negligible role in carbon dioxide transport.
Carbonic anhydrase is also found in skeletal muscle. Red oxidative muscle contains 3–5 times more zinc than does white glycolytic muscle (35); the zinc is found in carbonic anhydrase (36). Skeletal muscle contains 2 isoforms of carbonic anhydrase, III and IV (37). Isoform III protects against free radical damage and controls the intermediary metabolism of glucose and fat, whereas isoform IV facilitates carbon dioxide removal (38). Thus, the ubiquitous distribution of carbonic anhydrases in mammalian tissues and their heterogeneous roles in cellular energy metabolism provide a novel link between zinc and energy expenditure.
A low zinc intake, which is characteristic of certain groups of the US population [including adolescents, the elderly, and some physically active individuals (5, 7[, consumed for 9 wk resulted in significantly lower total and specific carbonic anhydrase isozyme activities in RBCs than did a diet supplemented with zinc. The decreased enzyme activity was found in parallel with impaired cardiorespiratory responses during intense and prolonged submaximal exercise. The blunted oxygen uptake and carbon dioxide elimination and decreased respiratory exchange ratio are consistent with the findings of Wada and King (39), who showed decreased resting energy expenditure and a reduced respiratory exchange ratio in young men fed 5.5 compared with 16.5 mg Zn. Thus, suboptimal zinc intake is associated with disturbances in energy metabolism and cardiorespiratory function.
The effect of low zinc on carbonic anhydrase activity may be useful in explaining previous reports of altered muscle function and exercise capacity in humans with low zinc status. Studies of adolescents and men with significant reductions in serum zinc concentrations and decreased muscle strength and work capacity may be explained by reduced carbonic anhydrase activity (9, 10). Similarly, men fed diets low in zinc responded with significant decreases in plasma zinc and reduced muscular strength (8). Conversely, increased strength gain and muscle endurance of older women supplemented with zinc (30 mg/d) may be the result of increased activity of tissue carbonic anhydrase (40). Collectively, these findings support a role for zinc, as related to carbonic anhydrase, in the promotion and maintenance of physical activity and performance and merit further research.
A previous study of young men fed a diet low in zinc (4 mg/d) and then supplemented with zinc (35 mg/d) failed to show any effect of zinc intake on peak work capacity, metabolic responses during exercise, zinc balance, or zinc status indicators (41). In contrast, the present study found net zinc loss, decreases in biochemical markers of zinc status, and altered metabolic responses to controlled exercise. Thus, reduced zinc status is associated with impaired physiologic responses during work.
Certain technical factors limited past success of carbonic anhydrase as a marker of human zinc nutritional status. Reliance on a colorimetric method (25) contributed to a general lack of precision and increased interobserver variability (26). However, the use of highly accurate and precise automated pH devices and electrodes has eliminated reproducibility as a problem (26). A second potential obstacle has been the use of short durations of dietary zinc interventions. Failure to use periods of 60 d, consistent with the half-life of RBCs, apparently reduced the likelihood of finding changes in carbonic anhydrase. Thus, future studies that seek to assess the sensitivity of this zinc metalloenzyme to dietary zinc might benefit from the use of cell-separation techniques that enable a comparison of carbonic anhydrase activity and apoenzyme protein expression in newly formed compared with older erythrocytes, as well as incorporation of more modern assay procedures.
In conclusion, the present study showed that low dietary zinc in amounts consumed by segments of the population regularly involved in regular physical activity reduces zinc status. Our results provide evidence in support of the use of RBC carbonic anhydrase activity as a useful marker of human zinc nutritional status and links its in vitro reduction in activity to functional impairments in respiratory and metabolic responses to different types of exercise. These findings provide the first hypothesis explaining previous observations of impaired muscle function with reduced circulating zinc concentrations in adolescents and adults and emphasize the potential of carbonic anhydrase measurement in future studies examining zinc intake and physical activity.
ACKNOWLEDGMENTS
The author gratefully recognizes the valuable contributions of other members of our human studies research team: BS Hoverson supervised diet development and preparations, SK Gallagher supervised clinical and mineral analysis laboratories, CB Hall and WA Siders administered the exercise tests and collected indirect calorimetry data, and KG Michelsen performed the carbonic anhydrase assays. I am particularly thankful for the dedication and conscientious participation of the men, without whose contributions this study would not have been successful.
HCL conceptualized and designed the study, supervised the data collection and analyses, interpreted the findings, and wrote the manuscript. The author had no conflict of interest to report.
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