PurposeTo evaluate the effect of nitric oxide (NO) donor, in combination with plasma volume expansion, on both fetal and maternal outcomes in pregnancies complicated by early-onset fetal growth restriction (FGR).MethodsA total of 40 pregnant women diagnosed with early onset FGR were recruited from Ain Shams University Maternity Hospital between June 2023 to December 2023. The patients were randomly assigned into two groups, 20 in each group. Group A received Nitroderm TTS ® 5 mg for 12 h daily with plasma volume expansion (PVE) in the form of 2.5 L of water per day. Group B represented the control group. The primary endpoint of the study, assessed after 2 weeks of treatment initiation, focused on fetal growth parameters as the primary outcome. In addition, amniotic fluid volume, umbilical artery Doppler changes, development of fetal complications, maternal vital signs, and any side effects, were recorded. At the time of delivery, the following also documented: timing, mode, and interval to delivery, along with neonatal outcomes.ResultsGroup A exhibit statistically significant enhancement in fetal growth compared to Group B in terms of estimated fetal weight, abdominal circumference, head circumference, biparietal diameter, femur length, amniotic fluid volume, and umbilical artery pulsatility index. Furthermore, Group A demonstrated more favorable outcomes in terms of gestational age at delivery, interval to delivery, birth weight, APGAR score and rates of NICU admission.ConclusionThe combination of NO donors and PVE has shown promising results in enhancing fetal growth and extending gestation. This study adds to the existing body of evidence supporting the effectiveness of NO donor therapy when used in conjunction with fluid management for managing FGR. Nonetheless, additional research is essential to validate these results and refine the treatment strategy for optimal outcomes in affected pregnancies.

Contrast-induced nephropathy (CIN) is associated with increased mortality and morbidity in patients undergoing coronary angiography (CAG) and percutaneous coronary intervention (PCI). We aimed to assess the latest evidence on the preventive effects of nitric oxide (NO) donors in CIN patients undergoing CAG/PCI. We conducted a comprehensive systematic review and meta-analysis of RCTs from PubMed, Web of Science, Scopus, Embase, and Cochrane searches until May 5th, 2024. Dichotomous data were pooled using risk ratio (RR), and continuous data were pooled using mean difference (MD), both with a 95% confidence interval (CI), using (R version 4.3). Our analysis included 13 RCTs encompassing 3,550 patients. NO donors were significantly associated with a decreased incidence of CIN compared to placebo either as an oral administration (RR: 0.33 with 95% CI [0.26, 0.42], P < 0.01) or IV infusions (RR: 0.56 with 95% CI [0.40, 0.78], P < 0.01). Moreover, NO donors were significantly associated with decreased serum creatinine levels compared to placebo either as an oral administration (MD: - 0.07 with 95% CI [- 0.10, - 0.04], P < 0.01) or IV infusions (MD: - 0.07 with 95% CI [- 0.09, - 0.04], P < 0.01). In terms of safety, NO donors were significantly associated with a decreased incidence of major adverse cardiac events (MACE) compared to placebo as an oral administration (RR: 0.64 with 95% CI [0.45, 0.89], P < 0.01). However, there was no significant difference between NO donors as IV infusions and placebo in MACE (RR: 0.68 with 95% CI [0.38, 1.21], P = 0.18). Finally, NO donors were significantly associated with a decreased incidence of all-cause mortality compared to placebo as an oral administration (RR: 0.58 with 95% CI [0.36, 0.94], P = 0.03). Nevertheless, there was no statistically significant difference in all-cause mortality between IV infusions of NO donors and placebo (RR: 1.84 with 95% CI [0.40, 8.52], P = 0.44). NO donors as adjunct therapy are associated with reduced incidence of CIN and decreased serum creatinine levels, either as an oral or IV administration. They were also associated with reduced incidence of MACE, all-cause mortality, and recurrent myocardial infarction as an oral administration, which makes this simple, low-cost intervention an important therapeutic option in patients undergoing CAG/PCI.

Background: Contrast-induced nephropathy (CIN) is associated with increased mortality and morbidity in patients undergoing coronary angiography (CAG) and percutaneous coronary intervention (PCI). We aimed to assess the latest evidence on the preventive effects of nitric oxide (NO) donors in CIN in patients undergoing CAG/PCI. Methods: We conducted a systematic review and meta-analysis of RCTs from PubMed, Web of Science, Scopus, Embase, and Cochrane searches until May 5th, 2024. Dichotomous data were pooled using risk ratio (RR), and continuous data were pooled using mean difference (MD), both with a 95% confidence interval (CI), using (R version 4.3). Results: Our analysis included 13 RCTs encompassing 3,550 patients. NO donors were significantly associated with a decreased incidence of CIN compared to placebo either as an oral administration (RR: 0.33 with 95% CI [0.26, 0.42], P&lt; 0.01) or IV infusions (RR: 0.56 with 95% CI [0.40, 0.78], P&lt; 0.01). Moreover, NO donors were significantly associated with decreased serum creatinine levels compared to placebo either as an oral administration (MD: -0.07 with 95% CI [-0.10, -0.04], P&lt; 0.01) or IV infusions (MD: -0.07 with 95% CI [-0.09, -0.04], P&lt; 0.01). In terms of safety, NO donors were significantly associated with a decreased incidence of MACE compared to placebo as an oral administration (RR: 0.64 with 95% CI [0.45, 0.89], P&lt; 0.01). However, there was no significant difference between NO donors as IV infusions and placebo in MACE (RR: 0.68 with 95% CI [0.38, 1.21], P= 0.18). Finally, NO donors were significantly associated with a decreased incidence of all-cause mortality compared to placebo as an oral administration (RR: 0.58 with 95% CI [0.36, 0.94], P= 0.03). However, there was no significant difference between NO donors as IV infusions and placebo in all-cause mortality (RR: 1.84 with 95% CI [0.40, 8.52], P= 0.44). Conclusion: NO donors as an adjunct therapy are associated with reduced incidence of CIN, and decreased serum creatinine levels either as an oral or IV administration. Also, it was associated with decreased incidence of MACE and all-cause mortality as an oral administration which make this simple low-cost intervention an important therapeutic option in patients undergoing CAG/PCI.

Unexplained infertility has been defined, by the Practice Committee of the American Society of Reproductive Medicine (ASRM), as inability of a couple to conceive for at least 12 months, with unremarkable standard infertility evaluation ((ASRM), 2006). Although estimates vary, the prevalence of unexplained infertility is approximately 15–30% Aim of the Work to evaluate the effect of nitroglycerine transdermal patches on uterine and subendometrial blood flow in women with unexplained infertility. Patients and Methods Transdermal nitroglycerine patch was administered to the allocated group from the 2nd day of cycle till maturation of the follicles ≥ 18 mm just for one cycle. Results The relatively underdeveloped endometrial thickness did not change significantly after treatment of women with unexplained infertility with nitroglycerin and remained significantly lower than women of the fertile group. Also, nitroglycerin treatment did not result in significant change in uterine artery Doppler indices. However, uterine artery indices of nitroglycerin-treated women did not also show a significant difference (P &amp;gt; 0.05) from fertile women. On the contrary, nitroglycerin treatment in women with unexplained infertility significantly increased subendometrial 3D power Doppler indices to mean values that did not differ significantly from the fertile women. According to the results of the current study, biochemical pregnancy was more commonly achieved in the nitroglycerin group compared to the control group (6.67% vs 3.33%), Yet this difference failed to prove statistical significance. Although adverse effects of nitroglycerin treatment in the current study, namely headache, blurring of vision and hypotension, were more common on the nitroglycerin group compared to the control and fertile groups; this difference couldn’t reach statistical significance (P &amp;gt; 0.05). Conclusion Doppler study of uterine hemodynamics can be considered in infertility work-up. Although still lacking evidence, women with suboptimal uterine perfusion may be offered therapies improving uterine blood flow, such as nitroglycerin.

BackgroundNitric oxide-releasing drugs are used for cardiovascular diseases; however, their effects on the tumor immune microenvironment are less clear. Therefore, this study explored the impact of nitric oxide donors on tumor progression in immune-competent mice.MethodsThe effects of three different nitric oxide-releasing compounds (SNAP, SNP, and ISMN) on tumor growth were studied in tumor-bearing mouse models. Three mouse tumor models were used: B16F1 melanoma and LL2 lung carcinoma in C57BL/6 mice, CT26 colon cancer in BALB/c mice, and LL2 lung carcinoma in NOD/SCID mice. After nitric oxide treatment, splenic cytokines and lymphocytes were analyzed by cytokine array and flow cytometry, and tumor-infiltrating lymphocytes in the TME were analyzed using flow cytometry and single-cell RNA sequencing.ResultsLow doses of three exogenous nitric oxide donors inhibited tumor growth in two immunocompetent mouse models but not in NOD/SCID immunodeficient mice. Low-dose nitric oxide donors increase the levels of splenic cytokines IFN-γ and TNF-α but decrease the levels of cytokines IL-6 and IL-10, suggesting an alteration in Th2 cells. Nitric oxide donors increased the number of CD8+ T cells with activation gene signatures, as indicated by single-cell RNA sequencing. Flow cytometry analysis confirmed an increase in infiltrating CD8+ T cells and dendritic cells. The antitumor effect of nitric oxide donors was abolished by depletion of CD8+ T cells, indicating the requirement for CD8+ T cells. Tumor inhibition correlated with a decrease in a subtype of protumor macrophages and an increase in a subset of Arg1-positive macrophages expressing antitumor gene signatures. The increase in this subset of macrophages was confirmed by flow cytometry analysis. Finally, the combination of low-dose nitric oxide donor and cisplatin induced an additive cancer therapeutic effect in two immunocompetent animal models. The enhanced therapeutic effect was accompanied by an increase in the cells expressing the gene signature of NK cell.ConclusionsLow concentrations of exogenous nitric oxide donors inhibit tumor growth in vivo by regulating T cells and macrophages. CD8+ T cells are essential for antitumor effects. In addition, low-dose nitric oxide donors may be combined with chemotherapeutic drugs in cancer therapy in the future.

Prophylactic Effect of Nitric Oxide Donors on Rat Models of EGFR Inhibitor‒Induced Cutaneous Toxicities

Co-culture hydrogel micro-chamber array-based plate for anti-tumor drug development at single-element resolution

Acute respiratory distress syndrome (ARDS) is characterized by acute hypoxemia, neutrophil-mediated inflammation, and lung edema formation. Whereas lung damage might be alleviated by nitric oxide (NO), goal of this study was to evaluate if intratracheal NO donor S-nitroso-N-acetylpenicillamine (SNAP) can positively influence the lung functions in experimental model of ARDS. New Zealand rabbits with respiratory failure induced by saline lavage (30 ml/kg, 9+/-3 times) were divided into: ARDS group without therapy, ARDS group treated with SNAP (7 mg/kg i.t.), and healthy Control group. During 5 h of ventilation, respiratory parameters (blood gases, ventilatory pressures) were estimated. After anesthetics overdosing, left lung was saline-lavaged and cell count, cell viability and protein content in bronchoalveolar lavage fluid (BALF) were measured. Right lung tissue was used for estimation of wet/dry weight ratio, concentration of NO metabolites, and histomorphological investigation. Repetitive lung lavage induced lung injury, worsened gas exchange, and damaged alveolar-capillary membrane. Administration of SNAP reduced cell count in BALF, lung edema formation, NO metabolites, and histopathological signs of injury, and improved respiratory parameters. Treatment with intratracheal SNAP alleviated lung injury and edema and improved lung functions in a saline-lavaged model of ARDS suggesting a potential of NO donors also for patients with ARDS.

Nitric oxide (NO) has been proven to be a key regulator in the mammalian immune response, such as the innate and adaptive immune responses to tumors. The messenger NO involves T helper cell differentiation and lymphocyte biofunctions. In this study, we employed N,N’-di-sec-butyl-N,N’-dinitroso-1,4-phenylenediamine as NO donor and released NO around tumor infiltrating lymphocytes in vitro by short-time blue light irradiation. The interferon-γ secretion of tumor infiltrating lymphocytes was investigated to study the functional changes caused by the accurate spatio-temporal delivery of NO. The downregulation of interferon-γ in tumor infiltrating lymphocytes after NO treatment indicates promising biological applications to potentially play a role in the treatment of autoimmune diseases. The study was approved by the Medical Ethics Committee of the Shenzhen Second People’s Hospital, the First Affiliated Hospital of Shenzhen University, China (approved No. 065) on February 12, 2018.

The role of aspirin, heparin, and other interventions in the prevention and treatment of fetal growth restriction.

The effects of an exogenous nitric oxide donor (sodium nitroprusside, SNP), a NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxode (PTIO) and carboxy-PTIO potassium salt (cPTIO) on the embryo germination of Sorbus pohuashanensis were studied in a petri dish test. SNP at 0.5–5 mmol L−1 increased germination percentage, mean time to germination, germination index and germination energy compared with the control to different degrees. Treatment with 2 mmol L−1 SNP improved germination most significantly; embryo germination percentage for mother tree 1 (91.11%) and mother tree 2 (64.44%) were much higher than the control. In addition, excessive SNP levels did not enhance embryo germination. Combined treatment with SNP and an NO scavenger delayed embryo germination. Treatment with cPTIO inhibited embryo germination; germination percentage was 42.22% and was lower than that of the control. These results show that low concentrations of exogenous NO can enhance the embryo germination of S. pohuashanensis, providing a simple, effective way for promoting germination of S. pohuashanensis.

The aim of this study is to investigate the effects of a nitric oxide (NO) donor S-nitrosoglutathione (GSNO) on themetabolism and improvement of preservation quality in apheresis platelets. A GSNO solution with a certain concentration was added into fresh apheresis platelets, and the parameters associated with platelet morphology, metabolism and function were temporally monitored for 7days. The results showed that the NO level in GSNO group were remarkably higher than those in the control group during the whole storage stage. No significant morphology or function difference was observed between the control and GSNO groups such as Platelet count, platelet distribution width, mean platelet volume and mitochondrial metabolic activity. But in metabolism there are something differences from morphology data: pCO2, pO2, cHCO3- were also found to have no clear difference between the control and GSNO groups; the lactic acid content, sugar consumption and Lactate dehydrogenase activity in the GSNO group were lower than that in the control group at some time point; and pH values in the GSNO group were higher than the control group. Our study discovered that the NO donor GSNO can reduce the metabolism and maintain the cellular characteristics of platelets in vitro during the platelet storage period.

Nitric oxide is a potent anti-apoptotic and cardioprotective molecule in healthy animals. However, recent study demonstrates that overexpression of eNOS exacerbates the liver injury in diabetic animals. whether diabetes may also alter NO's biologic activity in ischaemic/reperfused heart remains unknown. The present experiment was designed to determine whether the nitric oxide donor, S-nitrosoglutathione, may exert different effects on diabetic and non-diabetic myocardial ischaemia/reperfusion (MI/R) injury. Diabetic state was induced in mice by multiple intraperitoneal injections of low-dose streptozotocin (STZ). The control or diabetic mice were subjected to 30minutes ischaemia and 3 or 24hours reperfusion. At 10minutes before reperfusion, diabetic and non-diabetic mice were received an intraperitoneal injection of S-nitrosoglutathione (GSNO, a nitric oxide donor, 1μmol/kg). GSNO attenuated MI/R injury in non-diabetic mice, as measured by improved cardiac function, reduced infarct size and decreased cardiomyocyte apoptosis. In contrast, GSNO failed to attenuate but, rather, aggravated the MI/R injury in diabetic mice. Mechanically, the diabetic heart exhibited an increased nitrative/oxidative stress level, as measured by peroxynitrite formation, compared with non-diabetic mice. Co-administration of GSNO with EUK134 (a peroxynitrite scavenger) or MnTE-2-PyP5 (a superoxide dismutase mimetic) or Apocynin (a NADPH oxidase inhibitor) 10minutes before reperfusion significantly decreased the MI/R-induced peroxynitrite formation and the MI/R injury. Collectively, the present study for the first time demonstrated that diabetes may cause superoxide overproduction, increase NO inactivation and peroxynitrite formation, and thus convert GSNO from a cardioprotective molecule to a cardiotoxic molecule.

The effect of NO donor sodium nitroprusside (SNP) on salt resistance of 4-week-old Arabidopsis thaliana L. wild-type Columbia-0 (Col-0) plants and jin1 mutants defective in the jasmonate signaling have been investigated. As affected by 0.5 mM, SNP salt resistance of wild-type plants rose, which was exhibited in a smaller growth inhibition and preserving the pool of photosynthetic pigments after salt stress (200 mM NaCl). The positive effect of SNP leveled by treatment of plant with NO scavenger: 0.5 mM PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide). However, SNP treatment had no significant effect on the salt tolerance of jin1 genotype plants. In the case of wild-type plants but not jin1 mutants, treatment by NO donor increased activity of antioxidant enzymes—superoxide dismutase, guaiacol peroxidase, and catalase—in the leaves, which was especially noticeable in salt stress conditions. In wild-type plants treated by NO donor, proline content in the leaves after salt stress was lower and sugar content was higher than that in the untreated ones. In jin1 mutants, NO donor treatment resulted in a significant increase in proline content in the leaves under salt stress, without changing sugar content. A conclusion was made on the participation of transcript factor JIN1/MYC2 in NO-dependent induction of some plant defense responses to salt stress.

The nitric oxide donor, isosorbide dinitrate, induces a cephalic cutaneous hypersensitivity, associated with sensitization of the medullary dorsal horn

This study evaluated the effects of nitric oxide donor’s treatment on the pregnancy rate and uterine blood flow in patients with unexplained infertility undergoing clomiphene citrate stimulation and intrauterine insemination. A total of 120 patients were randomly allocated to a control group who received 100 mg clomiphene citrate daily from day 5 to 9 of cycle plus placebo vaginal tablets, and a study group received clomiphene citrate plus isosorbide mononitrate 10 mg vaginal tablets. Vaginal ultrasound was done before treatment and every other day starting from day 12 of cycle to count mature follicles and ovulation was triggered by IM injection of 10 000 IU hCG when one follicle measured 18 ≥ mm followed by intrauterine insemination after 36 h. The endometrial thickness, uterine arteries resistance and pulsation indices, and endometrial vascular flow and vascular flow indices were measured before treatment and at day of hCG injection. Results were analyzed after one cycle treatment using the Mean ± SD, the Student t test and the Fisher Exact test. Significant result was considered at p values <0.05. The study group had significant higher pregnancy rate/cycle, higher endometrial and lower uterine artery blood flow indices (p < 0.05).

Nitric oxide (NO) has been used as stimulating of the germination process for many species. However, there are few studies evaluating the effect of nitric oxide donor in the regulation of seed germination under salt stress, especially for native forest species. The objective was to evaluate the effects of SNP, an NO donor substance, on germination of Senna macranthera seeds under salt stress. The seeds were germinated at different osmotic potentials induced by NaCl solution (0.0, -0.1, -0.2, -0.3, -0.4 and -0.5 MPa). To evaluate the effect of the SNP, potentials -0.3 and -0,4 MPa were selected, applying SNP at different concentrations: 100, 200, 300 and 400 µM. Germination tests were conducted at 25 °C, with photoperiod of 8 hours. Percentage of radicle protrusion, radicle protrusion speed index, percentage of normal seedlings, shoots and roots length and dry matter were evaluated. Salt stress with NaCl is harmful to germination of S. macranthera seeds. SNP has the potential to recover germination under salt stress, especially in the concentration of 100 µM.

To study the effect of nitric oxide donor drug therapy on abnormal uterine artery Doppler indices after treatment in cases of recurrent unexplained implantation failure and to study the clinical pregnancy rate following treatment. 100 woman with history of failed ICSI trails, all women were subjected to pretreatment basic TVUS scan at mid-luteal period to exclude any anatomical gynecological problems, uterine artery blood flow was measured bilaterally and only abnormal uterine artery Doppler indices cases (PI = or >3 with or RI = or >0.9) were included into the study, after informed consent all patients took nitric-oxide donor (Nitromack retrard 2.5 mg capsule two cap/day) follow up of patients after 3 month by ultrasound scan mainly to study the changes on uterine artery Doppler indices and follow up ongoing ICSI cycles for detect clinical pregnancy rate. 100 woman of unexplained recurrent implantation failure following ICSI cycles, with their age (21–43 y), with normal anatomy, with abnormal Doppler study of uterine Doppler indices at mid-luteal phase before treatment shows PIwith mean (3.2 ± 0.72) and RI with mean (0.93 ± 0.03),with follow up after treatment for 3 month the Doppler indices shows improvement in about 69% of cases, with PI with mean (2.6 ± 0.92) and RI (resistive index) with mean (0.87 ± 0.07) and no improvement in 31 % of cases, about 80% of cases underwent ICSI trail after treatment with clinical pregnancy rate 59% of cases and about 20 % not went to ICSI cycle with spontaneous clinical pregnancy rate 12% within in this group. In cases of unexplained implantation failure or unexplained ICSI failure, altered uterine artery blood flow Doppler indices could be one of hidden causes, nitric oxide donor could have a beneficial role as potent vasodilator to improve uterine artery Doppler indices, in cases of unexplained ICSI failure using nitric oxide donor or unexplained cases not underwent ICSI clinical pregnancy rate could be improved after using nitric oxide donor drugs.

BackgroundMedullary thyroid cancer (MTC) is a C-cell neoplasm. Surgery remains its main treatment. Promising therapies based on tyrosine kinase inhibitors demand careful patient selection. We previously observed that two non-steroidal anti-inflammatory drugs (NSAID), indomethacin, celecoxib, and nitric oxide (NO) prevented tumor growth in a model of human MTC cell line (TT) in nude mice.MethodsIn the present study, we tested the NO donor: glyceryl trinitrate (GTN), at pharmacological dose, alone and in combination with each of the two NSAIDs on TT cells. We also assessed the anti-proliferative potential of NO-indomethacin, an indomethacin molecule chemically conjugated with a NO moiety (NCX 530, Nicox SA) on TT cells and indomethacin/GTN association in rMTC 6–23 cells. The anti-tumoral action of the combined sc. injections of GTN with oral delivery of indomethacin was also studied on subcutaneous TT tumors in nude mice. Apoptosis mechanisms were assessed by expression of caspase-3, TAp73α, TAp73α inhibition by siRNA or Annexin V externalisation.ResultsThe two NSAIDs and GTN reduced mitotic activity in TT cells versus control (cell number and PCNA protein expression). The combined treatments amplified the anti-tumor effect of single agents in the two tested cell lines and promoted cell death. Moreover, indomethacin/GTN association stopped the growth of established TT tumors in nude mice. We observed a significant cleavage of full length PARP, a caspase-3 substrate. The cell death appearance was correlated with a two-fold increase in TAp73α expression, with inhibition of apoptosis after TAp73α siRNA addition, demonstrating its crucial role in apoptosis.ConclusionAssociation of NO with NSAID exhibited amplified anti-tumoral effects on in vitro and in vivo MTC models by inducing p73-dependent apoptotic cell death.

Type 2 diabetic men commonly experience erectile dysfunction for whichphosphodiesterase-5 (PDE5) inhibitors like sildenafil (Viagra) are often recommended. Bypreventing degradation of cyclic guanosine monophosphate (cGMP) in vascular smooth muscle,these inhibitors also enhance arterial vasorelaxant effects of nitric oxide donors (whichstimulate cGMP synthesis). In the present work, we confirmed this enhancing effect afterco-administration of sildenafil with nitroprusside to freshly-isolated rat tail arterialtissues. However, in the same tissues we also observed that sildenafil does not enhancebut rather attenuates vasorelaxant effects of three commonly-used antidiabetic drugs, i.e.the biguanide metformin and the thiazolidinediones pioglitazone and rosiglitazone. Indeed,sildenafil completely blocked vasorelaxant effects of low concentrations of these drugs.In addition, we found that this same novel anti-vasorelaxant interaction of sildenafilwith these agents was abolished by either 1) omitting extracellular glucose or 2)inhibiting specific smooth muscle glycolytic pathways; pathways known to preferentiallyutilize extracellular glucose to fuel certain adenosine triphosphate (ATP)-dependent iontransporters: e.g. ATP-sensitive K channels, sarcoplasmic reticulum Ca-ATPase, plasmamembrane Ca-ATPase and Na/K-ATPase. Accordingly, we suspect that altered activity of oneor more of these ion transporters mediates the observed attenuating (anti-vasorelaxant)interaction of sildenafil with the antidiabetic drugs. The present results are relevantbecause hypertension is so common and difficult to control in Type 2 diabetes. The presentdata suggest that sildenafil might interfere with the known antihypertensive potential ofmetformin and the thiazolidinediones. However, they do not suggest that it will interactwith them to cause life-threatening episodes of severe hypotension, as can occur when itis co-administered with nitrates.