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Neutrophil-degranulation and lymphocyte-subset response after 48 hr of fluid and/or energy restriction

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The aim was to investigate the effects of 48 hr of fluid, energy, or combined fluid and energy restriction on circulating leukocyte and lymphocyte subset counts (CD3+, CD4+, and CD8+) and bacterially stimulated neutrophil degranulation at rest and
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  443 International Journal of Sport Nutrition and Exercise Metabolism, 2008, 18, 443-456  © 2008 Human Kinetics, Inc. Laing is with the Olympic Medical Institute, Northwick Park Hospital, Harrow, HA1 3UJ, UK. Oliver, Wilson, and Walsh are with the School of Sport, Health and Exercise Sciences, Bangor University, Bangor, LL57 2PZ, UK. Walters is with the Haematology Dept., Ysbyty Gwynedd, Bangor, LL57 2PW, UK. Bilzon is with Headquarters Army Recruiting and Training Division, Upavon, SN9 6BE, UK. Neutrophil-Degranulation and Lymphocyte-Subset Response After 48 hr of Fluid and/or Energy Restriction Stewart J. Laing, Samuel J. Oliver, Sally Wilson, Robert Walters, James L.J. Bilzon, and Neil P. Walsh The aim was to investigate the effects of 48 hr of fluid, energy, or combined fluid and energy restriction on circulating leukocyte and lymphocyte subset counts (CD3 + , CD4 + , and CD8 + ) and bacterially stimulated neutrophil degranulation at rest and after exercise. Thirteen healthy men (  M   ± SEM   age 21 ± 1 yr) participated in 4 randomized 48-hr trials. During control (CON) participants received their estimated energy (2,903 ± 17 kcal/day) and fluid (3,912 ± 140 ml/day) require-ments. During fluid restriction (FR) they received their energy requirements and 193 ± 19 ml/day water to drink. During energy restriction (ER) they received their fluid requirements and 290 ± 6 kcal/day. Fluid and energy restriction (F+ER) was a combination of FR and ER. After 48 hr, participants performed a 30-min treadmill time trial (TT) followed by rehydration (0–2 hr) and refeeding (2–6 hr). Circulating leukocyte and lymphocyte counts remained unchanged for CON and FR. Circulating leukocyte, lymphocyte, CD3 + , and CD4 +  counts decreased by ~20% in ER and ~30% in F+ER by 48 hr (  p  < .01), returning to within 0-hr values by 6 hr post-TT. Circulating neutrophil count and degranulation were unaltered by dietary restriction at rest and after TT. In conclusion, a 48-hr period of ER and F+ER, but not FR, decreased circulating leukocyte, lymphocyte, CD3 + , and CD4 +  counts but not neutrophil count or degranulation. Circulating leukocyte and lymphocyte counts normalized on refeeding. Finally, dietary restriction did not alter circulating leukocyte, lymphocyte, and neutrophil responses to 30 min of maximal exercise.  Keywords:  dietary restriction, immune, phagocyte, time trial Dietary restriction has the potential to weaken several aspects of immune function, potentially leaving individuals more susceptible to infection (Chandra, 1997). Periods of forced or voluntary fluid and energy restriction, often lasting for a number of days, are commonplace in athletes making weight or with eating disorders (Baum, 2006; Brownell, Steen, & Wilmore, 1987) and in occupational ORIGINAL RESEARCH  444  Laing et al. settings, for example, military personnel during survival training (Carins & Booth, 2002). Energy restriction might decrease immune-cell metabolism, pro-tein synthesis, cell replication, and antioxidant defenses directly through reduced substrate availability (Chandra). Alternatively, a decrease in substrate availabil-ity might weaken immune function indirectly through raised sympathoadrenal activity resulting in increased secretions of stress hormones (e.g., cortisol and catecholamines) known to have immunosuppressive effects (Gleeson, Nieman, & Pedersen, 2004).Little is known about the independent and combined effects of fluid and energy restriction on immune function at rest and after exercise. Cellular and humoral immunity were depressed in soldiers training in a tropical environment and surviving for 12 days on limited rations (1,800 kcal/day; Booth, Coad, Forbes-Ewan, Thomson, & Niro, 2003). A 36-hr fast decreased circulating lymphocyte counts (Walrand et al., 2001), and a 7-day fast decreased circulating T-lymphocyte (CD3 + ) and helper T-lymphocyte (CD4 + ) counts (Savendahl & Underwood, 1997). The mechanism responsible for lower circulating lymphocytes and lymphocyte subsets during fasting remains unclear, although a role for elevated circulating cortisol has been proposed (Mustafa, Ward, Treasure, & Peakman, 1997). Cir-culating neutrophil counts were lower after 7 days of exercise (3 hr/day at 75% VO 2max ) with a 25% energy deficit compared with when participants received 110% of their estimated daily energy requirements (Galassetti et al., 2006). Neutrophil chemotaxis was decreased after a 36-hr fast, but this was reversed after only 4 hr of refeeding (Walrand et al.). Impaired phagocytic activity has also been shown in athletes restricting food and fluid intake to make weight before competition (Kowatari et al., 2001; Suzuki et al., 2003). Unfortunately, these studies do not distinguish between energy-restriction and fluid-restriction effects on immune function. Studies have provided limited information about fluid intake, reported their participants to be hypohydrated after dietary restriction, or were performed in a multistressor setting (e.g., tropical climate, psychological stress). Therefore, it is difficult to assess whether the observed immune responses reflect energy-restriction effects, a combination of fluid and energy restriction, or effects of other uncontrolled variables. Indeed, elevated plasma stress hormones have been observed during dehydration in ruminants (Parker, Hamlin, Coleman, & Fitzpatrick, 2003) and during exercise with restricted fluid intake in humans (McGregor, Nicholas, Lakomy, & Williams, 1999). As such, a role for hypohy-dration in the observed decrease in immune function during dietary restriction warrants inquiry. Therefore, the purpose of the current study was to investigate the effects of a 48-hr period of fluid, energy, or combined fluid and energy restriction on circulating leukocyte and lymphocyte subset counts (CD3 + , CD4 + , and CD8 + ) and bacterially stimulated neutrophil degranulation at rest and after exercise. We hypothesized that fluid or energy restriction would decrease circulating leukocyte counts, lymphocyte subset counts, and neutrophil degranulation at rest and after exercise and that the effects of combined fluid and energy restriction would be additive.   Dietary Restriction, Exercise, and Immune Response 445 Methods Participants Thirteen recreationally active healthy men (  M   ± SEM   age 21 ± 1 years, height 1.79 ± 0.01 m, body mass 74.7 ± 1.3 kg, body fat 16.8% ± 1.5%, VO 2max  50.9 ± 1.2 ml · kg –1  · min –1 ) volunteered to participate in the study. All participants gave written informed consent before commencing the study, which received local ethics committee approval (Bangor University). There were no reported symptoms of infection, and participants did not take any medication or nutritional supplements in the 6 weeks before, or during, the study. Preliminary Measurements Before the main experimental trials, each participant completed a continuous incremental exercise test on a treadmill to determine maximal oxygen uptake (VO 2max ) as has been previously described (Oliver, Laing, Wilson, Bilzon, & Walsh, 2007). From the VO 2 –work-rate relationship, the work rate equivalent to 50% VO 2max  was estimated and used for submaximal exercise during the experimental trials. On a separate day, 7–10 days before beginning the experimental trials, par-ticipants returned to the laboratory for individual energy-expenditure estimation and familiarization. They arrived euhydrated at 8:00 a.m. after an overnight fast, having consumed water equal to 40 ml/kg of their body mass the previous day. On arrival and after voiding, height and nude body mass (NBM) were measured. After these measures, body composition was estimated using whole-body dual-energy X-ray absorptiometry (Hologic, QDR1500, software version 5.72, Bedford, MA, USA), and resting metabolic rate was estimated using a portable breath-by-breath system (Metamax 3B, Biophysik, Leipzig, Germany). After breakfast, participants performed a 1.5-hr treadmill walk at 50% VO 2max  during which energy expenditure was estimated (Cortex Metalyser 3B, Biophysik, Leipzig, Germany). For short periods during the day participants wore the portable breath-by-breath system (Metamax 3B, Biophysik, Leipzig, Germany) to estimate the energy expenditure incurred during habitual living in the laboratory environment. These additional energy-expenditure data were used, along with the data on resting metabolic rate, to estimate the energy intake required for the experimental trials. In addition, during this 24-hr period, fluid requirements were estimated by assessing changes in body mass at hourly intervals. Physical activity was standardized throughout the familiarization and all experimental trials by recording 24-hr step counts with pedometers (Digi-walker SW-200, Yamax, Tokyo, Japan). Experimental Trials Participants were required to complete four experimental trials separated by 7–10 days in a randomized order (Table 1). The four dietary interventions included a control trial (CON), a fluid-restriction trial (FR), an energy-restriction trial (ER),  446  Laing et al. and a combined fluid- and energy-restriction trial (F+ER). On the day before the experimental trial, to control nutritional and hydration status, participants were provided with their estimated energy requirements, 2,903 ± 17 kcal/day, of which 49%, 36%, and 15% were carbohydrate, fat, and protein, respectively, and with water equal to 40 ml/kg of body mass (Figure 1). Participants were also instructed to refrain from exercise. They arrived at the laboratory at 10:00 p.m. the evening before each trial. On the night before the completion of each trial participants slept for 8 hr in a temperate laboratory (19.7 ± 0.3 °C, 58.8% ± 1.9% relative humidity). The intervention began at 8:30 the following morning after participants had voided and an NBM was obtained. After 10–15 min seated rest, a baseline (0 hr) blood sample was obtained. Further blood samples were collected after 24 hr (8:30 a.m., Day 2) and 48 hr (8:30 a.m., Day 3). Participants performed a 1.5-hr treadmill walk at a set workload equivalent to 50% VO 2max  after breakfast on Days 1 and 2. During the 1.5-hr walks, water was consumed in CON and ER in an amount equal to fluid losses, whereas no fluids were provided in FR and F+ER. After lunch and the evening meals, participants also completed a 20-min walk. After providing a 48-hr sample participants performed a self-paced 30-min treadmill time trial (TT), the data from which are presented elsewhere (Oliver, Laing, Wilson, Bilzon, & Walsh, 2007). Participants were instructed to run as far as possible in 30 min and to control the speed of the treadmill (gradient set at 1%) as and when they felt appropriate. No fluids were consumed during the treadmill TT. Further blood samples were obtained immediately, 2 hr, and 6 hr post-TT. During the first 2 hr of recovery, fluid was provided as a citrus-flavored electrolyte-only solution (50 mmol/L sodium, Science in Sport, Blackburn, UK). The rehydration solution was divided evenly across the 2 hr in an amount equal to 100% body-mass loss (BML) or up to 29 m/kg of body mass, which reflects the approximate maximal gastric-emptying rate for this solution (Mitchell, Grandjean, Pizza, Starling, & Holtz, 1994). During 2–3 and 4–5 hr of recovery, participants consumed a total of 1,950 ± 37 kcal (49%, 36%, 15% were carbohydrate, fat, and protein, respectively) divided equally into two meals. Water was available ad libitum during these two meals. Table 1 Nutrient Intake for 24 hr, M   ± SEM   ( N   = 13) RestrictionControl Fluid EnergyFluid and energy Fluid consumed (ml)3,912 ± 140960 ± 153,893 ± 136962 ± 16 water to drink (ml)3,145 ± 134193 ± 193,816 ± 135885 ± 15 water in food (ml)767 ± 11767 ± 1177 ± 177 ± 1Energy consumed (kcal)2,903 ± 172,903 ± 17290 ± 6290 ± 6 carbohydrates (g)387 ± 8387 ± 839 ± 139 ± 1 fat (g)119 ± 3119 ± 212 ± 012 ± 0 protein (g)104 ± 1104 ± 110 ± 010 ± 0  Note . Macronutrient composition was the same across all trials and equal to 50%, 36%, 14% carbo-hydrate, fat, and protein, respectively. Adapted with permission from Oliver, Laing, Wilson, Bilzon, Walters, and Walsh (2007).   Dietary Restriction, Exercise, and Immune Response 447 Analytical Methods Blood samples were collected, without venostasis, by venipuncture from an antecubital vein into four Vacutainer tubes (Becton Dickinson, Oxford, UK), two 4-ml Vacutainers containing K 3 EDTA (1.6 mg EDTA/ml blood), and one 6-ml Vacutainer containing lithium heparin (1.5 IU heparin/ml blood). Blood taken from one K 3 EDTA tube was immediately centrifuged at 3,000 rpm for 10 min and the plasma aliquotted into snap-seal microcentrifuge tubes (1.5-ml capacity, Sarstedt, Germany) and stored at –80 °C. Blood collected in the second K 3 EDTA tube was stored at room temperature before hematological analysis within 6 hr of collection. Hematological analyses including hemoglobin and total and differential leukocyte counts were performed using an automated cell counter (Gen S, Beck-man Coulter, Fullerton, CA, USA). Standard flow-cytometric techniques were used to determine T-lymphocytes (CD3 + ), helper/inducer T-lymphocytes (CD4 + ), and cytotoxic/suppressor T-lymphocytes (CD8 + ) using the tetraCHROME murine monoclonal antibody reagent (Beckman Coulter Ltd., UK). A 100- µ l volume of whole blood from the K 3 EDTA tube was added to a tube containing 10 µ l reagent (FITC [CD45], PC5 [CD3], RD1 [CD4], and ECD [CD8]), and the tube was incubated in the dark at room temperature for 15 min and then lysed using a T-Q prep (Beckman Coulter). The stained cell suspensions were enumerated on an Epics XL flow cytometer (Beckman Coulter) with 10,000 events recorded. The lymphocyte population was determined by gating on CD45 versus side scatter. Figure 1  — Schematic of trial events. B-fast = breakfast; NBM = nude body mass. Reprinted with permission from Oliver, Laing, Wilson, Bilzon, Walters, and Walsh (2007).
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