Effects of 12-Week Low or Moderate Dietary Acid Intake on Acid–Base Status and Kidney Function at Rest and during Submaximal Cycling

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Effects of 12-Week Low or Moderate Dietary Acid Intake on Acid–Base Status and Kidney Function at Rest and during Submaximal Cycling Hietavala, Enni-Maria; Ihalainen, Johanna; Frassetto, Lynda A.; Schumann, Moritz; Eklund, Daniela; Pitkänen, Hannu; Häkkinen, Keijo; Mero, Antti Hietavala, E.-M., Ihalainen, J., Frassetto, L. A., Schumann, M., Eklund, D., Pitkänen, H., Häkkinen, K., & Mero, A. (2018). Effects of 12-Week Low or Moderate Dietary Acid Intake on Acid–Base Status and Kidney Function at Rest and during Submaximal Cycling. Nutrients, 10(3), Article 323. https://doi.org/10.3390/nu10030323 2018 nutrients Article Effects of 12-Week Low or Moderate Dietary Acid Intake on Acid–Base Status and Kidney Function at Rest and during Submaximal Cycling Enni-Maria Hietavala 1,*ID , Johanna K. Ihalainen 1, Lynda A. Frassetto 2, Moritz Schumann 3, Daniela Eklund 1, Hannu Pitkänen 4, Keijo Häkkinen 1and Antti A. Mero 1 1 Biology of Physical Activity, Faculty of Sport and Health Sciences, University of Jyväskylä, P.O. Box 35 (VIV), 40014 Jyväskylä, Finland; [email protected] (J.K.I.); [email protected] (D.E.); [email protected] (K.H.); [email protected] (A.A.M.) 2General Clinical Research Center, University of California San Francisco, 505 Parnassus Avenue, San Francisco, CA 94117, USA; L[email protected] 3Department of Molecular and Cellular Sports Medicine, German Sport University, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany; [email protected] 4Honka Holding, c/o Honkatarhat Oy, Kirkkokallio 20, 38950 Honkajoki, Finland; [email protected] *Correspondence: [email protected]; Tel.: +358-40-805-4733 Received: 28 January 2018; Accepted: 5 March 2018; Published: 8 March 2018 Abstract: Prolonged effects of dietary acid intake on acid–base status and kidney function have not yet been studied in an intervention study in healthy subjects. Dietary acid load can be estimated by calculating the potential renal acid load (PRAL) of foods. Effects of low-PRAL and moderate-PRAL diets on acid–base status and kidney function were investigated during a 12-week exercise training period. Healthy, 20–50-year-old men (n= 21) and women (n= 25) participated in the study and were randomly divided into low-PRAL and moderate-PRAL groups. Before (PRE), mid-phase (MID) and after the intervention (POST), the subjects participated in measurement sessions, where a 12-h urine sample and fasting blood samples were collected, and a submaximal cycle ergometer test was performed. Net acid excretion was significantly lower after 12 weeks of the low-PRAL diet as compared to the moderate-PRAL diet, both in men and women. In low-PRAL females, capillary pH and bicarbonate were significantly higher at 75% of VO 2max at POST as compared to PRE. Glomerular filtration rate decreased over the study period in moderate-PRAL men and women. The results of the present study suggest that an acidogenic diet and regularly training together may increase the acidic load of the body and start to impair the kidney function in recreationally active subjects. Keywords: dietary acid load; acid–base status; net acid excretion; exercise training; kidney function 1. Introduction Many biochemical reactions release and bind hydrogen ions (H + ) in the human body. Under normal physiological conditions, diet composition is the primary modifier of net endogenous acid production (NEAP) and it may further affect the acid–base status of the body [ 1 ]. Dietary acid load can be estimated by calculating the potential renal acid load (PRAL) of foods, which estimates the acidic potential of foodstuffs [ 2 ]. Digestion of large amounts of animal protein and grain products, but only small amounts of vegetables and fruits, leads to a net production of acids in the body [ 3 ]. H + concentrations in body fluids are regulated to remain in between rather narrow pH limits and thus, only minor changes occur in blood pH. In arterial blood at rest, pH is normally maintained strictly between 7.35–7.45 and extracellular buffering (i.e., mainly bicarbonate buffering) occurs concomitantly Nutrients 2018,10, 323; doi:10.3390/nu10030323 www.mdpi.com/journal/nutrients Nutrients 2018,10, 323 2 of 12 with any changes in plasma H + concentration. However, it has been shown that diet composition may cause acute changes inside the optimal blood pH range [ 4 ]. Urine pH can vary between 4.5 and 8.0, according to the amount of H + that needs to be excreted from the body by the kidneys. The systemic bicarbonate (HCO 3− ) concentration represents the metabolic component of acid–base balance, and the kidneys have a prevalent role in regulating it [5]. In the field of exercise physiology, effects of exercise on kidney function have not been studied very intensively. The kidney has an essential role in the homeostasis of the body at rest, but changes in renal function also occur with exercise. Under resting conditions, blood flow to the kidneys is among the highest to any organ. However, oxygen consumption is not increased in renal tissue during exercise, and blood flow is redistributed away from the kidney to skeletal muscles [ 6 ]. The glomerular filtration rate (GFR) is a measure of the amount of fluid filtered through the glomerular basement membrane in the kidneys, and it is considered to be the best overall assessment of kidney function [ 7 ]. With exercise loads up to 50% of VO 2max , GFR is slightly increased or unchanged, and at higher exercise intensities, GFR decreases at higher rates than renal blood flow [ 8 ]. The long-term effects of exercise training on kidney function have not been studied in healthy populations, but in patients suffering decreased kidney function there is evidence for an association between kidney function and exercise performance [ 9 ]. Moreover, in a study by Morales et al. [ 10 ], kidney function affected the physical performance of athletes, as VO 2max was lower and heart rate higher in a group of athletes with smaller GFR, compared to athletes with higher GFR. It was recently shown that dietary acid load may play a role in delaying fatigue during exercise as the effects of dietary acid load on acid–base status and physical performance were investigated during a 7-day diet period [ 11 ]. The data suggested that lower acid intake could help the kidneys to increase exercise capacity by maintaining a higher extracellular HCO3−concentration, which could delay the onset of fatigue caused by exercise-induced acidosis. To the best of our knowledge, the prolonged effects of dietary acid intake on acid–base status and kidney function, with or without exercise training, have not yet been studied in an intervention study in healthy subjects. The aim of the present research was to study how dietary acid load affects the acid–base status of the body during a 12-week combined endurance and strength training period at rest and during submaximal cycling. In addition, the effects of a diet and training intervention on kidney function were investigated at rest. The kidney function was assessed with GFR, serum urea-to-creatinine ratio (UCR) and serum urea. It was hypothesized that the lower acid intake would induce a less acidic blood acid–base status—that is, higher pH and HCO 3− —at rest and during submaximal exercise and would preserve kidney function at rest. 2. Materials and Methods 2.1. Subjects In total, 49 healthy men and women volunteered and were selected to participate in the study. The study participants were required to be 20–50 years old and recreationally physically active. Before inclusion into the study, the physical activity of the subjects was characterized by walking, cycling, team sports, or strength training at a light-to-moderate intensity, at a frequency of 1–3 times per week, but a lack of systematic engagement in any endurance or strength training. The female participants were allowed to use contraceptive pills during the study period, but any other medication was considered to be exclusion criteria. Also subjects whose body mass indexes were above 33 kg/m 2 or who had any relevant food allergy were excluded from the study. Ethical approval was obtained from the Ethical Committee of the University of Jyväskylä, and the study was in accordance with the Helsinki Declaration. Prior to the first testing, subjects were informed of the purpose and the methods of the study, and they signed a written informed consent. Additionally, the subjects completed questionnaires about their health, diet, and exercise background, and underwent a standardized resting electrocardiogram procedure, which was reviewed by a cardiologist. At the beginning of the study, the subjects were randomly divided into the low-PRAL and the moderate-PRAL diet groups and Nutrients 2018,10, 323 3 of 12 ate accordingly for the entire duration of the study period. Over the study period, there were three drop-outs, for reasons unrelated to the intervention. Baseline anthropometric characteristics of the subjects who completed the entire data collection are presented in Table 1. Table 1. Baseline anthropometric characteristics of the subjects in the low-potential renal acid load (PRAL) and moderate-PRAL diet groups. Parameters Women Men Low-PRAL Mod-PRAL Low-PRAL Mod-PRAL N13 12 9 12 Age (years) 34.3 ±6.9 32.0 ±5.9 32.0 ±9.6 31.3 ±5.1 Height (m) 1.67 ±0.07 1.66 ±0.06 1.78 ±0.07 1.77 ±0.06 Body mass (kg) 64.2 ±7.5 65.6 ±11.4 86.0 ±9.2 79.1 ±10.2 Body mass index (kg/m2)23.0 ±3.5 23.7 ±3.5 27.2 ±3.1 25.2 ±2.1 2.2. Study Design The study period lasted for 12 weeks. The subjects trained twice a week, and every training session consisted of both endurance and strength training (approximately 45 min + 45 min each). The measurement sessions took place before the intervention period (PRE), at the mid-phase during weeks 6–8 (MID) and after the intervention (POST). During each testing session a 12-h urine sample and fasting blood samples were collected. In addition, the subjects recorded their food intake via a 3-day food diary. At PRE and POST, the subjects also performed a submaximal cycle ergometer test during which the blood samples were obtained. One week before the start of the 12-week intervention, the VO 2max and maximal workloads of the subjects were measured via an incremental cycle ergometer test that was performed on a microprocessorcontrolled, eddy current brake equipped ergometer (Ergoline ergometrics 800, D-72475, Bitz, Germany). The initial workload was 50 W and it was increased by 25 W every 2 min until volitional exhaustion occurred. VO 2max was determined to be the highest 30-s VO 2 value during the test and coincided with at least one of the following two criteria: (a) respiratory exchange ratio >1.1; and/or (b) a plateau of oxygen uptake (less than 150 mL/min increase in VO 2 during the last 60 s of the test). Gaseous exchange was measured using a Jaeger Oxycon Pro breath-by-breath gas analyzer (VIASYS Healthcare GmbH, Hoechburg, Germany). The device was calibrated for volume and gas before every measurement. The VO 2max determined at baseline (PRE) was used in all subject groups to set the workloads for the submaximal cycling tests. Three days after the VO 2max test, the subjects performed a submaximal cycling test (PRE) that started with a 5-min warm-up, followed by a 4-min break. Thereafter, the subjects completed three 8 min trials at 35%, 55% and 75% of their VO 2max . All workloads were separated by 4-min rest periods, during which blood samples (CT35, CT55, CT75, respectively; CT = cycling test) were collected from a fingertip capillary and an antecubital vein. During the last 12 h before the start of the dietary intervention, subjects had a 12-h overnight fast and, at the same time, collected a 12-h urine sample. The next morning, in a laboratory, fasting blood samples (FAST) were drawn from a fingertip capillary and an antecubital vein. The blood samples were obtained at 7–10 a.m. and kept similar throughout the study. The 12-h urine collection commenced 12 h before FAST. Starting from PRE, the subjects followed either the low-PRAL or the moderate-PRAL diet, and the same urine and blood sampling sessions were repeated at MID and at POST. At PRE and POST, after the blood sampling, the body composition of the subjects was assessed by dual X-ray absorptiometry (DXA) (Lunar Prodigy Advance, GE Medical Systems, Madison, WI, USA). Total fat mass and total lean mass were automatically analyzed (enCORE software, version 14.10.022, GE Medical Systems, Madison, WI, USA). Thereafter, the subjects ate a light breakfast, which was consistent with their assigned diet. Resting blood samples were drawn once more (REST) before a submaximal cycle ergometer test was completed. Nutrients 2018,10, 323 4 of 12 2.3. Diets Dietary acid load can be estimated by calculating the potential renal acid load (PRAL) of foods, which represents the renal net acid excretion caused by a foodstuff [ 2 ]. The diets used in the present study were designed with the PRAL calculations to have low and moderate acid loads. The aim was that the low-PRAL diet would enhance the production of alkaline compounds in the body (PRAL < 0), whereas the moderate-PRAL diet was aimed to slightly increase the production of acid compounds (PRAL > 0). The PRAL values were calculated as follows: PRAL (mEq/100 g) = 0.49 × protein (g/100 g) + 0.037 × phosphorous (mg/100 g) − 0.021 × potassium (mg/100 g) − 0.026 × magnesium (mg/100 g) − 0.013 × calcium (mg/100 g) [ 2 ]. The nutrient contents of the food were taken from the Finnish Food Composition Database (Fineli, Finnish National Institute of Health and Welfare). Before the start of the 12-week intervention period, the subjects followed their normal diet and kept food diaries for 3 days. Appropriate dietary counselling was given for both diet groups based on the baseline dietary analysis, and the subjects were given instructions on how to follow the low- and moderate-PRAL diets. Both groups controlled nutritional intake according to the general dietary guidelines, but in the low-PRAL group, the subjects were advised to increase the consumption of fruits and vegetables up to 800–1000 g. On the other hand, in the moderate-PRAL group, the subjects were advised to limit their intake of fruits and vegetables to 200–300 g. The subjects kept food diaries for 3 days at PRE, MID, and POST. In addition, the subjects recorded their food intake during weeks 1–4 via a 3-day food diary, in order to check if the diet that the subjects were assigned was followed according to the instructions. The food diaries were analyzed for energy, protein, carbohydrate, fat, phosphorous, potassium, magnesium and calcium intake using Nutri-Flow software (Flow-Team Oy, Oulu, Finland). The average daily PRALs during the experimental diets were calculated according to the relevant dietary intake data. 2.4. Urine Sampling and Analysis The subjects collected 12-h urine samples at PRE, MID and POST. Each urine sample was collected in a sterile container and refrigerated until subjects came to the laboratory and brought the container with them. Upon receipt, urine pH was determined by dipping a pH strip into the urine (Combur-7 Test urinalysis test strips; Cobas, Roche, Germany). Urine electrolytes were analyzed by the direct ISE in vitro test (Ion Selective Microlyte Analyzer, Konelab 20 XTi; Kone Instruments, Espoo, Finland). Indirect Net acid excretion (NAE) was calculated as follows: NAE (mEq/day) = (Cl−+ Pi+ SO4−+ OA) −(Na++ K++ Ca2+ + Mg2+), where SO4−= 0.4875 ×dietary protein intake (g) OA (organic acids) = (BSA ×41)/1.73 where BSA (body surface area) = [(weight (kg) ×height (m))/3600]1 2[12]. 2.5. Blood Sampling and Analysis All fasting capillary and antecubital vein blood samples were drawn at the same time, in the morning, at all three sampling points. Li-heparinized whole blood samples (200 and 20 µ L) from a fingertip capillary were analyzed immediately after sampling for pH, and HCO 3− . The determination of pH was based on the principle of the ion selective electrode, whereas HCO 3− was determined computationally from pH and pCO 2 values (GEM Premier 3000, Instrumentation Laboratory, Lexington, MA, USA). The blood samples from the antecubital vein were drawn in vacuum tubes, stored at room temperature for 30 min and centrifuged for 10 min at 3500 rpm (2100 × g). The serum was separated, and creatinine and urea were analyzed by a KoneLab 20 XTi analyzer Nutrients 2018,10, 323 5 of 12 (Thermo Electron Corporation, Vantaa, Finland). Serum creatinine values were used to calculate the glomerular filtration rate (GFR) with the CKD-EPI equation [ 13 ]. Also, the serum urea to creatinine ratio (UCR) was calculated. 2.6. Training The training protocol has been described elsewhere [ 14 ]. A combination of aerobic and resistance training has been proposed to be the most effective strategy for maintaining and/or improving physical fitness and health [ 15 ]. Briefly, the endurance training was conducted on a cycle ergometer and the training program included mostly steady-state cycling of low to moderate intensity (below and above the aerobic threshold). The duration of endurance cycling increased progressively from 30 to 50 min. During the last 4 weeks of the training period, the intensity of cycling also increased from the aerobic to the anaerobic threshold and then further, until subjects were completing maximal aerobic workloads. The resistance training program included exercises for all major muscle groups with a focus on the lower extremities. During the first two weeks, training was performed as a circuit using low intensities. Thereafter, protocols aiming for muscle hypertrophy and maximal and explosive strength were performed. During the second half of the study, both training volume and frequency were increased. The overall duration of the strength training sessions was 30–50 min. 2.7. Statistical Analysis The main purpose of the present study was to determine if dietary acid intake has an effect on the primary outcome variable: acid–base status. NAE, capillary pH and capillary HCO 3− were analyzed to identify the possible differences in acid–base status. The secondary outcome of the study was to assess kidney function, measured by GFR, the serum urea-to-creatinine ratio (UCR) and serum urea. The effect of a 12-week intervention period on blood and urine variables, and variables of dietary intake analysis were examined by a two-way repeated measures analysis of variance (ANOVA). If a statistically significant difference was observed within one of the diet groups, or between groups, the comparison was continued with a suitable t-test. Data are presented as means ± SDs. The statistical difference was considered to be significant at the p< 0.05 level. 3. Results 3.1. Diets Dietary intake data are presented in Tables 2and 3. There were no significant differences in energy or macronutrient intakes within or between the diet groups, except in moderate-PRAL men, as their energy intake was significantly decreased from PRE to MID (p= 0.027). In both men and women, PRAL was significantly lower (p ≤ 0.001 in all) and the intake of fruits and vegetables (IFV) were higher (p ≤ 0.017 in all) in low-PRAL compared to moderate-PRAL at MID and POST. In low-PRAL men and women, PRAL was significantly lower (p ≤ 0.005 in all) and IFV higher (p ≤ 0.06 in all) at MID and POST, compared to PRE. There were no significant differences in PRAL and IFV between MID and POST in any of the groups. Nutrients 2018,10, 323 6 of 12 Table 2. Dietary intake data in men before (PRE), in the middle (MID) and after (POST) the 12-week diet period. Parameters Low-PRAL Moderate-PRAL PRE MID POST PRE MID POST PRAL (mEq/day) 23 ±32 −41 ±24 ††† −37 ±24 †† 8.1 ±16 10 ±12 *** 11 ±17 ** IFV (g/day) 250 ±140 900 ±300 800 ±380 300 ±250 250 ± 250 *** 230 ± 100 * Energy(kcal/day) 2670 ±910 1930 ±570 †1930 ±520 2220 ±630 2210 ±650 2180 ±690 Protein (g/kg/day) 1.5 ±0.8 1.0 ±0.3 1.1 ±0.2 1.3 ±0.5 1.4 ±0.5 1.4 ±0.4 CHO (g/kg/day) 3.4 ±1.6 2.2 ±1.0 2.5 ±1.7 2.9 ±0.9 3.2 ±0.9 3.1 ±0.9 Fat (g/kg/day) 1.2 ±0.5 0.8 ±0.3 0.8 ±0.3 1.2 ±0.4 1.2 ±0.4 0.9 ±0.4 CHO, carbohydrates; IFV, intake of fruits and vegetables. * p< 0.05, ** p< 0.01, *** p< 0.001 statistically significant difference between low- and moderate-PRAL at POST. † p< 0.05, †† p< 0.01, ††† p< 0.001 statistically significant difference between PRE and MID or PRE and POST in low-PRAL (two-way repeated measures ANOVA, a paired or independent t-test). Table 3. Dietary intake data in women before (PRE), in the middle (MID) and after (POST) the 12-week diet period. Parameters Low-PRAL Moderate-PRAL PRE MID POST PRE MID POST PRAL (mEq/day) −7.2 ±18 −51 ±19 ††† −56 ±40 †† −2.9 ±11 3.6 ±11 *** − 0.8 ± 17 *** IVF (g/day) 400 ±200 930 ±310 1070 ±630 250 ±80 210 ± 160 *** 260 ± 270 *** Energy (kcal/day) 2010 ±380 1880 ±360 1860 ±500 1900 ±280 1990 ±580 1870 ±340 Protein (g/kg/day) 1.3 ±0.5 1.1 ±0.2 1.1 ±0.3 1.2 ±0.2 1.4 ±0.4 1.1 ±0.2 CHO (g/kg/day) 3.6 ±0.8 3.6 ±0.8 3.8 ±1.3 3.6 ±0.7 3.8 ±1.6 3.2 ±0.8 Fat (g/kg/day) 1.3 ±0.3 1.1 ±0.4 1.0 ±0.8 1.1 ±0.4 1.2 ±0.5 1.0 ±0.2 CHO, carbohydrates; IFV, intake of fruits and vegetables. *** p< 0.001 statistically significant difference between low- and moderate-PRAL at POST. †† p< 0.01, ††† p< 0.001 statistically significant difference between PRE and MID or PRE and POST in low-PRAL (two-way repeated measures ANOVA, a paired or independent t-test). 3.2. Body Composition of the Subjects There were no significant changes in body mass, total lean mass or fat% in either of the subject groups over the 12-week study period (Table 4). Table 4. Body composition before (PRE), and after (POST) the 12-week diet period in low-PRAL and moderate-PRAL diet groups. Parameters Men Women Low-PRAL Mod-PRAL Low-PRAL Mod-PRAL PRE POST PRE POST PRE POST PRE POST Body mass (kg) 85.5 ±9.8 83.7 ±9.5 79.2 ±10.2 79.6 ±9.8 64.3 ±7.8 63.8 ±7.9 67.0 ±11.1 67.9 ±11.5 Lean mass (kg) 61.5 ±5.6 61.3 ±5.2 56.1 ±4.8 57.2 ±5.8 41.2 ±3.4 41.5 ±2.6 40.9 ±4.8 41.7 ±4.9 Fat % 23.9 ±7.4 22.0 ±7.9 25.3 ±6.9 23.8 ±5.9 31.0 ±7.0 30.4 ±6.6 33.2 ±9.3 33.8 ±9.0 3.3. Urine and Blood Acid-Base Status NAE was lower in low-PRAL compared to moderate-PRAL at POST in both men (p= 0.001) and women (p= 0.047) (Figure 1). There were no statistically significant changes in urine pH, which was estimated with the pH test strips over the study period, in either of the subject groups. The urine strip results are not presented. Nutrients 2018,10, 323 7 of 12 Nutrients 2018, 10, x FOR PEER REVIEW 6 of 12 Table 3. Dietary intake data in women before (PRE), in the middle (MID) and after (POST) the 12- week diet period. Parameters Low-PRAL Moderate-PRAL PRE MID POST PRE MID POST PRAL (mEq/day) −7.2 ± 18 −51 ± 19 ††† −56 ± 40 †† −2.9 ± 11 3.6 ± 11 *** −0.8 ± 17 *** IVF (g/day) 400 ± 200 930 ± 310 1070 ± 630 250 ± 80 210 ± 160 *** 260 ± 270 *** Energy (kcal/day) 2010 ± 380 1880 ± 360 1860 ± 500 1900 ± 280 1990 ± 580 1870 ± 340 Protein (g/kg/day) 1.3 ± 0.5 1.1 ± 0.2 1.1 ± 0.3 1.2 ± 0.2 1.4 ± 0.4 1.1 ± 0.2 CHO (g/kg/day) 3.6 ± 0.8 3.6 ± 0.8 3.8 ± 1.3 3.6 ± 0.7 3.8 ± 1.6 3.2 ± 0.8 Fat (g/kg/day) 1.3 ± 0.3 1.1 ± 0.4 1.0 ± 0.8 1.1 ± 0.4 1.2 ± 0.5 1.0 ± 0.2 CHO, carbohydrates; IFV, intake of fruits and vegetables. *** p < 0.001 statistically significant difference between low- and moderate-PRAL at POST. †† p < 0.01, ††† p < 0.001 statistically significant difference between PRE and MID or PRE and POST in low-PRAL (two-way repeated measures ANOVA, a paired or independent t-test). 3.2. Body Composition of the Subjects There were no significant changes in body mass, total lean mass or fat% in either of the subject groups over the 12-week study period (Table 4). Table 4. Body composition before (PRE), and after (POST) the 12-week diet period in low-PRAL and moderate-PRAL diet groups. Parameters Men Women Low-PRAL Mod-PRAL Low-PRAL Mod-PRAL PRE POST PRE POST PRE POST PRE POST Body mass (kg) 85.5 ± 9.8 83.7 ± 9.5 79.2 ± 10.2 79.6 ± 9.8 64.3 ± 7.8 63.8 ± 7.9 67.0 ± 11.1 67.9 ± 11.5 Lean mass (kg) 61.5 ± 5.6 61.3 ± 5.2 56.1 ± 4.8 57.2 ± 5.8 41.2 ± 3.4 41.5 ± 2.6 40.9 ± 4.8 41.7 ± 4.9 Fat % 23.9 ± 7.4 22.0 ± 7.9 25.3 ± 6.9 23.8 ± 5.9 31.0 ± 7.0 30.4 ± 6.6 33.2 ± 9.3 33.8 ± 9.0 3.3. Urine and Blood Acid-Base Status NAE was lower in low-PRAL compared to moderate-PRAL at POST in both men (p = 0.001) and women (p = 0.047) (Figure 1). There were no statistically significant changes in urine pH, which was estimated with the pH test strips over the study period, in either of the subject groups. The urine strip results are not presented. Figure 1. Net acid excretion (NAE) in the low-PRAL and the moderate-PRAL diet groups before (PRE), in the middle (MID) and after (POST) the 12-week diet period. * p < 0.05, ** p < 0.01 statistically significant difference between the diet groups at POST (two-way repeated measures ANOVA, an independent t-test). For capillary pH, there were no significant differences between the diet groups (Figure 2). In low-PRAL women, pH was significantly lower (p = 0.014) at 35% and higher (p = 0.020) at 75% of VO 2max at POST compared to PRE. In moderate-PRAL women, pH was significantly lower at 55% (p = 0.033) of VO 2max at POST compared to PRE. Figure 1. Net acid excretion (NAE) in the low-PRAL and the moderate-PRAL diet groups before (PRE), in the middle (MID) and after (POST) the 12-week diet period. * p< 0.05, ** p< 0.01 statistically significant difference between the diet groups at POST (two-way repeated measures ANOVA, an independent t-test). For capillary pH, there were no significant differences between the diet groups (Figure 2). In low-PRAL women, pH was significantly lower (p= 0.014) at 35% and higher (p= 0.020) at 75% of VO 2max at POST compared to PRE. In moderate-PRAL women, pH was significantly lower at 55% (p= 0.033) of VO2max at POST compared to PRE. Nutrients 2018, 10, x FOR PEER REVIEW 7 of 12 Figure 2. Capillary pH in men (A) and women (B) before (PRE) and after (POST) the 12-week diet period at rest (FAST, REST) and during submaximal cycling (CT35, CT55, CT75; CT = cycling test). * p < 0.05, statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). Values are mean ± SD. In low-PRAL women, HCO 3− was higher ( p = 0.006) at 75% of VO 2max at POST compared to PRE (Figure 3). In moderate-PRAL men, HCO 3− was higher ( p = 0.002) at 75% of VO 2max after the training period compared to PRE. The only significant differences in HCO 3− between the diet groups occurred in men, as HCO 3− was higher at FAST, REST and during cycling at 35% of VO 2max in low-PRAL compared to moderate-PRAL ( p = 0.015, p = 0.039, p = 0.010, respectively). Figure 3. Capillary bicarbonate in men (A) and women (B) before (PRE) and after (POST) the 12-week diet period at rest (FAST, REST) and during submaximal cycling (CT35, CT55, CT75). ** p < 0.01, statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). Figure 2. Capillary pH in men ( A ) and women ( B ) before (PRE) and after (POST) the 12-week diet period at rest (FAST, REST) and during submaximal cycling (CT35, CT55, CT75; CT = cycling test). *p< 0.05 , statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). Values are mean ±SD. In low-PRAL women, HCO 3− was higher (p= 0.006) at 75% of VO 2max at POST compared to PRE (Figure 3). In moderate-PRAL men, HCO 3− was higher (p= 0.002) at 75% of VO 2max after the training period compared to PRE. The only significant differences in HCO 3− between the diet groups occurred in men, as HCO 3− was higher at FAST, REST and during cycling at 35% of VO 2max in low-PRAL compared to moderate-PRAL (p= 0.015, p= 0.039, p= 0.010, respectively). Nutrients 2018,10, 323 8 of 12 Nutrients 2018, 10, x FOR PEER REVIEW 7 of 12 Figure 2. Capillary pH in men (A) and women (B) before (PRE) and after (POST) the 12-week diet period at rest (FAST, REST) and during submaximal cycling (CT35, CT55, CT75; CT = cycling test). * p < 0.05, statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). Values are mean ± SD. In low-PRAL women, HCO 3− was higher ( p = 0.006) at 75% of VO 2max at POST compared to PRE (Figure 3). In moderate-PRAL men, HCO 3− was higher ( p = 0.002) at 75% of VO 2max after the training period compared to PRE. The only significant differences in HCO 3− between the diet groups occurred in men, as HCO 3− was higher at FAST, REST and during cycling at 35% of VO 2max in low-PRAL compared to moderate-PRAL ( p = 0.015, p = 0.039, p = 0.010, respectively). Figure 3. Capillary bicarbonate in men (A) and women (B) before (PRE) and after (POST) the 12-week diet period at rest (FAST, REST) and during submaximal cycling (CT35, CT55, CT75). ** p < 0.01, statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). Figure 3. Capillary bicarbonate in men ( A ) and women ( B ) before (PRE) and after (POST) the 12-week diet period at rest (FAST, REST) and during submaximal cycling (CT35, CT55, CT75). ** p< 0.01, statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). 3.4. Renal Function GFR decreased in the moderate-PRAL men (p= 0.009) and women (p= 0.036) over the dietary intervention (Figure 4). There were no significant changes in the low-PRAL groups over the study period. Nutrients 2018, 10, x FOR PEER REVIEW 8 of 12 3.4. Renal Function GFR decreased in the moderate-PRAL men ( p = 0.009) and women ( p = 0.036) over the dietary intervention (Figure 4). There were no significant changes in the low-PRAL groups over the study period. Figure 4. Glomerular filtration rate (GFR) in the low-PRAL and moderate-PRAL groups before (PRE) and after (POST) the 12-week diet period. * p < 0.05, ** p < 0.01 statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). Serum urea decreased significantly in the low-PRAL men ( p = 0.037) and women ( p = 0.013) (Figure 5). Also, the serum urea to creatinine ratio decreased over the low-PRAL diet period in both men ( p = 0.030) and women ( p = 0.016) (Figure 5). No significant changes were observed in the moderate-PRAL diet groups for either of the variables. Figure 5. Serum urea and urea to creatinine ratio in the low-PRAL and moderate-PRAL groups before (PRE) and after (POST) the 12-week diet period. * p < 0.05 statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). 4. Discussion In the present study, recreationally active, healthy men and women followed either a low-PRAL or a moderate-PRAL diet for 12 weeks and participated in same session of combined endurance and strength training twice a week. Net acid excretion (NAE) was significantly lower after 12 weeks of Figure 4. Glomerular filtration rate (GFR) in the low-PRAL and moderate-PRAL groups before (PRE) and after (POST) the 12-week diet period. * p< 0.05, ** p< 0.01 statistically significant difference between PRE and POST within a diet group (two-way repeated measures ANOVA, a paired t-test). Serum urea decreased significantly in the low-PRAL men (p= 0.037) and women (p= 0.013) (Figure 5). Also, the serum urea to creatinine ratio decreased over the low-PRAL diet period in both men (p= 0.030) and women (p= 0.016) (Figure 5). No significant changes were observed in the moderate-PRAL diet groups for either of the variables.