An Update on Arginase Inhibitors and Inhibitory Assays

Arginase, which converts arginine into ornithine and urea, is a promising therapeutic target. Arginase is involved in cardiovascular diseases, parasitic infections and, through a critical role in immunity, in some cancers. There is a need to develop effective arginase inhibitors and therefore efforts to identify and optimize new inhibitors are increasing. Several methods of evaluating arginase activity are available, but few directly measure the product. Radiometric assays need to separate urea and dying reactions require acidic conditions and sometimes heating. Hence, there are a variety of different approaches available, and each approach has its own limits and benefits. In this review, we provide an update on arginase inhibitors, followed by a discussion on available arginase assays and alternative methods, with a focus on the intrinsic biases and parameters that are likely to impact results.

The Effect of Neutrophil-Derived Products on the Function of Leukocytes Obtained after Titanium Implantation in the Ovine Model

Titanium (Ti) is currently the most common biomaterial used for orthopedic implants; however, these implants may cause deleterious immune response. To investigate the possible mechanisms involved in excessive inflammation, we assessed the activity of neutrophils and monocyte-derived macrophages (MDMs) during the insertion of the Ti implant in a sheep model. The study was conducted on 12 sheep, 4 of which were control animals and 8 were in the experimental group with inserted Ti implant. Neutrophil secretory response was estimated at two time points T0 before surgery and T1 1 h after implantation and was based on the release of enzymes from neutrophil granules and reactive oxygen and nitrogen species (RONS) generation. MDM function was evaluated 5 months after implantation, on the basis of RONS generation arginase activity and morphological changes. Moreover, the influence of some autologous neutrophil derived products, namely, antimicrobial neutrophil extract (ANE) and neutrophil degranulation products (DGP) on leukocytes was estimated. Our study revealed that Ti implant insertion did not cause any adverse effects up to 5 months after surgical procedure. Stimulation of neutrophil cultures with ANE decreased the enzyme release as well as superoxide generation. Treatment of MDM with ANE diminished superoxide and NO generation and increased arginase activity. On the other hand, MDM stimulated with DGP showed elevated superoxide and NO generation as well as decreased arginase activity. To summarize, ANE exerted an anti-inflammatory and pro-resolving effect on studied leukocytes, whereas DGP acted as pro-inflammatory.

Urea Excretion and Arginase Activity as New Biomarkers for Nitrite Stress in Freshwater Aquatic Animals

Background: In recent years, the concern has been growing on increasing aquatic nitrite levels due to anthropogenic activities. Crustaceans and fish easily uptake nitrite via the chloride uptake system of gills. High nitrite body levels may interfere with nitric oxide (NO) production by nitric oxide synthase (NOS). The arginase, which catalyzes arginine conversion to ornithine and urea, is central to NO homeostasis. In vivo, changes in the arginase activity alter urea body levels and urea excretion and modulate NOS by altering arginine availability for NO synthesis. Excess arginase activity may uncouple NOS and induce oxidative stress. Methods: We tested muscle arginase activity and urea excretion in two fish species, zebrafish and convict cichlid, and the crustacean Yamato shrimp, under sub-lethal nitrite stress. Results: Exposure to nitrite (2 mM in the fish, 1 mM in the shrimp) significantly increased blood nitrite concentration in all species. Concomitantly, nitrite stress significantly increased arginase activity, urea excretion, and urea levels in the blood. In Yamato shrimp, urea levels also increased in muscle. Conclusion: Our results agree with the hypothesis that nitrite stress affects NO homeostasis by arginase stimulation and urea excretion. These parameters might function as markers of sub-lethal nitrite stress in freshwater fish and crustaceans.

CHANGES IN THE ACTIVITY OF INDUCIBLE NO-SYNTHASE AND ARGINASE, THEIR RELATIONSHIP WITH THE LEVEL OF PRO- AND ANTI-INFLAMMATORY CYTOKINES IN CERVICAL MUCUS IN PREGNANT WOMEN, WHO UNDERWENT IVF AND ARE AT RISK OF PRETERM CHILDBIRTH

Introduction. Nitric oxide (NO) produces a wide range of bioregulatory effects. Imbalance in the ratio of the activity of inducible NO-synthase and arginase in favor of iNOS can lead to a pro-inflammatory reaction. At the risk of preterm childbirth, the content of proinflammatory cytokines increases both at the local and systemic levels. The aim of this work is to study the relationship between changes in the content of pro- and anti-inflammatory cytokines and the activity of inducible NO-synthase and arginase in the cervical mucus of women with in vitro fertilization (IVF), who are at risk of preterm childbirth. Materials and methods. The test group (TG) included 37 women, who underwent IVF and presented prognostic signs, indicating a high risk of preterm birth in the future. The control group (CP) consisted of 20 healthy pregnant women, who did not undergo assisted reproductive technologies. We determined the indicators of inducible NO-synthase activity and total arginase activity, as well as the level of pro- and anti-inflammatory cytokines in the mucus of the cervical canal of the participants at their 28-34 weeks of pregnancy. Results and discussion. The activity of iNOS in TG significantly increased (in 2.57 times) compared to healthy pregnant women (CP), while the activity of arginase, on the contrary, decreased significantly (in 1.91 times). There was a significant increase in the levels of pro-inflammatory cytokines INF-γ and TNF-α in the TG women, while they demonstrated the significantly reduced concentration of anti-inflammatory cytokine IL-10. In cervical mucus taken from TG women, positive correlations were found between an increase in the concentration of the proinflammatory cytokine INF-γ and an increase in iNOS activity, between a decrease in IL-10 levels and arginase activity, and a negative relationship between an increase in iNOS activity and a decrease in arginase activity. Conclusions. The detected cytokine imbalance in pregnant women, who underwent in vitro fertilization and were at risk of preterm childbirth, together with a impaired ratio of iNOS and arginase, indicates an inappropriate level of immunosuppression. These immune changes, by activating cellular responses of maternal immunity, can contribute to preterme childbirth.

Elevated levels of arginase activity are related to inflammation in patients with COPD exacerbation

Introduction Within the pathogenesis of the chronic obstructive pulmonary disease (COPD) there are interactions between different inflammatory mediators that are enhanced during an exacerbation. Arginase is present in bronchial epithelial cells, endothelial, fibroblasts and alveolar macrophages, which make it a probable key enzyme in the regulation of inflammation and remodelling. We aimed to find a potential relationship between arginase activity, inflammatory mediators in COPD patients in stable phase and during exacerbations. Methods We performed a prospective, observational study of cases and controls, with 4 study groups (healthy controls, stable COPD, COPD during an exacerbation and COPD 3 months after exacerbation). We measured arginase, inflammation markers (IL-6, IL-8, TNF-∝, IFN-γ and C reactive protein), and mediators of immunity: neutrophils, monocytes, total TCD3 + lymphocytes (CD3ζ), CD4 + T cells, CD8 + T cells, NK cells. Results A total of 49 subjects were recruited, average age of 69.73 years (59.18% male). Arginase activity is elevated during an exacerbation of COPD, and this rise is related to an increase in IL-6 production. The levels of IL-6 and IL-8 remained elevated in patients with COPD at 3 months after hospital exacerbation. We did not find a clear relationship between arginase activity, immunity or with the degree of obstruction in COPD patients. Conclusions Arginase activity is elevated during an exacerbation of COPD, and it could be related to an increase in the production of IL-6. Levels of IL-6, IL-8, and arginase activity remain elevated in patients with COPD at 3 months after hospital exacerbation. Arginase activity could contribute to the development of COPD.

ANALYSIS OF THE EFFICIENCY OF COMPLEX TREATMENT OF PATIENTS WITH GENERALIZED PERIODONTITIS ACCORDING TO THE CHANGES IN THE ACTIVITY OF BLOOD SERUM ENZYMES

Introduction. The mechanisms of generalized periodontitis (GP) development and methods of its treatment remain obscure, so it is important to study changes in the activity of enzymes responsible for maintaining homeostasis, as well as the inclusion of medicines that regulate them into the complex treatment. Aim of research is to study the influence of comprehensive treatment in patients with GP on the dynamics of the activity of indicator blood serum enzymes in different observation periods. Methods. There were examined 29 people with a healthy periodontium and 143 patients with GP aged 19-45 years, somatically healthy, before, immediately after the treatment, after 6 and 12 months. Patients were divided into subgroups with chronic (A) and acute (B) course: IA and IB – the initial degree; IIA and IIB – the I degree; IIIA and IIIB – the II degree. The activity of lactate-dehydrogenase (LDG), arginase and sorbitol dehydrogenase (SDG) in blood serum has been studied. In addition to the basic periodontal therapy, the microalgal medicine Spirulina platensis was prescribed endogenously, and the paste with the same amount of spirulina powder and enterosorbent and 0.05% chlorhexidine bigluconate solution was exogenously prescribed. Results. In patients with IA and IB subgroups, LDG activity has increased in 1.37- and 1.48-times (p1 <0.01; p1 = 0.001). Under the influence of treatment, it has decreased in all patients, especially after 6 months – in 1.33- and 1.50-times (p2<0.001), but after 12 months it has been increased more (p2> 0.05; p2 <0.05). Arginase activity in IA and IB subgroups has reduced in 1.23- and 1.31-times (p1<0.05; p1=0.005). Due to the therapy, it has increased immediately, after 6 and 12 months, respectively in 1.23- and 1.26-times; in 1.21- and 1.25-times, and in 1.20-1.23-times (p2<0.05; p2> 0.05; p1> 0.05). In subgroups IA and IB, SDG activity has increased in 1.15- and 1.17-times (p1> 0.05), and after the treatment it has decreased immediately in 1.14- and 1.16-times (p2 <0.05); later it increased, but differed slightly from the norm (p1> 0.05). LDG activity in subgroups IIA and IIB has increased in 1.38- and 1.54-times (p1 <0.01; p1=0.001). After the treatment in subgroup IIA, it has decreased in 1.21-times immediately and after 6 months (p2<0.005), and a year later it has increased (p2>0.05; p1>0.05); in subgroup IIB it has decreased in 1.33-, 1.39- and 1.24-times (p2<0.05; p2<0.01; p2 <0.05 and p1> 0.05). In subgroups IIA and IIB, arginase activity has reduced in 1.32-times (p1=0.001). Immediately after the treatment in subgroup IIA, it has increased in 1.21-times (p2=0.005), and subsequently decreased (p2> 0.05). In the IIB subgroup, its increasing was 1.31-, 1.27- and 1.25-times (p2 <0.05), and the difference with the norm was insignificant. SDG activity in subgroups IIA and IIB has increased in 1.18- and 1.24-times (p1 <0.05; p1=0.01). After the treatment, it has decreased at all terms in both subgroups similarly: 1.13- and 1.16-times; 1.17- and 1.17-times; 1.12- and 1.10-times (p2 <0.05; p2 <0.05; p2> 0.05) and it differed slightly from normal one. The largest increase in LDG activity was found in subgroups IIIA and IIIB – 1.45- and 1.62-times (p1≤0.001). As a result of therapy immediately, after 6 and 12 months it has decreased in 1.18- and 1.20-times; 1.26- and 1.23-times; 1.13- and 1.15-times (p2 <0.05; p2 <0.05; p2> 0.05; p1> 0.05). In subgroups IIIA and IIIB, arginase activity has reduced in 1.32- and 1.37-times (p1≤0.005). Treatment has increased the indices in group IIIA in 1.22-, 1.22- and 1.18-times (p2<0.05), and in group IIIB it immediately increased in 1.25-times and then decreased (p2 <0.05; p2> 0.05). The activity of SDG in IIIA and IIIB subgroups has increased in 1.31-times (p1=0.001). Under the influence of therapy in subgroup IIIA, it has decreased immediately, after 6 and 12 months in 1.17-, 1.22- and 1.13-times (p2 <0.05; p1> 0.05), and in subgroup IIIB it initially decreased, but after a year it has increased (p2>0.05) and the difference with healthy people became significant. The altered indices of activity of enzymes studied in patients with GP did not exceed the reference values, but showed a violation of the enzyme system, which was regulated by the treatment. Prior to therapy, reliable (p<0.05-0.005) strong correlations were found between these parameters: LDG with SDG (r> 0.71) and arginase with SDG (r> -0.90). After the treatment, they were not found, six months later one correlation has restored, and a year later – both have restored, which indicates the necessity for the maintenance of endogenous therapy after six months. Conclusion. GP is accompanied by significant (p1<0.05-0.001) changes in the enzymes activity in the blood: in LDG and SDG, it is increased, and in arginase – it is reduced. Comprehensive treatment has regulated these disorders, especially immediately and after 6 months (p2<0.05-0.001). The activity of LDG and SDG of the initial and the I degree immediately and after 6 months and arginase at the initial degree after 6 and 12 months became the closest to norm. In the GP of the II degree, the data of healthy people were not achieved, but the difference with them was insignificant (p1> 0.05).

Altered amino acid profile in patients with SARS-CoV-2 infection

Low plasma arginine bioavailability has been implicated in endothelial dysfunction and immune dysregulation. The role of arginine in COVID-19 is unknown, but could contribute to cellular damage if low. Our objective was to determine arginine bioavailability in adults and children with COVID-19 vs. healthy controls. We hypothesized that arginine bioavailability would be low in patients with COVID-19 and multisystem inflammatory syndrome in children (MIS-C). We conducted a prospective observational study of three patient cohorts; arginine bioavailability was determined in asymptomatic healthy controls, adults hospitalized with COVID-19, and hospitalized children/adolescents <21 y old with COVID-19, MIS-C, or asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection identified on admission screen. Mean patient plasma amino acids were compared to controls using the Student’s t test. Arginine-to-ornithine ratio, a biomarker of arginase activity, and global arginine bioavailability ratio (GABR, arginine/[ornithine+citrulline]) were assessed in all three groups. A total of 80 patients were included (28 controls, 32 adults with COVID-19, and 20 pediatric patients with COVID-19/MIS-C). Mean plasma arginine and arginine bioavailability ratios were lower among adult and pediatric patients with COVID-19/MIS-C compared to controls. There was no difference between arginine bioavailability in children with COVID-19 vs. MIS-C. Adults and children with COVID-19 and MIS-C in our cohort had low arginine bioavailability compared to healthy adult controls. This may contribute to immune dysregulation and endothelial dysfunction in COVID-19. Low arginine-to-ornithine ratio in patients with COVID-19 or MIS-C suggests an elevation of arginase activity. Further study is merited to explore the role of arginine dysregulation in COVID-19.