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Nutrient Requirements and Optimal Nutrition

Several Indicators of Oxidative Stress, Immunity, and Illness Improved in Trained Men Consuming an Encapsulated Juice Powder Concentrate for 28 Weeks^1–3,

⁴ Styrian Health Association, 8010 Graz, Austria; ⁵ Institute of Physiological Chemistry, and ⁶ Institute of Physiology, Medical University of Graz, 8010 Graz, Austria; ⁷ Institute of Sports Science, Karl Franzens University of Graz, 8010 Graz, Austria; and ⁸ Human Performance Research^Graz, Karl Franzens University and Medical University of Graz, 8010 Graz, Austria

^* To whom correspondence should be addressed. E-mail: manfred.lamprecht{at}meduni-graz.at.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Phytonutrients from plant foods provide numerous antioxidants. We hypothesized that supplementation for 28 wk with a commercially available encapsulated juice powder concentrate (JPC) could influence indicators of oxidative stress, immunity, and illness. Trained men (n = 41; 34 ± 5 y; maximum oxygen uptake = 55 ± 7 mL·kg^–1·min^–1) from a homogenous police Special Forces unit were randomly assigned in a double blind manner to either JPC (n = 21) or placebo (n = 20). We used multiple 7-d food records to assess dietary intake and found inadequate mean daily fruit and vegetable consumption (3.2 ± 1.2 servings). The group physician documented all duty days lost due to illness. We collected plasma at baseline and study wk 4, 8, 16, and 28 for analysis of carbonyl groups on protein (CP) and TNF. Over the 28-wk investigation, CP was lower in the JPC group, with both a treatment and a time x treatment interaction (P < 0.05). Concentrations of both CP and TNF at 16 and 28 wk were lower in the JPC than in the placebo group (P < 0.001). TNF increased during the first 8 wk followed by a decrease in both groups for the following 20 wk (P < 0.001). Over the final 20 wk of the study, the placebo group tended to have more days of illness than the JPC group (P = 0.068). These data suggest beneficial JPC effects with regard to reduction of duty days lost due to illness and reduction of CP and TNF concentrations in this group of trained men over 28 wk.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Some benefits of increased produce consumption may be obtained by adding a juice powder concentrate (JPC) to the daily diet. For example, a pilot study found that JPC reduced plasma CP and malondialdehyde concentrations in trained male cyclists (8) and others have reported reduced CP in aerobically trained men after controlled exercise periods (9). A study in untrained subjects found 11 wk of JPC intake positively influenced several markers of immune function and oxidative stress, specifically increasing -T cells and plasma oxygen radical absorbance capacity while decreasing the plasma cytokine, IFN- (10).

Stress likely has detrimental effects on immunity and may increase susceptibility to infectious agents (11). Regular exercise training can adapt the endogenous antioxidant enzyme systems [e.g. increasing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx)]. This could result in decreased oxidative damage and increased resistance to oxidative stress (12–14). The influence of antioxidant supplementation and exercise training on these enzyme activities is unclear (15).

The objectives of this study were to evaluate JPC effects, compared with placebo, on blood concentrations of oxidative stress and immunity biomarkers, in addition to the functional effects (monitored as duty days lost due to illness), in a homogeneous cohort of trained men over an extended period of time (28 wk). These subjects were a trained and disciplined cohort living in a group environment following a standardized diet and training regimen.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Study design. This was a randomized, double blind, placebo-controlled study. The study term was a 28-wk experimental period from November 2005 to June 2006 (Supplemental Table 1). We conducted anthropometric measurements and physical activity and dietary questionnaires, and determined maximum oxygen uptake (VO₂max) 4–6 wk prior to the start of this investigation, followed by a minimum 4-wk washout period from all dietary supplements. Dietary supplement use was prohibited during the study period. On study d 1, subjects were randomly assigned to receive either JPC or placebo capsules. Those randomized to JPC (n = 21) received capsules containing primarily blended fruit, vegetable, and berry JPC derived from: apple, beet, bilberry, blackberry, black currant, blueberry, broccoli, cabbage, carrot, cherry (acerola), Concord grape, cranberry, elderberry, kale, orange, peach, papaya, parsley, pineapple, raspberry, red currant, spinach, and tomato (Juice Plus+, NSA). Six JPC capsules provided 7.5 mg ß-carotene, 200 mg vitamin C, 60 mg vitamin E in the form of RRR--tocopherol, 600 µg folate, and 63 kJ/d. The study subjects randomized to placebo (n = 20) received capsules identical in appearance containing microcrystalline cellulose. All subjects took 6 capsules daily with meals for 28 wk (3 capsules in the morning and evening, except on training days: 3 capsules with the last meal before training and 3 capsules with the first meal after training). Capsule compliance was >85% in both groups, estimated from returned capsule counts and questionnaires.

Blood was collected 3 h after a standardized breakfast (providing 4222 kJ: 32–34 g protein, 144–150 g carbohydrate, and 28–30 g fat) for biochemical analysis. Duty and training obligations did not allow for a meal after the laboratory visits for blood collection. The standardized meal limited nutrient variation due to self-selection on the mornings scheduled for blood draws. Prospective subjects were healthy, nonsmoking men in the Graz region of the Austrian Special Forces Cobra unit (n = 46). All trained aerobically at least 3 d/wk for a minimum of 1 y prior to study participation and had a minimum level of aerobic fitness as assessed with maximal testing (VO₂max > 45 mL·kg^–1·min^–1) 4–6 wk prior to baseline. Each volunteer provided written informed consent prior to participating in this investigation. This study was conducted in accordance with the guidelines of the Declaration of Helsinki for Research on Human Subjects and was approved by the Human Ethics Committee at the Medical University of Graz, Austria. Exclusion criteria included use of tobacco products, chronic or excessive alcohol consumption, recent surgery or severe injury or illness, and use of prescription or over-the-counter medications. The Cobra unit physician documented and evaluated any new medications or injuries occurring during the study. One potential participant was excluded prior to randomization due to an exceptionally high VO₂max > 65 mL·kg^–1·min^–1 at the initial evaluation. Four randomized participants did not complete the investigation (3 were transferred to the Vienna area Cobra unit and 1 required hospitalization unrelated to the investigation). Therefore, 41 subjects were included in the data analysis.

    Dietary records. Dietary data were collected according to guidelines of the Austrian Nutrition Society. The same researcher instructed all subjects regarding portion sizes and proper recording of foods and beverages consumed. Food records for 7 d prior to each blood collection allowed for nutrient intake assessment and diet duplication. Subjects received copies of their previous 7-d food record and were instructed to replicate that diet prior to their next blood collection and to otherwise maintain their habitual diet throughout the entire study period. All participants had lunch at the Cobra group house and often breakfast and dinner, depending on individual duty assignments. The 7-d food records were analyzed twice (at baseline and after 28 wk) for average number of servings by food group (Supplemental Table 2) and for energy and nutrient content using Opti Diet software (version 3.1.2, GOEmbH), then compared with the recommended dietary allowance established by the German and Austrian Nutrition Societies (16) (Supplemental Table 3).

    Body fat determination. Baseline body fat content and distribution were estimated using a computerized optical device (Lipometer, Moeller Messtechnik), which allows rapid, safe, and noninvasive determination of subcutaneous adipose tissue thickness (17).

    Screening for VO₂max. Step test VO₂max and respiratory gas determination was conducted using a bicycle ergometer (Schiller ERG 900S Ergometer). To qualify, 46 potential study participants performed VO₂max tests with additional blood lactate analysis to determine exercise performance. Respiratory variables were measured using a portable open-air spirometry system (MetaMax I, Cortex).

    Medical monitoring. The Cobra unit physician documented all duty days lost due to illness, injury, mission assignments, and other stressors such as circadian imbalance (jet lag). Detailed symptom information was documented for all illnesses.

    Physical activity records. The training program, including intensity, duration, extent, and frequency, for each subject was recorded and summarized weekly. This program included both endurance and weight training 2–3 times per week along with additional special training for specific missions. Each subject was instructed to not perform weight training or high intensity/long duration endurance training 4 d prior to blood collections. They were allowed to perform daily cardiovascular exercise at a maximum heart rate of 130 beats/min for up to 30 min during the 4 d prior to blood draw.

    Blood collection and sample preparation. Collection of capillary blood (600 µL) used EDTA-coated vials (Sarstedt). After centrifugation at 3000 x g for 10 min plasma was removed and samples were frozen at –70°C for analysis of CP, TNF, and IL-6. Erythrocytes were washed 3 times with isotonic solution and then lysed with Millipore H₂O. After centrifugation at 3000 x g, the supernatant was frozen at –70°C for analysis of hemoglobin (Hb), GPx, and SOD.

    Blood chemistry panel. Baseline and final standard blood chemistry used EDTA plasma from peripheral venous blood. Analysis used routine methods and the clinical chemistry analyzer "Eurolyser" (Dia Team, Diagnostica und Arzneimittel Großhandel). Assessment of Hb and iron concentrations used the Advia clinical analyzer (Fa. Bayer).

    Analysis of CP, IL-6, and TNF. Determination of CP required preparation of oxidized bovine serum albumin for standardization, as described elsewhere (18). Measurement of CP used chemiluminescent detection (Lumistar, BMG) after derivatization with dinitrophenyl-hydrazine. Assessment of plasma protein concentration used the bicinchoninic assay (Pierce). Quantitation of IL-6 and TNF used an ELISA from Biosource with a spectrophotometric reader (Power Wave, Bio-TEK Instruments). The analytical inter-assay CV were 3.6% for CP, 9.6% for IL-6, and 8.2% for TNF.

    Analysis of SOD and GPx activity. Determination of GPx activity from erythrocyte lysate was performed indirectly by a coupled reaction with glutathione reductase utilizing the ZeptoMetrix assay kit adapted to 96-well plates, with results expressed in units/g Hb. Assessment of SOD activity used erythrocyte lysate with xanthine oxidase in the start reagent, as previously described (19), expressed in units/mg Hb. Analytical inter-assay CV were 5.6% for SOD and 9.2% for GPx.

    Statistical analysis. We used SPSS version 12.0 for statistical analysis. Data are presented as means ± SD. Significance was set at P < 0.05. Power analysis and sample size calculation based on previous work with CP (8) indicated 20 subjects per group were adequate for statistical power of 85% with = 0.05. Comparison of group baseline characteristics and food intake data used a t test. Group comparison of nutrient analysis data used 1-way ANOVA and duty days lost due to illness used the binomial test. We analyzed data obtained for CP, TNF, IL-6, SOD, and GPx using a 5 (time) x 2 (treatment) repeated measures ANOVA with grouping factors to show group differences over time. When equal variance failed, a comparison was made by the Mann-Whitney rank sum test. To evaluate differences between treatments at specific time points, additional analysis used t tests. We used Pearson correlation coefficient and regression analysis to evaluate bivariate relationships.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
At baseline, the 2 randomized groups did not differ in age, height, weight, total body fat, lean body mass, VO₂max, maximum workload, or clinical chemistry variables (Table 1). Baseline clinical blood chemistry parameters, including glucose, albumin, cholesterol, triglycerides, C-reactive protein, Hb, and iron concentrations and liver enzyme activity, were within normal limits for this population and did not change significantly in any subject throughout the study period (data not shown). The groups had equivalent dietary intake by both food group consumption pattern (Supplemental Table 2) and diet nutrient analysis (Supplemental Table 3). The entire cohort consumed fewer than 4 servings daily of fruit and nonstarchy vegetables.

The group physician documented duty days lost to illnesses (cold/flu symptoms) over the 28-wk observation period, with 134 d lost in the placebo group and108 d lost in the JPC group (P = 0.089). Duty days lost due to illness did not differ between the groups (P = 0.713) from baseline to wk 8 (placebo, 73 d; JPC, 67 d). Duty days lost to illness tended to be fewer (P = 0.068) in the JPC group (41 d) than in the placebo group (61 d) over the remaining 20 wk (Table 2).

View this table: [in this window] [in a new window]   TABLE 3 Comparison of plasma CP, TNF, and RBC SOD and GPx in physically active men consuming JPC or placebo for 28 wk1

During the 28-wk study period, there was a positive correlation between SOD and GPx enzyme activity (P = 0.01; r² = 0.982). SOD activity changed over time (P < 0.001); it decreased from baseline to wk 8, increased from wk 8 to wk 28, and then returned to baseline values (Table 3). GPx activity, similar to SOD, also showed a time effect (P = 0.011), decreasing from baseline to wk 8, then increasing from wk 8 to wk 28, returning to baseline values (Table 3).

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
This was an investigation of a homogenous population of fit men who shared many group meals and often slept in the Cobra group house. The results of this study indicate daily supplementation with JPC for 28 wk protected plasma proteins against free radical-induced oxidative stress compared with a placebo, as demonstrated by decreased CP concentrations. The immune and inflammatory marker, TNF, was also reduced in the JPC group. The difference was most pronounced from wk 8 to wk 28 (January to June) in conjunction with the additional stress of increased duty hours. The stable activities of antioxidant enzymes GPx and SOD across the study term, combined with reduced concentrations of CP, suggest enhanced antioxidant status in the JPC group as previously reported by others (10,21).

The combination of fruit and nonstarchy vegetable servings averaged 3.2/d in both groups (Supplemental Table 2), below the lowest public health recommendation (1). Dietary intake analysis found folate was low but equivalent in the groups at 60–61% of reference intake amount, whereas dietary intake of antioxidant vitamins C and E was adequate and equivalent in both groups. Intake of ß-carotene was also equivalent in both groups, between 80 and 88% of the reference intake amount (Supplemental Table 3).

Peters et al. (22) demonstrated that antioxidant supplementation could attenuate upper-respiratory tract infections in runners. Because running was included in the cohorts' training program twice weekly, we documented illnesses with cold and flu symptoms, such as sore throat and fever. We found a trend for fewer duty days lost due to illness in the JPC group across the entire 28 wk (P = 0.089), which was more pronounced during the final 20 wk of the study term (P = 0.068), in agreement with previous findings (10). The mean number of duty hours increased from 1140 to 1620 in both groups from the baseline to wk 28, representing additional stress for the subjects. It is possible that the additional nutrients provided in the JPC group contributed to modified immune function, resulting in fewer illness days and reduction of circulating cytokines in blood (22).

Leucocytes such as monocytes, macrophages, neutrophils, and natural killer cells produce TNF in addition to endothelium and adipocytes. TNF is a central mediator of systemic inflammatory and immune response. There is little influence of exercise alone on this parameter (23,24). The significant increase observed at wk 8 in both groups might be due to the illnesses in the entire cohort before the wk 8 (January) follow-up visit (a total of 140 d; Table 2). After the initial increase of TNF in both groups, the JPC group had significantly lower concentrations toward the end of the study than the placebo group (Table 3; Supplemental Fig. 1B). As previously mentioned, there were also fewer documented days of illness during this time (wk 8–wk 28) in the JPC group (Table 2).

IL-6 is a cytokine primary produced by fibroblasts, endothelium cells, and leucocytes like monocytes, macrophages, lymphocytes, and granulocytes. Like TNF, IL-6 is a mediator of systemic inflammatory and immune reactions, but as opposed to TNF, IL-6 is released in higher concentrations after exercise (24). IL-6 mediates many aspects of the exercise-induced acute phase response, including upregulation of antioxidant defenses. Synthesis is regulated in part by oxidative stress (25). Sacheck et al. (25) reported no changes from baseline IL-6 after a 12-wk period of supplementation with vitamin E (1000 IU/d) in healthy, trained young and old men. Childs et al. (26) found no significant effect of vitamin C and N-acetyl-cysteine supplementation on IL-6 concentrations. Nantz et al. (10) also did not observe any effect on IL-6 after 11 wk of JPC supplementation in an untrained population. These studies illustrate supplemental antioxidants may not influence IL-6 concentrations.

Protein oxidation can result in loss of enzyme and protein structural functions (27). Protein attack by free radicals, such as reactive oxygen and nitrogen species, free metal ions, and lipid peroxidation end products (including hydroxynonenal or malonedialdehyde) can generate CP (28). We observed a significant decrease in CP in the JPC group, whereas the concentrations in the placebo group were constant over the first 16 wk (Table 3; Supplemental Fig. 1A). This reduction of plasma CP concentrations in response to JPC treatment is in agreement with previous results (6,9). The significant differences in CP between the study groups after 16 and 28 wk indicate a different profile of oxidative stress in these groups. Whereas the concentrations in the JPC group decreased further for the remaining study term, the placebo group increased in CP from wk 16 to 28. The etiology of this increased CP in the placebo group is unknown, although the stress profile due to the increased amount of duty hours (in both groups) may have contributed to this observation.

SOD are a family of mineral-containing antioxidant enzymes that scavenge superoxide radicals and are regarded as important enzymes in the antioxidant defense system (29,30). GPx are a family of selenium-containing antioxidant enzymes catalyzing the reduction of reactive oxygen and nitrogen species, such as hydrogen peroxide or lipid peroxides, in the presence of reduced glutathione (31,32). As demonstrated previously (33), GPx activities in erythrocytes decreased when large amounts of ß-carotene, vitamin E, vitamin C, and selenium were taken for 15 d. In comparison, the JPC used in this longer study provided modest amounts of these nutrients and the findings did not indicate any effect on SOD or GPx activity. It has been postulated in a review and several studies (30,34–37) that physically active people have adapted antioxidant enzyme systems. Therefore, the stable enzyme activities observed here might be the result of adaptation to chronic aerobic training. However, a time effect occurred across the study term, with significant decreases of both enzyme activities from baseline to wk 8 followed by significant increases in both groups over the remaining time. Both SOD and GPx were correlated over time, suggesting a higher level of oxidative stress over the winter months, with no difference between the study groups.

In conclusion, the groups did not differ in relation to IL-6, SOD, and GPx. However, the JPC group showed positive changes in CP and TNF concentrations and a trend toward fewer duty days lost due to illness compared with the placebo group. We can postulate that intervention may have been helpful in maintaining resistance against illness in this specific cohort of chronically stressed and exercising men. Applicability of these findings to other populations will require additional study in a larger, more diverse group.

	ACKNOWLEDGMENTS

 
The authors thank the Einsatzkommando Cobra Süd Commander, Colonel Manfred Komericky, for cooperation and access to this cohort.

	FOOTNOTES

 
¹ Supported by a cooperative international research grant from NSA (Collierville, TN) to the Medical University of Graz (project no. 679) and the Styrian Health Association, Graz, Austria.

² Author disclosures: M. Lamprecht, K. Oettl, G. Schwaberger, P. Hofmann, and J. F. Greilberger, no conflicts of interest.

³ Supplemental Tables 1–3 and Supplemental Figure 1 are available with the online posting of this paper at jn.nutrition.org.

⁹ Abbreviations used: CP, carbonyl groups on protein; GPx, glutathione peroxidase; Hb, hemoglobin; JPC, juice powder concentrate; SOD, superoxide dismutase; VO₂max, maximum oxygen uptake.

Manuscript received 19 June 2007. Initial review completed 11 July 2007. Revision accepted 18 September 2007.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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This Article Abstract Full Text (PDF) Online Supporting Material Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lamprecht, M. Articles by Greilberger, J. F. Search for Related Content PubMed PubMed Citation Articles by Lamprecht, M. Articles by Greilberger, J. F.