Cross-Talk Between Nitrosative Stress, Inflammation and Hypoxia-Inducible Factor in Patients with Adrenal Masses

Figures & data

Table 1 Clinical and Laboratory Characteristics of the Controls, Incidentaloma, Pheochromocytoma, and Cushing’s/Conn’s Adenoma Patients. Results are Presented as Median with 25% and 75% Percentiles

	Controls (n=50)	Incidentaloma (n=25)	Pheochromocytoma (n=25)	Cushing’s/Conn’s Adenoma (n=25)	ANOVA
Age	60 (53–65)	61 (54–65)	60 (51–64)	59 (52–65)	p=0.8913
BMI (kg/m^2)	23.1 (22.6–23.7)	29.3**** (26.4–30.1)	26.7***^~~ (24.7–29.1)	29.9**** (29.1–30.2)	p<0.0001
Glucose (mg/dL)	76 (72–80)	101****^^ (86.5–107)	84^~ (78–93)	98**** (86–110)	p<0.0001
Serum cortisol before 10 a.m. (µg/dL)	12.25 (9.05–17)	12.3 (8.25–18.9)	12.5 (10.35–18.95)	16.7 (11.65–18.55)	p=0.1369
Aldosterone (ng/dL)	12.3 (7.6–21.2)	11.8^~~ (6.7–21.5)	15.2 (11.1–21.3)	19.03** (14.3–36.9)	p=0.0025
Urine metanephrine (µg/24h)	100 (87–177)	97^^^^^ (76–177)	401**** (321–518)	122^^^^^ (89–202)	p<0.0001
Urine normetanephrine (µg/24h)	207.2 (144.5–319.3)	235^^^^^ (186–340)	567**** (421–772.5)	391**^^ (277–439)	p<0.0001
K^+ (mmol/L)	4.4 (4.1–4.7)	4.5 (4.3–4.8)	4.6 (4.3–4.8)	4.3 (3.9–4.6)	p=0.0688
Na^+ (mmol/L)	139 (138–141)	141 (139–141)	139 (137–141)	139 (137–141)	p=0.1078
CRP (mg/L)	5.5 (5.27–5.65)	5.6^^ (2.35–8.1)	8.35* (5.72–10.1)	5.8 (2.31–9.81)	p=0.0151
WBC (10^3/μL)	7.55 (6.57–8.3)	7.08 (4.97–9.01)	7.08 (6.35–8.41)	7.18 (6.67–8.81)	p=0.9292
RBC (10^6/μL)	4.6 (4.47–4.87)	4.79^^ (4.49–5.06)	4.4 (4.15–4.6)	4.66 (4.36–4.8)	p=0.0206
HGB (g/dL)	13.8 (13.3–14.1)	14.3^^ (13.5–14.8)	13.2 (12.5–14.2)	14.1 (13.5–14.6)	p=0.013
PLT (10^3/μL)	295 (277–300)	237** (200–289)	283^~~~ (227–316)	197**** (158–253)	p<0.0001

Notes: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate significant differences from the controls; ^ p<0.05, ^^^^ p<0.0001 indicate significant differences from the pheochromocytoma group; ~p<0.05, ~~p<0.01, ~~~p<0.001 indicate significant differences from the Cushing’s/Conn’s group.

Abbreviations: BMI, body mass index; CRP, C reactive protein; HGB, hemoglobin; PLT, platelet count; RBC, red blood cell count; WBC, white blood cell count; UA, uric acid.

Figure 1 Violin plots of plasma total NO (A), ONOO⁻ (B), S-nitrosothiols (C) and nitrotyrosine (D) of the control, incidentaloma, pheochromocytoma and Cushing’s Conn’s adenoma patients. Results are presented as median with 25% and 75% percentiles. **p<0.01, ***p<0.001, ****p<0.0001 indicate significant differences from the controls; ~p<0.05, ~~p<0.01, ~~~p<0.001 indicate significant differences from the Cushing’s Conn’s adenoma group; total nitric oxide (NO), peroxynitrite (ONOO⁻).

Figure 2 Violin plots of serum MPO (A), plasma IL-1β (B), TNF-α (C) and HIF-1α (D) of the control, incidentaloma, pheochromocytoma and Cushing’s Conn’s adenoma patients. Results are presented as median with 25% and 75% percentiles. **p<0.01, ***p<0.001, ****p<0.0001 indicate significant differences from the controls; myeloperoxidase (MPO), interleukin 1 beta (IL-1β) and tumor necrosis factor α (TNF-α).

Figure 3 Correlation heat map between the analyzed nitrosative stress, inflammation and clinical parameters in serum and plasma of the patients with adrenal masses; body mass index (BMI), C reactive protein (CRP), nitric oxide (NO), myeloperoxidase (MPO), interleukin 1 beta (IL-1β), tumor necrosis factor α (TNF-α) and hypoxia-inducible factor 1 alpha (HIF-1α).

Table 2 Multifactorial Regression Analysis of Nitrosative Stress and Inflammatory Biomarkers in Adrenal Tumor Patients

Biomarker	β1: Cortisol			β2: Metanephrine			β3: Normetanephrine			β4: Aldosterone
	Estimate	95% CI	P value	Estimate	95% CI	P value	Estimate	95% CI	P value	Estimate	95% CI	P value
Total NO	−0.9032	−1.950 to 0.1439	0.0897	−0.01666	−0.04279 to 0.009469	0.2076	0.002921	−0.03319 to 0.03903	0.8722	0.1679	−0.3978 to 0.7336	0.5556
Peroxynitrite	−0.7573	−1.481 to −0.03359	0.0405	−0.003478	−0.02154 to 0.01458	0.7019	−0.00785	−0.03281 to 0.01711	0.5323	0.5457	0.1547 to 0.9367	0.0069
S-nitrosothiols	−0.1579	−0.4363 to 0.1206	0.2619	−0.0001034	−0.007052 to 0.006846	0.9764	−0.00007228	−0.009675 to 0.009530	0.9881	−0.0276	−0.1780 to 0.1228	0.7155
Nitrotyrosine	−0.1827	−0.8154 to 0.4499	0.5663	0.0001806	−0.01561 to 0.01597	0.9819	−0.003021	−0.02484 to 0.01880	0.7831	0.3514	0.009547 to 0.6932	0.0441
MPO	−0.000006629	−0.0007959 to 0.0007827	0.9867	−0.000001015	− 0.00002071 to 0.00001868	0.9184	0.000003794	− 0.00002343 to 0.00003101	0.7817	0.0000716	−0.0003548 to 0.0004980	0.7386
IL-1β	−0.04327	−0.08314 to −0.003399	0.0339	0.0001613	−0.0008315 to 0.001154	0.7467	−0.001012	−0.002385 to 0.0003614	0.146	0.01017	−0.01149 to 0.03184	0.352
TNF-α	−0.0101	−0.1585 to 0.1383	0.8924	0.0005452	−0.003159 to 0.004249	0.7699	−0.003281	−0.008400 to 0.001838	0.2053	0.02296	−0.05723 to 0.1032	0.5696
HIF-1α	−1.521	−15.05 to 12.01	0.8232	0.09888	−0.2388 to 0.4366	0.5609	−0.3156	−0.7823 to 0.1510	0.1816	11.16	3.846 to 18.47	0.0033

Abbreviations: NO, nitric oxide; MPO, myeloperoxidase; IL-1β, interleukin 1 beta; TNF-α, tumor necrosis factor α; HIF-1α, hypoxia-inducible factor 1 alpha.

Table 3 Area Under the Curve of Nitrosative Stress and Inflammatory Biomarkers Between Adrenal Tumor Patients and the Controls

Biomarker	Control Group	Study Group	P value	AUC	95% CI	Cut Off	Sensitivity%	95% CI	Specificity%	95% CI
NO	14.24 (12.06–15.93)	17.78 (7.907–42.07)	0.1538	0.5755	0.4705 to 0.6805	< 15.40	68	54.19% to 79.24%	54.67	43.45% to 65.43%
Peroxynitrite	25.35 (22.08–27.9)	29.32 (22.95–41.07)	0.0019	0.6643	0.5698 to 0.7587	< 26.56	58	44.23% to 70.62%	57.33	46.05% to 67.90%
S-nitrosothiols	18.47 (14.11–21.87)	25.48 (21.65–29.77)	<0.0001	0.8379	0.7662 to 0.9095	< 21.19	72	58.33% to 82.53%	82.67	72.57% to 89.58%
Nitrotyrosine	12.43 (9.802–15.12)	24.44 (17.41–34.66)	<0.0001	0.8867	0.8293 to 0.9440	< 15.18	76	62.59% to 85.70%	82.67	72.57% to 89.58%
MPO	0.1608 (0.1535–0.1664)	0.2268 (0.2121–0.2365)	<0.0001	0.9909	0.9731 to 1.000	< 0.1862	100	92.87% to 100.0%	98.67	92.83% to 99.93%
IL-1β	2.109 (1.717–2.373)	2.733 (2.25–3.37)	<0.0001	0.7935	0.7163 to 0.8707	< 2.358	74	60.45% to 84.13%	71.62	60.48% to 80.63%
TNF-α	11.79 (10.62–13.63)	15.48 (13.25–17.62)	<0.0001	0.8387	0.7717 to 0.9057	< 13.35	72	58.33% to 82.53%	73.33	62.37% to 82.02%
HIF-1α	710.7 (650.1–775.1)	801.8 (772.3–1004)	<0.0001	0.8229	0.7448 to 0.9011	< 773.6	74	60.45% to 84.13%	72	60.96% to 80.90%

Note: Results are presented as median with 25% and 75% percentiles.

Abbreviations: NO, nitric oxide; MPO, myeloperoxidase; IL-1β, interleukin 1 beta; TNF-α, tumor necrosis factor α; HIF-1α, hypoxia-inducible factor 1 alpha.