Epigenetics is generally defined as reversible changes in gene expression that are not due to changes in DNA sequence (Lacal and Ventura, 2018[55]; Ling and Rönn, 2019[62]). According to the Minkowskian cone view, assuming conception as the zero point, indirect epigenetics includes all adaptations in parental life that precede conception (across indirect epigenetics) or occur during the gestational period (within indirect epigenetics), and direct epigenetics includes plastic processes that can occur after birth (Lacal and Ventura, 2018[55]). Epigenetic inheritance refers to the transmission of certain epigenetic changes to offspring, and its central concept is that “information about the environment is passed to the next generation” (Lacal and Ventura, 2018[55]).

Modifications to chromatin density (DNA methylation, acetylation, and phosphorylation), histone modification, and non-coding RNAs (ncRNAs) are major mechanisms underlying epigenetic inheritance (Lacal and Ventura, 2018[54]; Suárez et al., 2023[104]). DNA methylation, the primary mechanism of epigenetic inheritance, is the covalent binding of a methyl group to the cytosine residue that is catalyzed by DNA methyltransferases (DNMTs) and alters gene expression; methylation at the gene promoter is associated with gene silencing, and methylation of the transcribed region increases transcriptional activity (Lacal and Ventura, 2018[55]). ncRNAs, that are not translated to proteins, may have < 200 nucleotides (short or small ncRNAs, sncRNAs) or > 200 nucleotides (long ncRNAs, lncRNAs) (Lacal and Ventura, 2018[55]). ncRNAs participate in epigenetic regulation through different mechanisms, including transcriptional regulation, RNA stability, and protein complex recruitment (Lacal and Ventura, 2018[55]).

Epigenetic dysregulation contributes to many diseases, including obesity, type 2 diabetes, and cardiovascular diseases (Lacal and Ventura, 2018[55]; Ling and Rönn, 2019[62]; Suárez et al., 2023[104]; Yang et al., 2011[128]). Diet is one of the factors that trigger epigenetic changes (Lacal and Ventura, 2018[55]; Tzika et al., 2018[110]), and a healthy diet helps prevent non-communicable diseases through epigenetic mechanisms (Ling and Rönn, 2019[62]). The contribution of inorganic nitrate to the prevention of chronic diseases through epigenetic inheritance has been addressed in a few animal (Crowe-White et al., 2025[21]; Serrano-Nascimento, 2021[95]) and human (Gonzalez-Nahm et al., 2017[37]; Jönsson et al., 2021[48]) studies, which will be discussed below and summarized in Figure 4(Fig. 4).

It has been reported that high maternal adherence to a Mediterranean Diet is associated with hypomethylation of the maternally expressed gene 3:intergenic differentially methylated region (MEG3-IG MDR) in offspring, which may be protective against type 2 diabetes (Gonzalez-Nahm et al., 2017[37]). Results of a genome-wide epigenetic analysis of cord blood from pregnant women with obesity indicate that adherence to a lifestyle intervention (physical activity with or without the Mediterranean Diet) during pregnancy is associated with greater lean mass in offspring (Jönsson et al., 2021[48]). A recent study by Crowe-White et al. report that nitrate supplementation (40 mg/day translated to 600 mg/day in humans that is provided by the DASH diet) in the HFD-induced obese female rats during the periconceptional (4 weeks before mating) and prenatal (during pregnancy) window has chronic imprinting effects on offspring (Crowe-White et al., 2025[21]); Compared to offspring from mothers on HFD, those from mothers on HFD + nitrate had lower fat mass, lower serum glucose, and lower serum triglycerides at post-natal day 65. In addition, maternal HFD decreased the protein expression of uncoupling protein 1 (UCP1) and c-subunit of mitochondrial ATP synthase in the brown adipose tissue of offspring, which decreases energy expenditure. Treatment of mothers with nitrate increased the expression of both proteins, indicating the epigenetic effect of nitrate on energy expenditure (Crowe-White et al., 2025[21]). It should be noted that epigenetic modifications are tissue- and even cell-specific; thus, it is important to study tissues relevant to a particular intervention (Ling and Rönn, 2019[62]). Nitrate is a competitive inhibitor of iodide uptake by thyroid; it has been shown that nitrate administration (20 and 50 mg/L in drinking water) to pregnant mice increases histone methylation and decreases histone acetylation, resulting in decreased expression of thyroid transcription factors and thyroid differentiation markers and disrupts thyroid development, as measured at gestational day 16.5 (Serrano-Nascimento, 2021[95]).

In summary, epigenetics links environmental factors (such as dietary habits) to altered gene activity and disease phenotypes (such as obesity) in the next generations (Ling and Rönn, 2019[62]). Epigenetic modulation has the potential to play a significant role in preventing non-communicable diseases. Further studies are needed to demonstrate the potential of dietary nitrate for preventing non-communicable diseases through epigenetic mechanisms.

All three NOS isoforms are expressed in salivary glands. nNOS has been found in human salivary duct epithelium (Soinila et al., 2006[97]), rat parotid gland acinar cells (Mitsui and Furuyama, 2000[71]), rat submandibular gland acinar and duct cells (Xu et al., 1997[125]), and to a lesser extent, duct cells in rabbits (Sugiya et al., 2001[105]). eNOS has been found in the human salivary duct epithelium (Bentz et al., 1998[13]; Soinila et al., 2006[97]). iNOS has been reported in the human salivary duct epithelium (Brennan et al., 2000[17]) and in rat submandibular gland acinar and duct cells (Xu et al., 1997[125] ). Collectively, these findings suggest that nNOS is the main NO-producing NOS isoform in the salivary glands and also highlight species-dependent differences in NOS expression (Hezel and Weitzberg, 2015[41]; Looms et al., 2002[64]; Soinila et al., 2006[97]). However, data on NOS expression in salivary glands are not entirely consistent. For example, it has been reported that none of the NOS isoforms are expressed in human salivary gland acinar cells (Bentz et al., 1998[13]; Soinila et al., 2006[97]), and that eNOS and iNOS are not expressed in rat submandibular gland acinar and duct cells (Xu et al., 1997[125]).

Despite nitrate uptake at physiological extracellular nitrate concentrations (50-1,000 μM), human submandibular gland cells produce NO and cyclic guanosine monophosphate (cGMP) only at high, non-physiological nitrate concentrations (≥ 15 mM). This contrasts with the liver, where a much lower extracellular nitrate concentration (500 μM) increases intracellular NO production, indicating that, under physiological conditions, salivary gland cells primarily accumulate nitrate for transport and concentration in saliva rather than for NO generation (Qin et al., 2012[89]). Another factor supporting the limited contribution of nitrate-derived NO in the salivary glands is the low nitrate reductase activity in these glands. In eukaryotic cells, the enzyme xanthine oxidoreductase (XOR) catalyzes the reduction of nitrate to nitrite, which can subsequently be reduced to NO under physiological conditions (Jansson et al., 2008[46]). XOR gene expression in the salivary glands (3.8 TPM, transcript per million) is intermediate between that of the liver (26.6 TPM) and skeletal muscle (0.08 TPM), as indicated by data from the Genotype-Tissue Expression (GTEx) portal (https://gtexportal.org).

In mammals, XOR exists in two interconvertible forms derived from the same gene product: xanthine dehydrogenase (XDH) and xanthine oxidase (XO) (Hille and Nishino, 1995[42]). Under metabolic or oxidative stress, XDH can be post-translationally converted to XO (Hellsten et al., 1996[40]; Williams et al., 2023[121]). Both forms catalyze the oxidation of hypoxanthine to xanthine and xanthine to uric acid, but XDH and XO use NAD⁺ and O₂ as electron acceptors, respectively (Hille and Nishino, 1995[42]). Total XOR activity in mouse liver (3.3 nmol/min/mg protein) is approximately twofold higher than in salivary glands (1.6 nmol/min/mg protein); however, the proportion of the enzyme present in the XO form is substantially higher in the salivary glands (73 %) than in the liver (14 %) (Kusano et al., 2023[54]). This high XO activity in salivary glands contrasts with most other tissues, where XOR is predominantly expressed as XDH (Kusano et al., 2023[54]), the predominant form in vivo (Godber et al., 2001[35]). Importantly, XDH is about 50 times more efficient than XO in reducing nitrite to NO (Godber et al., 2000[36]). Collectively, these data indicate minimal, if any, conversion of nitrate to NO in the salivary glands.

NO production in skeletal muscle is mainly NOS-dependent. In support of this, decreased NO release has been reported following NOS inhibition in resting diaphragm fibers (67 %, from 2.8 to 0.9 pmol/min/mg muscle) (Kobzik et al., 1994[53]; Reid et al., 1998[91]) and extensor digitorum longus (EDL) muscle (68 %, from 1 to 0.3 pmol/min/mg muscle) (Balon and Nadler, 1994[11]) in male Sprague-Dawley rats, as well as in tibialis anterior (TA) skeletal muscle (57-77 %) in rabbits (Sutherland et al., 2001[106]).

Human skeletal muscle fibers express all three isoforms of NOS, with nNOS being the most abundant isoform (Bahadoran et al., 2024[7]; Frandsen et al., 1996[28]; Stamler and Meissner, 2001[101]), and its mRNA expression is even higher in human skeletal muscle than in the brain (Nakane et al., 1993[72]). Under physiologic conditions, nNOS appears to be the principal NOS isoform responsible for NO production in skeletal muscle (Baldelli et al., 2014[10]), with little or no contribution from eNOS (Gilliard et al., 2018[34]; Hirschfield et al., 2000[43]) or iNOS (Sutherland et al., 2001[106]). In support of this notion, the rate of NO release from diaphragm (14.9 ± 9.1 vs. 18.6 ± 4.8 pmol/min/mg muscle) or soleus muscle (12.8 ± 6.8 vs. 15.6 ± 2.2 pmol/min/mg muscle) did not differ between wild-type and eNOS-deficient mice (Hirschfield et al., 2000[43]), indicating that eNOS does not contribute to resting NO release from skeletal muscle (Hirschfield et al., 2000[43]) or may have only a minor role (Gilliard et al., 2018[34]). Furthermore, Ca²⁺ removal completely blocked NO production in TA muscle in rabbits, indicating that iNOS does not contribute to NO production in skeletal muscle under normal conditions (Sutherland et al., 2001[106]). Consistent with these findings, GTEx portal data indicate that mRNA expression of nNOS in skeletal muscle is about 5-fold and 35-fold higher than that of eNOS and iNOS, respectively.

In skeletal muscle, nitrate can also be reduced to NO. The presence of both XOR and aldehyde oxidase (AO), which are molybdopterin-containing nitrate reductase enzymes, has been documented in human skeletal muscle (Hellsten et al., 1996[40]; Wylie et al., 2019[124] ). In rats, XOR appears to be the major enzyme responsible for nitrate reduction in skeletal muscle, as oxypurinol, an XOR inhibitor, completely blocks this reaction in the hind legs of adult male Wistar rats (Piknova et al., 2016[82]). However, nitrate reductase activity in skeletal muscle is relatively low (Piknova et al., 2015[84]), with about 1 % of nitrate uptake converted to nitrite (Srihirun et al., 2020[100]). At 2 % oxygen, nitrate reductase activity in rat hindlimb skeletal muscle has been reported to be 0.4, 0.5, and 0.9 nmol/h/g tissue at nitrate concentrations of 100, 300, and 500 μM, respectively (Piknova et al., 2015[84]). These values are approximately 4-, 10-, and 12-fold lower than those observed in the liver, indicating that nitrate reductase activity in skeletal muscle is comparatively limited (Piknova et al., 2015[84]).

eNOS is constitutively expressed in human hepatocytes under normal conditions, with an activity of 0.39 ± 0.14 pmol/min/mg protein in liver homogenates (McNaughton et al., 2002[69]). Constitutive expression of iNOS in the normal human hepatocytes has been reported (Leifeld et al., 2002[57]; McNaughton et al., 2002[69]), and iNOS activity in liver homogenates is 0.44 ± 0.16 pmol/min/mg protein (McNaughton et al., 2002[69]). Data regarding nNOS expression in the liver are inconclusive. Low levels of nNOS protein have been detected in mouse liver homogenates (Schild et al., 2006[94]), and selective nNOS inhibition blocks acetaminophen toxicity in mouse hepatocytes (Banerjee et al., 2015[12]). However, nNOS is not expressed in normal human hepatocytes (McNaughton et al., 2002[69]), and nNOS knockout does not alter nitrate or nitrite levels in the liver of mice (Piknova et al., 2015[84]). Previous reviews have suggested that eNOS is the principal NOS isoform contributing to NO production in hepatocytes under normal physiological conditions (Bahadoran et al., 2020[6]; Farahani et al., 2025[25]).

The liver exhibits relatively high XOR activity (Jansson et al., 2008[46]; Piknova et al., 2015[84]; Qin et al., 2012[89]; Srihirun et al., 2020[100]). In the rat liver, XOR is the primary nitrate reductase, as inhibition by oxypurinol results in approximately 51 % decrease in nitrate reductase activity (Piknova et al., 2015[84]). In mammals, nitrate reductase activity is most abundant in the gastrointestinal tract and liver, with the rank order colon > stomach > kidney > small intestine > liver > heart > lung (Jansson et al., 2008[46]). At 2 % oxygen, nitrate reductase activity in rat liver has been reported to be 1.5, 4.8, and 10.9 nmol/h/g tissue at 100, 300, and 500 μM nitrate, respectively (Piknova et al., 2015[84]). Compared with anaerobic conditions (0 % oxygen), nitrate reduction in liver homogenates decreases by approximately 25 % in the presence of 6 % oxygen, which approximates normal tissue oxygenation (~60 μM, 45 mm Hg) (Jansson et al., 2008[46]).

The characteristics of NO production and nitrate metabolism in the salivary glands, skeletal muscle, and liver are summarized in Table 1(Tab. 1) (References in Table 1: Carreau et al., 2011[18]; Frandsen et al., 1996[28]; Ghasemi, 2022[29]; Jansson et al., 2008[46]; Kusano et al., 2023[54]; Mitsui and Furuyama, 2000[71]; Piknova et al., 2015[84]; Piknova et al., 2023[85]; Piknova et al., 2024[86]; Piknova et al., 2025[83]; Qin et al., 2012[89]; Snyder et al., 1975[96]; Srihirun et al., 2020[100]; Stamler and Meissner, 2001[101]; Sugiya et al., 2001[105]; Xu et al., 1997[125]). NO production in the salivary glands is primarily NOS-dependent, with nNOS serving as the main NO-producing enzyme, and nitrate reductase activity in these glands is minimal. Skeletal muscle can generate NO via both NOS-dependent (predominantly nNOS-mediated) and NOS-independent pathways, although its nitrate reductase activity is relatively low. In the liver, eNOS is likely the principal NO-producing NOS isoform, and relatively high XOR activity enables effective conversion of nitrate to NO. mRNA expression of the XOR gene in the liver and salivary glands is approximately 330-fold and 50-fold higher, respectively, than in skeletal muscle. Nevertheless, the overall order of nitrate reductase activity is liver > skeletal muscle > salivary glands, as XOR in the salivary glands is predominantly expressed as XO (Kusano et al., 2023[54]), which is less efficient in reducing nitrite to NO than XDH (Godber et al., 2000[36]).

Substantial inter-individual differences in circulating nitrate concentrations have been reported (Bescos et al., 2025[14]; Ghasemi et al., 2008[31]; Ghasemi et al., 2010[32]; Tsikas et al., 2006[109]). Measurements of plasma nitrate in humans have yielded values ranging from 29 to 66 nmol/g (Jonvik et al., 2016[49]; Kadach et al., 2023[50]; Kadach et al., 2022[51]; Nyakayiru et al., 2017[73]; Wylie et al., 2019[124] ). Plasma nitrate levels in rats appear lower, ranging from 12.5 to 29.2 nmol/g (Ferguson et al., 2015[27]; Park et al., 2023[79]). The weighted mean plasma nitrate concentration in healthy adults is approximately 35 μM (Bahadoran et al., 2019[8]), consistent with other reports (Bahadoran et al., 2019[8]; Bescos et al., 2025[14]; Kapil et al., 2020[52]; Tsikas, 2008[108]). A systematic review and meta-analysis including data from 40 studies reported a mean fasting plasma nitrate concentration of 33.9 μM (95 % confidence interval: 29.9-37.9 μM) in healthy adult men and women (Bescos et al., 2025[14]). Overall, the average circulating nitrate concentration in healthy adults is about 35 μM, with reported values ranging from 12 to 76 μM (Ghasemi et al., 2010[32]).

Substantial inter-individual differences in basal salivary nitrate concentrations have also been reported (Bescos et al., 2025[14]; Granli et al., 1989[39]). A systematic review and meta-analysis that included data from 12 studies reported a mean fasting salivary nitrate concentration of 535.9 μM (95 % confidence interval: 384.2-687.6 μM) (Bescos et al., 2025[14]). However, basal salivary nitrate concentrations of 200 μM (Lundberg and Govoni, 2004[68]), 249 μM (Bahadoran et al., 2021[5]), and 720 μM (Govoni et al., 2008[38]) have also been reported. Overall, basal salivary nitrate concentrations in humans are approximately 500 μM, with reported values ranging from 200 to 700 μM.

At baseline, in healthy individuals, the saliva-to-plasma nitrate concentration ration has been reported to be 9 (Cortas and Wakid, 1991[20]), 10 (Bahadoran et al., 2021[5]), 16 (Bescos et al., 2025[14]), 60 (Govoni et al., 2008[38]), and 100 (Srihirun et al., 2020[100]). These findings indicate that the saliva-to-plasma nitrate concentration ratio generally ranges from approximately 10- to 100-fold (Bahadoran et al., 2021[5]; Bescos et al., 2025[14]; Cortas and Wakid, 1991[20]; Govoni et al., 2008[38]; Lundberg and Govoni, 2004[68]; Srihirun et al., 2020[100]; Weitzberg et al., 2010[119]), with values of 10-20 appearing to be reasonable estimates (Weitzberg et al., 2010[119]).

Table 2(Tab. 2) (References in Table 2: Gilliard et al., 2018[34]; Ibrahim and Ashour, 2011[44]; Kadach et al., 2022[51]; Kadach et al., 2023[50]; Long et al., 2020[63]; Nyakayiru et al., 2017[73]; Park et al., 2019[78]; Park et al., 2021[80]; Park et al., 2023[79]; Piknova et al., 2015[84]; Piknova et al., 2016[82]; Piknova et al., 2023[85]; Piknova et al., 2024[86]; Troutman et al., 2018[107]; Upanan et al., 2024[111]; Wylie et al., 2019[124] ) summarizes basal nitrate content in different skeletal muscles as measured in humans (Kadach et al., 2023[50]; Kadach et al., 2022[51]; Nyakayiru et al., 2017[73]; Wylie et al., 2019[124]), rats (Gilliard et al., 2018[34]; Ibrahim and Ashour, 2011[44]; Long et al., 2020[63]; Park et al., 2023[79]; Park et al., 2021[80]; Piknova et al., 2016[82]; Piknova et al., 2015[84]; Piknova et al., 2023[85]; Troutman et al., 2018[107]), mice (Park et al., 2019[78]; Piknova et al., 2015[84]; Upanan et al., 2024[111]), and pigs (Piknova et al., 2024[86]); values presented graphically were extracted as described previously (Gheibi et al., 2019[33]). In humans, all measurements have been conducted in the vastus lateralis, yielding basal nitrate concentrations ranging from 35 to 226 nmol/g. Most studies have been conducted in rats, reporting values ranging from 12 to 222 nmol/g, with the lowest concentrations observed in the tibialis anterior (TA) and the highest in the soleus. Studies in mice using mixtures of hindlimb muscles reported concentrations ranging from 17 to 113 nmol/g, whereas the only study in pigs reported a concentration of 103 nmol/g. Considerable variation exists in basal nitrate levels both within the same muscle among different individuals and among different skeletal muscles. In healthy young adults, large inter-individual variability in baseline muscle nitrate concentration has been reported, with values ranging from 30 to 1,036 nmol/g tissue (Wylie et al., 2019[124]). In addition, nitrate content in the gluteus muscle (43.4 nmol/g) was 3.6-fold higher than in TA (12.1 nmol/g) (Park et al., 2021[80]), indicating that different skeletal muscles store different amounts of nitrate (Park et al., 2021[80]; Piknova et al., 2021[87]). Despite this variability, an approximate average value of 100 nmol/g (range: 12-226 nmol/g) can be considered for overall skeletal muscle nitrate concentration.

Differences in nitrate concentration among skeletal muscles may be related to differences in predominant muscle fiber type (Kadach et al., 2022[51]; Park et al., 2021[80]), differential expression of nitrate transporters, differences in nitrate production capacity, and variation in XOR expression or activity (Piknova et al., 2021[87]). In rats, the percentages of type II fibers in the extensor digitorum longus (EDL), gluteus, TA, vastus lateralis, gastrocnemius, and soleus are 100 %, 100 %, 99.6 %, 98.8 %, 94 %, and 20 %, respectively (Eng et al., 2008[23]). Species differences are also evident: in rats, the vastus lateralis muscle contains approximately 98.8 % type II fibers and only 1.2 % type I fibers (Eng et al., 2008[23]), whereas in humans, the vastus lateralis contains approximately 40 % and 44 % type I fibers in healthy young men and women, respectively (Staron et al., 2000[102]). In rats, higher nitrate concentrations have been observed in skeletal muscles with a higher proportion of type I (slow-twitch) fibers compared with type II (fast-twitch) fibers, with nitrate content in the soleus muscle being 3.5-fold higher than in the vastus lateralis (Long et al., 2020[63]). In addition, basal microvascular PO₂ was higher in soleus (32 ± 3 mm Hg) than in gastrocnemius (24 ± 2 mm Hg) in rats (Ferguson et al., 2015[27]). However, this relationship requires further investigation, as the opposite pattern has also been reported, with nitrate content in the gluteus muscle being 1.9-fold higher than in the soleus muscle (Park et al., 2021[80]).

As shown in Table 2(Tab. 2), the skeletal muscle-to-plasma ratio of nitrate varies across skeletal muscles, with reported values ranging from below 1 to above 1. In humans, four studies conducted in the vastus lateralis muscle yield a mean skeletal muscle-to-plasma nitrate ratio of approximately 2.1. In rats, reported ratios are 1.3 for the TA and gastrocnemius muscles, 1.4 for the EDL, 2.2 for the vastus lateralis, 2.3 for the gluteus, and 3.8 for the soleus muscle.

Overall, nitrate content in skeletal muscle varies substantially across muscle types and species, an issue that warrants further investigation.

As shown in Table 3(Tab. 3) (References in Table 3: Gilliard et al., 2018[34]; Park et al., 2019[78]; Park et al., 2021[80]; Park et al., 2023[79]; Piknova et al., 2015[84]; Piknova et al., 2016[82]; Piknova et al., 2023[85]; Piknova et al., 2024[86]; Upanan et al., 2024[111]), nitrate content in the liver has been measured in rats (Gilliard et al., 2018[34]; Park et al., 2023[79]; Park et al., 2021[80]; Piknova et al., 2016[82]; Piknova et al., 2015[84]; Piknova et al., 2023[85]), mice (Park et al., 2019[78]; Piknova et al., 2015[84]; Upanan et al., 2024[111]), and pigs (Piknova et al., 2024[86]). Reported values in rats range from 4.7 to 12.7 nmol/g, which are similar to those measured in mice (7.4-16.9 nmol/g) and pigs (15.7 nmol/g) (Piknova et al., 2024[86]). One study reported higher nitrate levels in rat liver (46.8 ± 9.0 nmol/g tissue) (Gilliard et al., 2018[34]). In addition, the liver-to-plasma nitrate ratio is approximately 0.24 in rats, 0.40 in pigs, and 0.48 in mice. Overall, a reasonable estimate of hepatic nitrate content is approximately 10 nmol/g (range: 5-15 nmol/g), with the liver-to-plasma nitrate ratio being consistently below 1 across all species studied.

Table 4(Tab. 4) (References in Table 4: Bahadoran et al., 2021[5]; du Toit et al., 2024[22]; Govoni et al., 2008[38]; Jonvik et al., 2016[49]; Kadach et al., 2022[51]; Kadach et al., 2023[50]; Lundberg and Govoni, 2004[68]; Pannala et al., 2003[77]; Webb et al., 2008[117]; Wylie et al., 2013[123]; Wylie et al., 2019[124]) summarizes increases in plasma and salivary nitrate concentrations following nitrate ingestion in healthy humans. Interventions includes sodium nitrate (Govoni et al., 2008[38]; Jonvik et al., 2016[49]; Lundberg and Govoni, 2004[68]), potassium nitrate (du Toit et al., 2024[22]; Kadach et al., 2023[50]; Kadach et al., 2022[51]), or nitrate-rich foods such as beetroot (Bahadoran et al., 2021[5]; Webb et al., 2008[117]; Wylie et al., 2013[123]; Wylie et al., 2019[124]), lettuce (Pannala et al., 2003[77]), and green leafy vegetables (du Toit et al., 2024[22]). Nitrate doses ranged from 3.1 to 21.5 mg/kg, and total nitrate intake ranged from 222 to 1,395 mg/day.

Human studies indicate that following nitrate ingestion, plasma nitrate concentration increases by approximately 2-20-fold and typically peaks at around 90 min (range: 30-160 min). In addition, salivary nitrate concentration increases by approximately 3- to 85-fold and generally peaks at around 90 min (range: 30-180 min). Data presented in Table 4(Tab. 4) indicate that following nitrate ingestion, plasma and salivary nitrate concentrations peak simultaneously (at approximately 90 min), consistent with a previous report (Lundberg and Govoni, 2004[68]). However, findings on this issue are not entirely consistent. Some studies have reported that plasma nitrate peaks before salivary nitrate (Kadach et al., 2023[50]; Pannala et al., 2003[77]; Webb et al., 2008[117]), whereas others have reported that plasma nitrate peaks after salivary nitrate (Bahadoran et al., 2021[5]; Kadach et al., 2022[51]).

Increased plasma nitrate concentrations following nitrate consumption have also been reported in animal studies, including beetroot supplementation (62 mg/kg for 5 days) (Ferguson et al., 2015[27]) and sodium nitrate administration (1g/L in drinking water for 5 days) (Piknova et al., 2023[85]) in rats, resulting in 2.7-fold (29 ± 6 to 79 ± 17 nmol/g) and 3.9-fold (15.1 ± 3.9 to 58.8 ± 30.8 nmol/g) increases, respectively. In pigs, ingestion of Na¹⁵NO₃ (9.3 mg/kg, single dose) increased plasma nitrate concentration approximately 5-fold (39.8 ± 19.7 to 199 ± 35.6 μM) (Piknova et al., 2024[86]).

It has been shown that plasma nitrate concentration increases from a baseline value of 34 ± 8 μM by 4-, 8-, and 17-fold following ingestion of 3.1, 6.8, and 13.6 mg/kg nitrate from beetroot, respectively, at 2.5 h post-ingestion in healthy men (Wylie et al., 2013[123]). Figure 6(Fig. 6) illustrates changes in plasma and salivary nitrate concentrations as a function of ingested nitrate dose in humans, based on studies in which both circulating and salivary nitrate concentrations were measured simultaneously (Bahadoran et al., 2021[5]; du Toit et al., 2024[22]; Kadach et al., 2023[50]; Kadach et al., 2022[51]; Lundberg and Govoni, 2004[68]; Pannala et al., 2003[77]; Webb et al., 2008[117]). Increased plasma and salivary nitrate concentrations following beetroot ingestion are dose-dependent; for example, when an individual consumes 10 mg/kg nitrate, circulating nitrate concentration increases by approximately 10-fold, whereas salivary nitrate concentration increases by approximately 18-fold.

Nitrate ingestion increases skeletal muscle nitrate content in humans (Kadach et al., 2022[51]; Wylie et al., 2019[124] ) and animals (Gilliard et al., 2018[34]; Piknova et al., 2023[85]; Piknova et al., 2024[86]) by approximately 3-5-fold and 1-2-fold, respectively (Table 5(Tab. 5); References in Table 5: Gilliard et al., 2018[34]; Kadach et al., 2022[51]; Kadach et al., 2023[50]; Park et al., 2023[79]; Piknova et al., 2023[85]; Piknova et al., 2024[86]; Wylie et al., 2019[124]). Studies employing a single dose of nitrate ingestion measured skeletal muscle nitrate concentrations at 2-3 h post-ingestion (Kadach et al., 2022[51]; Piknova et al., 2024[86]; Wylie et al., 2019[124]). A study by the Piknova group indicates that increases in skeletal muscle nitrate content following nitrate ingestion vary among muscle types, with the TA and EDL showing the lowest and highest increases, respectively (Piknova et al., 2023[85]).

Animal studies indicate that liver nitrate content increases following nitrate ingestion in rats (Gilliard et al., 2018[34]; Park et al., 2023[79]; Piknova et al., 2023[85]) and pigs (Piknova et al., 2024[86]) by about 2-3-fold (Table 6(Tab. 6); References in Table 6: Gilliard et al., 2018[34]; Park et al., 2023[79]; Piknova et al., 2023[85]; Piknova et al., 2024[86]).

This study was supported by Shahid Beheshti University of Medical Sciences (Grant number: 43018291-1), Tehran, Iran. KK is supported in part by the National Institutes of Health, USA, grant number 2U54MD017979-01A1.

The authors declare that they have no conflict of interest.

Artificial intelligence was not used in the preparation of this manuscript.