Determination of nitric oxide using fluorescence spectroscopy

Ribonucleotide reductase (RNR) is a multi-subunit enzyme responsible for catalyzing the rate-limiting step in the production of deoxyribonucleotides essential for DNA synthesis and repair. The active RNR complex is composed of multimeric R1 and R2 subunits. The RNR catalysis involves the formation of tyrosyl radicals in R2 subunits and thiyl radicals in R1 subunits. Despite the quaternary structure and cofactor diversity, all the three classes of RNR have a conserved cysteine residue at the active site which is converted into a thiyl radical that initiates the substrate turnover, suggesting that the catalytic mechanism is somewhat similar for all three classes of the RNR enzyme. Increased RNR activity has been associated with malignant transformation, cancer cell growth, and tumorigenesis. Efforts concerning the understanding of RNR inhibition in designing potent RNR inhibitors/drugs as well as developing novel approaches for antibacterial, antiviral treatments, and cancer therapeutics with improved radiosensitization have been made in clinical research. This review highlights the precise and potent roles of NO in RNR inhibition by targeting both the subunits. Under nitrosative stress, the thiols of the R1 subunits have been found to be modified by S-nitrosylation and the tyrosyl radicals of the R2 subunits have been modified by nitration. In view of the recent advances and progresses in the field of nitrosative modifications and its fundamental role in signaling with implications in health and diseases, the present article focuses on the regulations of RNR activity by S-nitrosylation of thiols (R1 subunits) and nitration of tyrosyl residues (R2 subunits) which will further help in designing new drugs and therapies.

Vitamin C may enhance nitric oxide (NO) production through stepwise reduction of dietary nitrate (NO₃) to nitrite (NO₂) to NO. The combined effect of vitamin C and NO₃ supplementation is relatively unexplored in untreated hypercholesterolemia.

We aimed to examine whether co-administration of vitamin C and nitrate for 4-weeks would improve endothelial function (primary outcome), plasma NO metabolites, oxidative stress, and blood lipids (secondary outcomes).

Subjects 50–70 years of age with low density lipoprotein (LDL) > 130 mg/dL and RHI ≤2 were enrolled in this randomized double-blind crossover study. Subjects were assigned to two 4-week supplementation treatments starting with 70 ml of concentrated beetroot juice (CBJ) with 1000 mg of vitamin C (NC) or CBJ with matched placebo (N), then switched to alternate treatment following 2-week washout. The change in reactive hyperemia index (RHI), sum of plasma NO metabolites (NO₂ + NO₃ (NOx)), oxidized LDL (oxLDL), and serum lipids were assessed at baseline and at 4-weeks of each treatment period.

Eighteen subjects (11M:7F) completed all study visits. No significant treatment differences were observed in RHI change (N: 0.21 ± 0.12; NC: 0.20 ± 0.17; p = 0.99). Secondary analysis revealed that a subgroup of NC subjects who started with a baseline RHI of <1.67 (threshold value for ED) had greater improvements in RHI compared to subjects with RHI >1.67 (1.23 ± 0.15 to 1.96 ± 0.19; n = 8 vs. 1.75 ± 0.11 to 1.43 ± 0.10; n = 8; p = 0.02). Compared to N, NC experienced a significant increase in plasma NOx (N: 94.2 ± 15.5 μmol/L; NC: 128.7 ± 29.1 μmol/L; p = 0.01). Although there was no significant difference in oxLDL change between treatments (N: −1.08 ± 9.8 U/L; NC: −6.07 ± 9.14 U/L; p = 0.19), NC elicited significant reductions in LDL (N: 2.2 ± 2; NC: −10.7 ± 23; p = 0.049), triglycerides (N: 14.6 ± 43; NC: −43.7 ± 45; p = 0.03), and no change in serum high density lipoprotein. Within treatment group comparisons showed that only NC reduced oxLDL significantly from baseline to 4 weeks (p = 0.01).

No between intervention differences were observed in RHI. RHI only improved in NC subjects with ED at intervention baseline. Four weeks of NC enriched the NO pool and promoted reduction of blood lipids and oxidative stress in subjects with hypercholesterolemia. These preliminary findings highlight a supplementation strategy that may reduce the progression of atherosclerotic disease and deserves further attention in studies using flow mediated dilation methods.

www.clinicaltrials.gov (NCT04283630).

Nitric oxide (NO) produced in the oral cavity is a powerful resource for the human body, especially when NO-syntethase production is not adequate. The role of oral microbiome in determining blood pressure levels has been linked to the active role of some bacterial species involved in the nitro-reducing process. In the present study we investigated the correlation between selected oral microbiome characteristics, nitric oxide (NO) concentration in saliva and their association with hypertension.

A case-control study including 48 (25 normotensive and 23 hypertensive subjects), subjects between 50 and 70 years old, was carried out at the dental clinic of an Italian teaching hospital. Characteristics of participants have been evaluated by means of a physical examination, and by an assisted interview. A real-time polymerase chain reaction in samples of saliva and plaque was used to detect Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tannerella forsythia, Porphyromonas gingivalis, Treponema denticola, Streptococcus mutans, Streptococcus sanguinis, Veillonella dispar and Neisseria subflava as well as total bacterial count. Nitric oxide in saliva was evaluated by the ELISA method.

Normotensive subjects, compared with hypertensive subjects, had significantly higher concentration of NO (165.77 ± 61.7 vs 57.49 ± 19.61 μmol/l; p = 0.023), and higher bacterial concentration of the supragingival plaque (4.73E+07 ± 4.33+07 vs 4.02E+07 ± 4.00+07; p = 0.024). Bacterial species, usually associated to good oral health status, such as Neisseria subflava, were significantly more present in normotensive subjects than in hypertensive ones (9090.88 ± 5481.49 vs 4791.35 ± 4349.37; p < 0.001). considering the concentration of bacteria as a biomarker of the development of hypertension.

The results support the association between hypertension, oral microbiome and salivary nitric oxide, in fact do the results allow us to establish any biomarkers (microbial or biochemical, NO) that allow early therapeutic intervention.

The effects of deoxyhemoglobin (Hb) and albumin on the NO-donor activity of the anionic tetranitrosyl iron complex with thiosulfate ligands (1) were studied for the first time. It was shown that Hb significantly stabilizes complex 1; in its presence, NO generation from the complex proceeds at a noticeably slower rate. A similar effect is observed when complex 1 is bound to albumin, in which case complex 1 decomposes 27 times slower than in the absence of albumin in the solution. The observed effects provide a prolonged action of complex 1 as NO-donor, which may enhance its potential pharmacological efficacy.

The role of nitric oxide (NO) in cancer progression has largely been studied in the context of tumor NOS2 expression. However, pro- versus anti-tumor signaling is also affected by tumor cell-macrophage interactions. While these cell-cell interactions are partly regulated by NO, the functional effects of NO flux on proinflammatory (M1) macrophages are unknown. Using a triple negative murine breast cancer model, we explored the potential role of macrophage Nos2 on 4T1 tumor progression. The effects of NO on macrophage phenotype were examined in bone marrow derived macrophages from wild type and Nos2^−/− mice following in vitro stimulation with cytokine/LPS combinations to produce low, medium, and high NO flux. Remarkably, Nos2 induction was spatially distinct, where Nos2^high cells expressed low cyclooxygenase-2 (Cox2) and vice versa. Importantly, in vitro M1 polarization with IFNγ+LPS induced high NO flux that was restricted to cells harboring depolarized mitochondria. This flux altered the magnitude and spatial extent of hypoxic gradients. Metabolic and single cell analyses demonstrated that single cell Nos2 induction limited the generation of hypoxic gradients in vitro, and Nos2-dependent and independent features may collaborate to regulate M1 functionality. It was found that Cox2 expression was important for Nos2^high cells to maintain NO tolerance. Furthermore, Nos2 and Cox2 expression in 4T1 mouse tumors was spatially orthogonal forming distinct cellular neighborhoods. In summary, the location and type of Nos2^high cells, NO flux, and the inflammatory status of other cells, such as Cox2^high cells in the tumor niche contribute to Nos2 inflammatory mechanisms that promote disease progression of 4T1 tumors.

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS^−/− mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection.