Effects of plant- and animal-based-protein meals for a day on serum nitric oxide and peroxynitrite levels in healthy young men (2025)

Abstract

Plant-based diets that replace animal-based proteins with plant-based proteins have received increased attention for cardiovascular protection. Nitric oxide (NO) plays an essential role in the maintenance of endothelial function. However, under higher oxidative stress, NO generation produces peroxynitrite, a powerful oxidant and vasoconstrictor. Diet-replaced protein sources has been reported to decrease oxidative stress. However, the effects of plant-based protein on NO and peroxynitrite have not yet been clarified. Therefore, this study aimed to compare the effects of plant- and animal-based-protein meals for a day on NO, peroxynitrite, and NO/peroxynitrite balance. A crossover trial of two meal conditions involving nine healthy men was performed. Participants ate standard meals during day 1. On day 2, baseline measurements were performed and the participants were provided with plant-based-protein meals or animal-based-protein meals. The standard and test meals consisted of breakfast, lunch, and dinner and were designed to be isocaloric. Plant-based-protein meals contained no animal protein. Blood samples were collected in the morning after overnight fasting before and after the test meals consumption. In the plant-based-protein meal condition, serum NOx levels (the sum of serum nitrite and nitrate) significantly increased, while serum peroxynitrite levels did not change significantly. Animal-based-protein meals significantly increased serum peroxynitrite levels but showed a trend of reduction in the serum NOx levels. Furthermore, serum NO/peroxynitrite balance significantly increased after plant-based-protein meals consumption, but significantly decreased after animal-based-protein meals consumption. These results suggest that, compared with animal-based-protein meals, plant-based-protein meals increase NO levels and NO/peroxynitrite balance, which reflects increased endothelial function.

VASCULAR ENDOTHELIAL DYSFUNCTION is well known as an early step in the progression toward cardiovascular disease (CVD) and is closely associated with the occurrence of several diseases, such as hypertension, arteriosclerosis, heart failure, and metabolic syndrome [1-5]. Nitric oxide (NO) is a potent vasodilator and plays an essential role in maintaining and regulating endothelial function [2, 6-8]. Endothelium-derived NO is generated from L-arginine by endothelial nitric oxide synthase (eNOS) [8]. However, under high levels of oxidative stress, superoxide is generated instead of NO by eNOS uncoupling [8-10]. Moreover, superoxide reacts with NO to produce peroxynitrite, which is one of the most powerful oxidants in the biological milieu and has vasoconstrictive effects [11]. Higher peroxynitrite levels considerably shift the redox balance in the endothelium and negatively affect vascular homeostasis [12, 13]. This process is the primary pathological mechanism underlying the reduction in NO bioavailability and the first indication of endothelial dysfunction [1, 8, 14]. Therefore, increasing NO levels and inhibiting peroxynitrite generation, namely elevation of the balance between NO and peroxynitrite levels, are necessary to maintain and improve endothelial function.

Diet modification is considered as a potentially important method for the prevention of endothelial dysfunction [15-17]. Recently, there is a heightened interest in plant-based diets that replace animal-based protein with plant-based protein [17-19]. Because it has been shown that different dietary protein sources affect oxidative stress [20-22], consuming plant-based protein instead of animal-based protein may affect endothelial function. Legumes, the main source of plant-based protein, are rich in polyphenols, which are well known to reduce oxidative stress owing to reduced free radical reactivity [23-25]. In contrast, a higher intake of animal-based proteins elevates oxidative stress by producing more reactive oxygen species (ROS), because heme iron in animal-based proteins has pro-oxidative properties [26, 27]. Several studies have reported that diets with plant-based proteins decrease oxidative stress markers and increase antioxidant capacity compared with diets with animal-based proteins [20-22]. Oxidative stress plays an important role in NO production and decreases peroxynitrite [8-10]. Based on these considerations, a meal high in plant-based protein may increase NO levels, decrease peroxynitrite levels, and thereby elevate NO/peroxynitrite balance compared with a meal high in animal-based protein. However, whether a diet with plant-based protein affects NO and peroxynitrite levels and their balance still needs to be elucidated.

This study aimed to compare the effects of plant-based-protein meals and animal-based-protein meals for a day on serum NO and peroxynitrite levels and NO/peroxynitrite balance. We hypothesized that plant-based-protein meals would increase serum NO levels and NO/peroxynitrite balance and decrease peroxynitrite levels compared with animal-based-protein meals.

Materials and Methods

Participants

Participants were recruited by snowball sampling. The eligibility criteria were as follows: (a) male sex; (b) age 20–29 years; (c) no history or symptoms of cardiovascular disease; (d) non-smoking; and (e) no regular exercise training for the past 6 months. In total, ten healthy young men (aged 22–28 years) were enrolled. All participants were students from University of Tsukuba. One participant dropped out between the first period for personal reasons. Therefore, nine participants completed both conditions, and included in the analysis. The study protocol was approved by the Ethical Committee of the Institute of Health and Sports Sciences of the University of Tsukuba (Approval No., Tai021-19). This study was conducted following the principles outlined in the Declaration of Helsinki, and all participants provided written informed consent before inclusion in this study.

Study protocol

This was a crossover trial of two diet conditions. The study design is illustrated in Fig. 1A. The 10 participants were assigned to two sequences using computer-generated random numbers. Specifically, in period Ⅰ, six participants were assigned to the plant-based-protein meal (PPM) condition and four participants were assigned to the animal-based-protein meal (APM) condition. Each period was separated by a 2-week washout. Fig. 1B shows the experimental protocol. On day 1, the participants were provided with standard meals to eat thrice daily: breakfast, lunch, and dinner. In the early morning of day 2, the participants visited the laboratory in an overnight fasted state (~10 h) and underwent baseline measurements. All measurements were performed in an environmentally controlled laboratory. Body height and mass were measured barefoot and with light clothing. Blood samples were obtained by venipuncture to measure serum NO and peroxynitrite levels and blood biochemistry. After 15 min of rest on a bed in the supine position, brachial blood pressure was measured. The participants were then provided either PPMs or APMs to eat thrice daily. Measurements for day 2 were repeated in the morning of day 3. Each condition was separated by 2 weeks of washout. The participants were instructed not to eat or drink anything other than the meals and beverages provided, except for water, and to refrain from vigorous exercise from days 1 to 3 of the measurement.

Diet

The standard and test meals were designed to be isocaloric and have equal protein, fat, and carbohydrate (PFC) balance (Table 1). Moreover, PFC balance was designed to be percentages of daily calories (e.g., 15% of calories as protein and 22% as fat). APMs (control meals) contained 11% animal-based proteins, such as pork, poultry, beef, and milk. This ratio corresponds to the intake of the Japanese population with the highest intake of animal-based protein [28]. In the PPM condition, we used commercially available plant-based foods containing no animal protein. These foods provided a menu similar to APMs. Except for boiled eggs, the standard and test meals were provided frozen or chilled.

Body composition and hemodynamics

Height was measured to the nearest 0.1 cm using a stadiometer (AD-6227R; A&D, Tokyo, Japan). Body weight, body fat mass, skeletal muscle mass and appendicular skeletal muscle mass (ASMM) were measured to the nearest 0.1 kg on a calibrated digital scale (InBody 770; InBody Japan, Tokyo, Japan) and adjusted for the estimated clothing mass by subtracting 0.5 kg. Skeletal muscle mass index (SMI) was calculated from the ASMM and height according to the following formula: SMI = ASMM/height². The participants were placed in the supine position, and an automated sphygmomanometer (SphygmoCor XCEL; ATCOR Medical, Sydney, Australia) was applied to measure brachial systolic (SBP) and diastolic (DBP) blood pressures after resting quietly for 15 min.

Blood biochemistry

Each blood sample was placed in a serum and plasma separator tube and centrifuged at 3,000 rpm for 15 min at 4°C. The blood samples were stored at –80°C until the assay. Serum concentrations of triglycerides, total cholesterol (TC), high-density lipoprotein cholesterol (HDLC), and low-density lipoprotein cholesterol (LDLC) and plasma concentrations of fasting blood glucose were determined using standard enzymatic techniques.

Serum NO and peroxynitrite levels

NO has a very short half-life; therefore, the end-products of NO, such as nitrite and nitrate, are used as indicators of NO production [29]. The sum of serum nitrite and nitrate is referred to as serum NOx. Serum NOx levels were measured using the Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit based on the Griess reaction (R&D Systems, Minneapolis, MN, USA). The detection limits were 0.25 μmol/L. All assays were performed according to the manufacturer’s instructions.

Peroxynitrite is a short-lived and reactive biological oxidant; therefore, it was measured using 3-nitrotyrosine. Because 3-nitrotyrosine is generally considered a footprint of NO-ROS interactions [11], it was used as a marker of peroxynitrite in this study. Serum 3-nitrotyrosine levels were determined in duplicate using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Nitrotyrosine ELISA; Immundiagnostik AG, Bensheim, Germany) according to the manufacturer’s instructions.

The balance between NO and peroxynitrite levels (NO/peroxynitrite) is used as an indicator of endothelial function [30]. Therefore, we evaluated the ratio of serum NOx to 3-nitrotyrosine as NO/peroxynitrite.

Statistical analysis

Row data are reported as mean ± standard deviation or standard error. Linear mixed model was used to determine the effects of PPM and APM conditions, including time (before or after consumption), period (first or second), and sequence (PPM–APM or APM–PPM) as fixed effects. Subject was included as a random effect. Post hoc multiple pairwise comparisons were corrected using Bonferroni’s method. Changes in serum NOx, peroxynitrite levels, and NO/peroxynitrite balance were compared between the meal conditions using analysis of Covariance (ANCOVA) to adjust for sequence and period. To evaluate a carryover and period effect was used by unpaired t-test [31]. All statistical analyses were performed using SPSS 28.0 (IBM Inc., Chicago, IL, USA). Statistical significance was set at p < 0.05.

Results

Participant characteristics are presented in Table 2. The mean age was 25 ± 2 years, and the mean BMI was 22.9 ± 1.9 kg/m². None of the participants had blood pressure values that exceeded the criteria for hypertension (averaged three measurements of SBP ≥140 or DBP ≥90 mmHg confirmed).

Table 3 shows the hemodynamics and biochemical parameters before and after PPMs and APMs consumption. No interaction was observed between the PPM and APM conditions in terms of SBP, DBP, triglyceride, TC, HDLC, LDLC, and fasting blood glucose levels. Fasting blood glucose level significantly decreased after the test meal consumption in both conditions.

Fig. 2A and Supplementary Table S1 present the serum NOx levels. The linear mixed model analysis indicated a significant interaction between condition and time (p = 0.005). After consuming PPMs, the serum NOx levels significantly increased (16.0 ± 1.3 vs. 20.1 ± 1.5 μmol/L, p = 0.014), but tended to decrease after consuming APMs (17.8 ± 1.6 vs. 15.1 ± 1.5 μmol/L, p = 0.091). Fig. 2B presents changes in serum NOx levels compared between the two conditions. The changes in serum NOx levels were significantly greater for the PPM condition than for the APM condition (4.2 ± 1.0 vs. –2.7 ± 1.9 μmol/L, p = 0.019).

Fig. 3A and Supplementary Table S1 demonstrate the serum 3-nitrotyrosine (peroxynitrite) levels. The time effect and interaction for serum peroxynitrite levels was close to significance (p = 0.064, p = 0.072). Specifically, serum peroxynitrite levels did not change (320 ± 27 vs. 321 ± 27 nmol/L, p = 0.966) in the PPM condition but significantly increased in the APM condition (324 ± 28 vs. 346 ± 34 nmol/L, p = 0.014). Compared with the PPM condition, the APM condition showed greater changes in serum peroxynitrite levels, although the difference was not significant (0.35 ± 4.11 vs. 22.41 ± 10.69 nmol/L, p = 0.170; Fig. 3B).

The serum NOx/3-nitrotyrosine (NO/peroxynitrite) balance showed significant interactions with condition and time (p = 0.002) (Fig. 4A, Supplementary Table S1). Specifically, serum NO/peroxynitrite balance significantly increased after PPMs consumption (52.2 ± 5.4 vs. 65.5 ± 6.0, p = 0.009), but significantly decreased after APMs consumption (56.0 ± 4.7 vs. 45.7 ± 4.5, p = 0.037). Additionally, after the test meals consumption, serum NO/peroxynitrite balance was higher in the PPM condition than in the APM condition (p = 0.034). Changes in the serum NO/peroxynitrite balance were greater in the PPM condition than in the APM condition (13.3 ± 3.9 vs. –10.3 ± 5.1, p = 0.009) (Fig. 4B).

No carryover effects were observed for all measurements, including serum NOx, peroxynitrite levels, and NO/peroxynitrite balance. On the other hand, there was no period effects, except for LDLC, TC, and fasting blood glucose levels. In the first PPM condition, LDLC and TC levels were significantly higher, and fasting blood glucose level was lower than in the first APM condition.

Discussion

In the present study, we compared the effects of PPMs and APMs for a day on serum NOx and peroxynitrite levels and NO/peroxynitrite balance. Serum NOx levels significantly increased in the PPM condition. Conversely, in the APM condition, serum NOx levels tended to decrease. Serum peroxynitrite levels increase significantly under the APM condition, whereas no significant increase was observed under the PPM condition. Furthermore, the NO/peroxynitrite balance significantly increased in the PPM condition, but significantly decreased in the APM condition. These results indicate that replacing animal-based protein with plant-based protein in the diet affects the serum NOx and peroxynitrite levels and NO/peroxynitrite balance.

This study assessed not only serum NOx and peroxynitrite levels but also their balance to evaluate endothelial function, an early step in the progression toward CVD [1, 8, 14]. Loss of NO production has been linked to various vascular diseases, including atherosclerosis and hypertension [1, 3, 7]. However, under higher oxidative stress levels, endothelial NO generation may be accompanied by the production of peroxynitrite [8-10]. Therefore, both high NO and low peroxynitrite levels are needed to improve endothelial function, and an increase in serum NO/peroxynitrite balance indicates an increase in endothelial function [13, 30]. We demonstrated that PPMs for a day significantly increased serum NO/peroxynitrite balance. This finding suggests that plant-based-protein diets may prevent endothelial dysfunction compared with animal-based-protein diets, thereby reducing the risk of cardiovascular events.

The antioxidant properties of plant-based proteins likely elevate NO/peroxynitrite balance. Particularly, the use of more soy products could have affected our results. The first reason is that soy isoflavones directly increase NO production in endothelial cells. In vitro studies reported that soy isoflavones enhanced the expression of eNOS and elevated NO synthesis [32, 33]. It has been shown that soy isoflavones increase NO production in animal studies [20, 33]. Mahn et al. demonstrated that eNOS gene expression was reduced in animals fed a soy-deficient diet, but this was reversed after the animals were fed a soy-rich protein diet [20]. In addition, Squadrito et al. found that in healthy postmenopausal women, long-term supplementation with soy isoflavones increased circulating NO levels and endothelial function [34]. In this way, soy isoflavones activate eNOS transcription and increase NO production. Therefore, the use of soy products may have increased the NO levels in this study.

The second reason is that soy proteins may be more effective than other plant-based proteins in inhibiting increased peroxynitrite levels. Pivovarova-Ramich et al. investigated the effects of a 6-week plant- or animal-based-protein diet on oxidative stress in individuals with type 2 diabetes. The results showed that although the antioxidant status was higher in plant-based-protein diet than in animal-based-protein diet, circulating peroxynitrite levels increased in both diets [35]. However, in the present study, serum peroxynitrite levels were lower in the PPM condition than in the APM condition. This difference may be related to the different types of plant-based proteins. In the previous study, a plant-based-protein diet consisted mainly of pea protein [35]. On the other hand, the PPMs in the present study included a higher soy protein level because we used not only plant-based foods but also soy products, such as natto, tofu, and soy milk. Recent research has shown that pea proteins have antioxidant properties, but their antioxidant activity was less than that of soy [25]. Therefore, the use of soy protein in this study may explain the lower serum peroxynitrite levels compared with the PPM condition. From the above, in order to prevent endothelial dysfunction, consuming soy products may be a good choice as a plant-based protein.

Meanwhile, serum peroxynitrite levels significantly increased after APMs consumption, which may be attributed to increased oxidative stress from digestion and metabolization of meat products. Animal-based protein is an excellent source of heme iron, but it catalyzes ROS formation and promotes oxidative stress [26, 27]. Moreover, as meat and meat products undergo oxidative changes during storage and processing, their excessive consumption generates a large amount of ROS [36-38]. In healthy adults, high intake of meat and meat products is associated with higher oxidative stress [39]. It has been reported that plasma malondialdehyde levels increase after consumption of red turkey meat [37]. In this study, standard and test meals were composed of frozen or chilled foods, and the amount of animal-based protein in APMs was a little higher than that in standard meals (54 g vs. 43 g). Therefore, the elevation of oxidative stress introduced by meat products may have contributed to the increase in serum peroxynitrite levels in the APM condition.

In a Japanese cohort study, replacing 3% of the energy in red and processed meat with plant-based proteins reduced the risk of total and cardiovascular disease-related mortality [28]. Thus, consuming plant-based protein instead of animal-based protein may improve cardiovascular health. However, it is unclear whether this observed reduction in the CVD risk is driven by the protein itself because these observational studies were affected by confounding factors, such as healthy eating habits and behaviors. Mariotti recommends further studies on the impact of replacing animal-based protein with plant-based protein on various CVD risk factors, such as endothelial function [19]. Therefore, this study focused on endothelial function and experimentally compared the effects of PPMs and APMs on NO and peroxynitrite levels and NO/peroxynitrite balance. The present study showed that PPMs increase serum NOx levels and NO/peroxynitrite balance compared with APMs. These results may partly support the observational studies showing that replacing animal-based protein with plant-based protein may improve cardiovascular health.

The strength of the present study is that all meals were provided to unify total energy and PFC balance. In particular, providing standard meals can unify the total nitrite and nitrate intake, as some foods containing nitrates or nitrite are strongly related to circulating NOx levels [40]. This study is unique because it focused on different dietary protein sources and examined them experimentally. However, it also had some limitations. First, the intervention period was short because we distributed all meals. Second, as estrogen affects NO and peroxynitrite levels, only young male participants were included to control for internal validity. Estrogen can enhance NO generation by increasing activation of eNOS and can reduce oxidative stress by increasing antioxidant enzyme expression [41, 42]. Therefore, women were excluded in this study to avoid the effect of estrogen. Further studies including long-term intervention and/or female participants are needed to clarify the effects of plant-based protein on cardiovascular health. Third, the sample size of this study was not calculated based on the estimated effect size owing to the lack of prior research on the acute effects of plant-based diet on circulating NO and peroxynitrite levels. Despite these limitations, this study provides important information for future studies investigating the beneficial effects of replacing animal-based proteins with plant-based proteins on health.

In conclusion, our study revealed that PPMs increased serum NOx levels without altering serum peroxynitrite levels and thus increased serum NO/peroxynitrite balance compared to APMs. These findings indicates that PPMs improve endothelial function and that replacing animal-based protein in the diet with plant-based protein may prevent endothelial dysfunction.

Acknowledgments

The authors would like to thank all participants in the study. We also thank S.M. and K.K.’s laboratory members at the University of Tsukuba for their technical assistance.

Disclosure

The authors have no financial, consultant, institutional, or other relationships that might lead to bias or a conflict of interest.

Contributions

T Kaneko, M Yoshioka, and K Kosaki designed and conceived the study. T Kaneko, M Yoshioka, and F Kawahara performed the experiments. T Kaneko, M Yoshioka, J Park and T Tarumi analyzed the data. T Kaneko drafted the manuscript, and K Kosaki and S Maeda revised the manuscript. N Nishitani and S Mori provided advice on the design of the diet menus. M Yoshioka, F Kawahara, N Nishitani, S Mori and T Tarumi reviewed and edited the manuscript. All authors approved the final version of the manuscript. T Kaneko, M Yoshioka, and K Kosaki provided research funds.

Funding

This work was supported in part by Green Culture as a supplier of plant-based meal and Mishima Kaiun Memorial Foundation. The funder did not participate in the design, collection, analysis, or interpretation of the data. Keisei Kosaki was the recipient of the MEXT Leading Initiative for Excellent Young Researchers Grant Number JPMXS0320200234. Masaki Yoshioka and Shoya Mori were recipients of a Grant-in-Aid for Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists (21J10316 and 21J10952). Tomoko Kaneko and Natsumi Nishitani were recipients of a Grant-in-Aid for Research Fellowships of Japan Science and Technology (JPMJSP2124).

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