Ergogenic and Antioxidant Effects of Spirulina Supplementation in Humans

Maria Kalafati; Athanasios Z. Jamurtas; Michalis G. Nikolaidis; Vassilis Paschalis; Anastasios A. Theodorou; Giorgos K. Sakellariou; Yiannis Koutedakis; Dimitris Kouretas

Disclosures

Med Sci Sports Exerc. 2010;42(1):142-151. 

In This Article

Materials and Methods

Subjects

Nine healthy moderately trained men (age = 23.3 ± 1.7 yr, height = 174.3 ± 1.7 cm, weight = 70.7 ± 1.9 kg, body fat = 9.8 ± 1.3%, maximal oxygen consumption (V·O2max) = 52.2 ± 1.8 mL·kg−1·min−1) volunteered to participate. The subjects were recreational runners and had trained for at least 1 yr (3.4 ± 1.1 yr), at least two times per week (3.1 ± 0.9 times per week), at least 45 min per session (56 ± 10 min per session). All subjects were informed thoroughly about the risks, the possible discomforts, and the benefits of the study before signing a written informed consent. All subjects completed a medical and supplementation history and physical activity questionnaire to determine eligibility. No subject was a smoker or taking supplements or anti-inflammatory drugs. The procedures were in accordance with the Helsinki Declaration of 1975 and approved by the institutional review board.

Baseline Measurements

One to two weeks before the first exercise trial, subjects visited the laboratory for baseline measurements. Body mass was measured to the nearest 0.5 kg with subjects lightly dressed and barefoot (Beam Balance 710; Seca, Birmingham, United Kingdom) and standing height was measured to the nearest 0.5 cm (Stadiometer 208; Seca). Percentage body fat was calculated from seven skinfold measurements using a Harpenden skinfold caliper (John Bull, British Indicators Ltd, St. Albans, United Kingdom) according to published guidelines.[4] To establish that all subjects ran at similar exercise intensity, V·O2max was determined using a treadmill test to exhaustion. The protocol began at 10 km·h−1 and was increased by 1 km every 2 min until V·O2max was reached. V·O2max test was terminated when three of the following four criteria were met: (i) subject exhaustion, (ii) a < 2 mL·kg−1·min−1 increase in V·O2 with an increase in work rate, (iii) a respiratory exchange ratio ≥1.10, and (iv) an HR within 10 bpm of the theoretical maximum HR (220 - age). Respiratory gas variables were measured using a metabolic cart (Vmax29; SensorMedics, Yorba Linda, CA), which was calibrated before each test using standard gases of known concentration. Exercise HR was monitored by telemetry (Tester S610™; Polar, Electro Oy, Finland).

Study Design

A double-blind, placebo-controlled, counterbalanced crossover design was used (i.e., half of the subjects were given the spirulina first and the other half were given the placebo and the reversed). Each subject participated in four exercise trials (Fig. 1). In the first exercise trial, subjects visited the laboratory 7-14 d after V·O2max determination (between 08:00 and 10:00), where they ran on a treadmill at an intensity corresponding to 70%-75% of their V·O2max for 2 h. After the 2-h run, the speed of the treadmill was increased to elicit the 95% V·O2max, and exercise was terminated at exhaustion.[31] Fatigue was considered to have occurred when the required speed could not be maintained by the subject or when the subject stopped voluntarily. The time to reach volitional fatigue was recorded and used as an index of aerobic performance. Expired gas samples were obtained every 10 min to ensure the prescribed exercise intensity and to calculate the fat and carbohydrate oxidation rates. Water (250 mL) was given to the volunteers every 20 min during exercise. After the end of the initial exercise trial, each subject consumed two capsules (1 g each) containing either S. platensis manufactured by Algae AC (Serres, Greece) or 100% egg protein (placebo). The capsules were consumed before meals three times per day for 4 wk. The daily dosage of spirulina that was used (6 g·d−1) was close to other relevant human studies (7.5 [15] and 8 [25] g·d.−1 One day after the end of the 4-wk supplementation period, subjects came back to the laboratory to perform the second exercise bout with identical conditions as the first exercise trial. A 2-wk washout period occurred between the second and the third exercise trials to avoid possible carryover effects. After the washout period, the subjects came back for a third and fourth times, where the exercise conditions of the first and second exercise trials were followed. The first and third exercise trials were performed to ensure that the 2-wk washout period was adequate to have similar physiological and biochemical values before the two periods of supplementation. We are aware of only one study that investigated the effects of spirulina supplementation on humans using a crossover design.[6] In this study, a 2-wk washout period was also used. In addition, taking into account the short supplementation period used in the present study (i.e., 4 wk), we considered that the 2-wk washout period would be long enough for any effects of placebo or spirulina to disappear.

Figure 1.

Study design. Arrows indicate blood sampling.

The basic composition of dry spirulina is as follows: 63.3% protein, 7.1% lipid, and 15.2% carbohydrate[50]), 101 mg of vitamin C[5]), 15 mg of vitamin E, and 0.13 mg of selenium per 100 g[50]), as well as 43.6% palmitic acid, 17.2% linoleic acid, and 21.7% γ-linolenic acid of total fatty acids.[33]

Fat and Carbohydrate Oxidation

Fat and carbohydrate oxidation rates (g·min−1) were calculated indirectly by monitoring the rate of O2 consumption (L·min−1) and CO2 production (L·min−1) using the following stoichiometric equations[18]), assuming that protein oxidation during exercise was negligible:

Blood Collection and Handling

Blood samples were drawn from a forearm vein at rest and after exercise (immediately after exercise and at 1, 24, and 48 h after exercise). Directly after taking the blood sample, 0.5 mL of blood was placed in a tube containing EDTA for the determination of hematocrit and hemoglobin. Whole-blood lysate was produced by adding 5% trichloroacetic acid (TCA) to whole blood (1:1 v/v) collected in EDTA tubes for reduced GSH and oxidized glutathione (GSSG) analysis. The whole-blood samples were centrifuged at 4000g for 10 min at 4°C, and the supernatant was removed and centrifuged again at 28,000g for 5 min at 4°C. The clear supernatant was collected in Eppendorf tubes and stored at -80°C until GSH and GSSG determination. Another portion of blood was collected in plain tubes, left on ice for 20 min to clot, and centrifuged at 1500g for 10 min at 4°C for serum separation. Serum was transferred in Eppendorf tubes and was used for the determination of creatine kinase, thiobarbituric acid-reactive substances (TBARS), protein carbonyls, catalase, and total antioxidant capacity (TAC). Serum samples were stored in multiple aliquots at -80°C and were thawed only once before analysis.

Assays

A slightly modified version of Reddy et al.[40]) was used to measure GSH, which is originally based on Beutler et al.[7] Twenty microliters of whole blood treated with TCA was mixed with 660 μL of 67 mM sodium potassium phosphate (pH 8.0) and 330 μL of 1 mM 5,5-dithiobis-2-nitrobenzoate (DTNB). The samples were incubated in the dark at room temperature for 45 min, and the absorbance was read at 412 nm. A standard curve was constructed by using GSH as a standard at concentrations of 0, 0.25, 0.50, and 1 mM. GSSG was determined according to Tietze.[49] Two hundred and sixty microliters of whole blood treated with TCA was neutralized up to pH 7.0-7.5 with NaOH. Four microliters of 2-vinyl pyridine was added, and the samples were incubated for 2 h at room temperature. Five microliters of whole blood treated with TCA was mixed with 600 μL of 143 mM sodium phosphate (6.3 mM EDTA, pH 7.5), 100 μL of 3 mM nicotinamide dinucleotide phosphate (NADPH), 100 μL of 10 mM DTNB, and 194 μL of distilled water. The samples were incubated for 10 min at room temperature. After the addition of 1 μL of glutathione reductase, the change in absorbance at 412 nm was read for 3 min. A standard curve was constructed by using GSSG as a standard at concentrations of 0, 0.025, 0.050, and 0.100 mM. The GSH/GSSG ratio was calculated for each subject, and the means of these ratios for each time point are presented.

TBARS were measured according to Keles et al.[22] One hundred microliters of serum was mixed with 500 μL of 35% TCA and 500 μL of Tris-HCl (200 mM, pH 7.4) and incubated for 10 min at room temperature. One microliter of 2 M Na2SO4 and 55 mM thiobarbituric acid solution was added, and the samples were incubated at 95°C for 45 min. The samples were cooled on ice for 5 min and were vortexed after adding 1 mL of 70% TCA. Finally, the samples were centrifuged at 15,000g for 3 min, and the absorbance of the supernatant was read at 530 nm. A standard curve was constructed by using malondialdehyde as a standard at concentrations of 0, 1.25, 2.5, 5, and 10 μM.

Protein carbonyls were measured according to Patsoukis et al.[36] In 50 μL of serum, 50 μL of 20% TCA was added, incubated in the ice bath for 15 min, and centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 500 μL of 10 mM 2,4-dinitrophenylhydrazine (in 2.5N HCl) for the sample, or 500 μL of 2.5N HCl for the blank, was added to the pellet. The samples were incubated in the dark at room temperature for 1 h, with intermittent vortexing every 15 min, and were centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 1 mL of 10% TCA was added, vortexed, and centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 1 mL of ethanol-ethyl acetate (1:1 v/v) was added, vortexed, and centrifuged at 15,000g for 5 min at 4°C. The washing step was repeated two more times. The supernatant was discarded, and 1 mL of 5 M urea (pH 2.3) was added, vortexed, and incubated at 37°C for 15 min. The samples were centrifuged at 15,000g for 3 min at 4°C, and the absorbance was read at 375 nm. Protein carbonyls values were obtained by using the extinction coefficient of 2,4-dinitrophenylhydrazine (22 mM·cm−1.

Catalase activity was measured according to Aebi.[1] In 20 μL of serum, 2975 μL of 67 mM sodium potassium phosphate (pH 7.4) was added, and the samples were incubated at 37°C for 10 min. Five microliters of 30% hydrogen peroxide was added to the samples, and the change in absorbance was immediately read at 240 nm for 1.5 min. Catalase activity was obtained by using the extinction coefficient of hydrogen peroxide (43.6 M·cm−1.

TAC was measured according to Janaszewska and Bartosz.[17] For TAC, in 20 μL of serum, 480 μL of 10 mM sodium potassium phosphate (pH 7.4) and 500 μL of 0.1 mM 2,2-diphenyl-1 picrylhydrazyl (DPPH) were added and incubated in the dark for 30 min at room temperature. The samples were centrifuged for 3 min at 20,000g, and the absorbance was read at 520 nm. TAC values were obtained by calculating the number of DPPH molecules scavenged per minute.

Serum creatine kinase was determined spectrophotometrically using a commercially available kit (Spinreact, Sant Esteve, Spain). Total protein in serum was assayed using a Bradford reagent. Postexercise plasma volume changes were computed based on hematocrit and hemoglobin. Hematocrit was measured by microcentrifugation, and hemoglobin was measured using a kit from Spinreact. Each assay was performed in duplicates, except for GSSG, which was performed in triplicates. The intra-assay coefficient of variation for each measurement was as follows: GSH 4.0%, GSSG 6.5%, TBARS 3.9%, protein carbonyls 5.5%, catalase 6.7%, TAC 3.7%, and creatine kinase 2.9%.

Dietary Analysis

To factor the effect of the diet on the outcome measures of the study and to establish that participants had similar levels of macronutrient and antioxidant intake during the period of data collection, they were asked to record their diet for 3 d preceding their first visit to the laboratory and to repeat this diet before their next three visits to the laboratory. Each subject had been provided with a written set of guidelines for monitoring dietary consumption and a record sheet for recording food intake. Diet records were analyzed using the nutritional analysis system ScienceFit Diet 200A (ScienceFit, Athens, Greece).

Statistical Analysis

The distribution of all dependent variables was examined by the Shapiro-Wilk test and was found not to differ significantly from normal. First, to ensure that the 2-wk washout period was adequate, the data from the first and the third trials were analyzed through two-way (trial × time) ANOVA with repeated measures on time. Second, to evaluate the effects of supplementation and exercise, the data from the second and the fourth trials were analyzed through two-way (group × time) ANOVA with repeated measures on time. If a significant interaction was obtained, pairwise comparisons were performed through simple main effect analysis. Differences in diet among trials or groups were examined through one-way ANOVA. Aerobic performance at the second and fourth exercise trials was examined by paired t-test. Carbohydrate and lipid oxidation rates during the 2-h run at the second and fourth exercise trials were also examined by paired t-test. Statistical significance was considered when P < 0.05. The SPSS version 15.0 was used for all analyses (SPSS, Inc., Chicago, IL). Data are presented as mean ± SEM.

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