Gout is one of the most common inflammatory arthropathies, characterized by the deposition of monosodium urate crystals in joint tissues [1, 2]. The key factor in the development of gout is hyperuricemia (elevated blood uric acid levels and xanthine oxidase activity) [3]. Uric acid is a potent extracellular antioxidant; however, at high intracellular concentrations, it acquires pro-oxidant properties, contributing to oxidative stress [4–6]. Thus, excess uric acid promotes oxidative stress, inflammation, and endothelial dysfunction [3]. Endothelial NO synthase (eNOS, NOS3), which produces nitric oxide (NO), plays an important role in vascular function. NO regulates vascular tone, inhibits platelet aggregation, and suppresses leukocyte adhesion [3]. When uric acid levels rise, eNOS activity decreases—manifested by reduced eNOS binding to calmodulin and decreased NO production [5]. Therefore, in gout, impaired eNOS function may exacerbate vascular and anti-inflammatory mechanisms.

Lipopolysaccharide-binding protein (LBP) is an acute-phase protein involved in immune responses triggered by bacterial cell wall components [6]. Disruption of the intestinal barrier and alterations in the gut microbiota may increase the risk of bacterial translocation and chronic inflammation, which is thought to be associated with elevated LBP [6, 7]. Elevated plasma LBP levels are linked to chronic inflammation and metabolic syndrome [6, 7]. Patients with metabolic syndrome, pre-existing cardiovascular disease, and obesity exhibit higher LBP levels [6].

Bactericidal/permeability-increasing protein (BPI) is a component of innate immunity with antibacterial and anti-inflammatory activity. BPI (55 kDa) can eliminate gram-negative bacteria and neutralize endotoxin (LPS) [8]. In experimental models, BPI has been shown to significantly reduce the inflammatory response in arthritis: for example, in mice with monosodium urate-induced arthritis, BPI administration reduced leukocyte infiltration and cytokine production [2]. Based on these data, we hypothesized that BPI (or the BPI family) may exert a protective effect in gout by reducing LPS translocation and partially counteracting the oxidative stress associated with xanthine oxidase and uric acid. In this context, the role of endogenous anti-inflammatory proteins such as BPI in modulating endothelial function in gout remains unexplored.

To study the relationship between BPI levels and the concentration of endothelial NO synthase (NOS3) in patients with gout.

The study included 41 patients with a confirmed diagnosis of gout according to the EULAR/ACR 2015 classification criteria, and 33 control subjects matched for age and sex. Age distribution did not follow a normal pattern; therefore, quantitative data are presented as medians and interquartile ranges: Me [Q1; Q3]. The median age of patients was 60 years [52.5; 65.2], and that of the control group was 52 years [45; 59] (p = 0.41).

Exclusion criteria were: age < 18 years; acute infectious, autoimmune, or oncological diseases; chronic kidney disease stages 4–5; acute and chronic gastrointestinal diseases; use of antibiotics or probiotics within one month prior to the study; pregnancy; refusal to participate; and hereditary gout. All participants provided written informed consent. The study was conducted in accordance with the 1964 Helsinki Declaration of the World Medical Association, which establishes ethical principles for medical research involving human subjects, including the collection and use of data and biological material. The study protocol was approved by the Biomedical Ethics Committee of Vernadsky Crimean Federal University (Meeting No. 6, dated 04.09.2025).

Figure 1. Patient flow chart (STROBE)

The flowchart illustrates the participant flow (Fig. 1). A total of 74 individuals were included in the analysis: 41 patients with confirmed gout (according to EULAR/ACR 2015 criteria) and 33 control subjects. All enrolled patients completed the study and were included in the final analysis. No losses to follow-up occurred.

Additionally, the study group was subdivided as follows:

Biomarker levels (LBP, BPI, and NOS3) were compared to exclude the influence of these diseases on biomarker concentrations.

Venous blood was collected from patients in the rheumatology department of the N.A. Semashko Republican Clinical Hospital and centrifuged (3000 rpm, 10 min). Plasma concentrations of LBP, NOS3 (eNOS), and BPI were measured by solid-phase enzyme-linked immunosorbent assay (ELISA) (Cloud-Clone Corp., China) using a Synergy H1 microplate reader (BioTek). Quantitative data are presented as medians and interquartile ranges: Me [Q1; Q3], where Me is the median, Q1 is the first quartile (25th percentile), and Q3 is the third quartile (75th percentile). Linear regression was used to assess the relationship between BPI and NOS3 levels, and the Pearson correlation coefficient (r), coefficient of determination (R²), and significance level (p) were calculated. Differences between groups in BPI and NOS3 levels were analyzed using the Mann–Whitney U test. IBM SPSS Statistics 26 software was used.

The minimum sample size was calculated prior to the study. Based on an expected Pearson correlation coefficient of r = 0.5, with a significance level of α = 0.05 (two-sided) and power of 80%, the minimum required sample size was 29 individuals per group. The actual sample size (n = 41 in the gout patient group and n = 33 in the control group) exceeds the calculated minimum and provides sufficient statistical power to detect a correlation of this magnitude.

LBP and high-sensitivity CRP levels were significantly higher in gout patients compared to controls (p < 0.001), indicating systemic activation of the inflammatory response, possibly related to enhanced translocation of intestinal endotoxin (lipopolysaccharide of gram-negative flora) into lymph and portal blood. In the main group of gout patients, BPI levels were significantly lower (p < 0.001), suggesting an imbalance in endotoxin-limiting systems under chronic oxidative stress conditions due to high uric acid concentrations; these factors likely "deplete" BPI reserves. Males predominated in both the main (78%) and control (73%) groups, with no significant difference (p = 0.62).

ParameterGout Patients (n=41)Control Group (n=33)pSex   Male, n (%)32 (78%)24 (73%)0.620Female, n (%)9 (22%)9 (27%) Age, years60.5 [52.5; 65.2]52 [45; 59]0.410Disease duration, years1 [1; 4.25]––Obesity, n (%)15 (42%)––T2DM, n (%)16 (46%)––AH, n (%)25 (61%)––BMI, kg/m²26.1 [24.3; 30.2]21.1 [19.1; 24.3]0.006LBP, ng/mL14.25 [10.9; 18.5]1.14 [0.43; 2.82]<0.001*hsCRP, mg/mL3.7 [3.05; 4.46]0.17 [0.09; 0.33]<0.001*NOS3, ng/mL0.28 [0; 3.125]0.046 [0.01; 0.1]0.924BPI, pg/mL68.2 [45.2; 116.2]395.0 [145.0; 895.0]<0.001*

Note: * — significant at p < 0.05; T2DM — type 2 diabetes mellitus; AH — arterial hypertension; BMI — body mass index; LBP — lipopolysaccharide-binding protein; hsCRP — high-sensitivity C-reactive protein; NOS3 — endothelial nitric oxide synthase 3; BPI — Bactericidal/Permeability-Increasing Protein.

Regression analysis revealed a positive correlation between BPI and NOS3 levels. The observed relationship is described by the equation:

YₑNOS = 0.148 + 0.022 × X_BPI

For each 1 pg/mL increase in BPI concentration, NOS3 is expected to increase by approximately 0.022 ng/mL. The correlation coefficient was r = 0.605, which, according to the Chaddock scale, indicates a substantial strength of association. The significance level was p < 0.001, indicating statistical significance. The coefficient of determination R² = 0.366 indicates that approximately 36.6% of the variance in NOS3 levels is explained by changes in BPI concentration. It should be noted that plasma NOS3 concentrations did not show statistically significant differences between groups (median 0.28 [0; 3.125] ng/mL in gout patients vs. 0.046 [0.01; 0.1] ng/mL in the control group; p = 0.924); that is, absolute NOS3 levels did not differ between the compared groups. However, the identified significant positive correlation between BPI and NOS3 within the gout patient group (r = 0.605; 95% CI: 0.365–0.769; p < 0.001) indicates a relationship between these markers at the individual level in the context of chronic inflammation, regardless of absolute concentrations.

When comparing biomarker levels (LBP, BPI, and NOS3) between groups with and without AH and T2DM, the following results were obtained (Table 2). For most compared pairs, no significant statistical differences were found (p > 0.05). For LBP, a tendency toward higher levels was noted in patients with AH compared to patients without AH (medians 14.35 and 11.45, respectively; p = 0.066), although the significance level did not reach the threshold. For other biomarkers, differences between groups by AH and T2DM were absent (p values ranged from 0.15 to 0.53). Thus, in this sample, no associations were found between the studied biomarkers and the presence of AH or T2DM.

BiomarkerGroup 0 (n=17)Group 1 (n=16)pLBP12.4014.180.377BPI84.8064.600.149NOS31.400.03350.244

BiomarkerGroup 0 (n=8)Group 1 (n=25)pLBP11.4514.350.066BPI75.6080.200.531NOS31.550.500.443

Note: * — statistically significant at p < 0.05.

This study is the first to identify a significant positive correlation between BPI and NOS3 levels in patients with gout, which is of particular clinical interest. This association reflects a complex interaction between innate immune systems and endothelial function in the pathogenesis of gout. BPI, as a key component of the antibacterial response, and NOS3, which regulates vascular tone and endothelial function, form an integrative axis reflecting the degree of vascular inflammation and endothelial dysfunction. The clinical significance of the identified BPI–NOS3 correlation lies in the prospects for using these biomarkers for comprehensive assessment of vascular complications in gout. Combined determination of BPI and NOS3 may serve as a basis for individualizing therapeutic approaches, allowing the identification of patients at high risk of developing cardiovascular complications. Moreover, this biomarker panel opens opportunities for monitoring early vascular damage, which is critically important for timely therapy adjustment and prevention of atherosclerotic progression.

The protective vascular effect of BPI may be mediated through its anti-inflammatory action, including binding and inactivation of lipopolysaccharide (LPS) in the blood, as well as antagonistic effects on lipopolysaccharide-binding protein (LBP) [9–11]. Literature data indicate that BPI can reduce IL-8 secretion by epithelial cells infected with E. coli; furthermore, the anti-inflammatory effect of BPI may be realized through inactivation of the intracellular inflammatory cascade upon interaction of the endotoxin + LBP complex with CD14/TLR4 receptors on monocyte-macrophage lineage cells [12]. It should also be considered that BPI belongs to the BPI-like protein family, which participates in lipid metabolism and the pathogenesis of certain cardiovascular diseases [9]. The specific isomer BPIFB4 (bactericidal/permeability-increasing fold-containing family B member 4), particularly the longevity-associated variant (LAV-BPIFB4), exerts significant effects on the endothelium and eNOS activity [13]. LAV-BPIFB4 is phosphorylated at Ser75, after which it binds to 14-3-3 and Hsp90 proteins, forming a BPIFB4–HSP90–14-3-3 complex that promotes eNOS activation (specifically through phosphorylation of eNOS at Ser1177) [13]. This complex induces phosphorylation of eNOS at Ser1177, leading to nitric oxide (NO) production in endothelial cells [13]. Classical NOS3 activation requires extracellular Ca²⁺; however, alternative pathways also exist: for example, BPI may promote vasorelaxation mediated by endothelial-derived hyperpolarizing factor (EDHF) [14]. Additionally, BPI acts as an anti-angiogenic factor, inducing apoptosis in endothelial cells and binding to vascular endothelial growth factor (VEGF) [15]. Data also indicate that BPI influences compensatory mechanisms triggered by endotoxemia and oxidative stress: BPI attenuates LPS-induced changes in vascular reactivity by inhibiting iNOS expression, leading to restoration of vascular sensitivity to vasoconstrictors [16].

Studies demonstrate a close relationship between alterations in gut microbiota, endotoxemia, and the development of systemic inflammation in gout [17]. Intestinal dysbiosis leads to increased intestinal barrier permeability and translocation of lipopolysaccharide (LPS) into the systemic circulation, initiating a cascade of inflammatory responses through activation of Toll-like receptors, particularly TLR4 [17, 18]. Lipopolysaccharide-binding protein (LBP) serves as a key mediator of this process, facilitating endotoxin binding and presentation to the CD14/TLR4/MD2 complex on immune cells, which triggers a pro-inflammatory signaling cascade [17, 18]. Endotoxemia promotes oxidative stress, endothelial dysfunction, and activation of pro-inflammatory cytokines, exacerbating metabolic disturbances and vascular complications in gout [17, 18]. In this context, BPI and LBP represent integrative markers reflecting the state of the "gut–endotoxemia–systemic inflammation" axis, determining their high clinical value for assessing disease severity and predicting vascular complications [17, 18].

It can be hypothesized that the elevated LBP levels observed in gout patients represent a response to bacterial component translocation, while the decreased BPI concentration may result from desynchronization of the LBP/BPI ratio. This hypothesis is consistent with data showing significantly higher affinity of BPI for LPS compared to LBP (Kd BPI ≈ 2.6 nM vs. Kd LBP ≈ 58 nM for lipid A). Consequently, the reduction in BPI levels may potentially be related to its "consumption" for inactivation of pro-inflammatory LPS properties, as well as to the general oxidative stress characteristic of hyperuricemia [17, 18].

By neutralizing endotoxins [19, 20], BPI may indirectly protect endothelial cells from excessive activation and LPS-induced damage [21]. BPI has been shown to suppress inducible NO synthase (iNOS) expression, contributing to restoration of vascular sensitivity to vasoconstrictors and normalization of vascular tone. Furthermore, BPI inhibits release of pro-inflammatory cytokines (TNF-α, IL-1, IL-6) in response to LPS, which also reduces endothelial dysfunction [19, 21]. Thus, patients with a higher compensatory response in the form of BPI production likely maintain better endothelial function, which may be reflected by our regression model. Comparison of the control and study groups shows that plasma BPI concentration in gout patients is reduced relative to the control group. It can be assumed that in this case, BPI is "consumed" in processes related to LPS inhibition and anti-inflammatory effects, consistent with the competitive relationship between BPI and LBP, where BPI has higher affinity for LPS [20, 22].

We found that in patients with gout, there is a statistically significant positive correlation between BPI (bactericidal/permeability-increasing protein) concentration and endothelial NO synthase (NOS3/eNOS) levels. This indicates a close relationship between innate immunity factors and markers of vascular function in gout. The obtained data suggest that BPI may have a protective effect on the endothelium under conditions of chronic inflammation and oxidative stress caused by hyperuricemia, consistent with BPI's known ability to neutralize LPS-induced endothelial cell damage and suppress inducible NO synthase (iNOS) expression. However, the mechanisms underlying the identified correlation between BPI and eNOS require further investigation. The identified association highlights the potential utility of a combined biomarker panel (BPI + NOS3) for monitoring vascular complications and individualizing therapy in gout patients. Thus, BPI may be considered a potential vasoprotective marker reflecting compensatory protective mechanisms in gout patients, opening new avenues for further research in the prevention and treatment of vascular complications in gout.