
==== Front
Medicine (Baltimore)
Medicine (Baltimore)
MD
Medicine
0025-7974
1536-5964
Lippincott Williams & Wilkins Hagerstown, MD

39312385
MD-D-24-08300
00086
10.1097/MD.0000000000039742
3
7100
Research Article
Observational Study
Efficacy of N-acetylcysteine in reducing inflammation and oxidative stress to prevent complex regional pain syndrome type 1
https://orcid.org/0000-0002-3002-5028
Dinç Mustafa MD a*
Soydemir Ömer Cevdet MD dromer77@hotmail.com
a
a Bursa City Hospital, Orthopedics and Traumatology Clinics, Bursa, Turkey.
* Correspondence: Mustafa Dinç, Bursa City Hospital, Orthopedics and Traumatology Clinics, Doğanköy, Doğanköy iç yolu, 16110 Nilüfer/Bursa, Turkey (e-mail: drindianster@gmail.com).
20 9 2024
20 9 2024
103 38 e3974221 7 2024
23 8 2024
27 8 2024
Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.

This study aimed to evaluate the effectiveness of N-acetylcysteine (NAC) in preventing complex regional pain syndrome type 1 (CRPS-1) by reducing proinflammatory cytokines and oxidative stress markers in patients with distal radius fractures. A retrospective single-center study at Bursa City Hospital involves patients over 50 years of age with distal radius fractures treated between January 2021 and December 2023. A total of 60 patients (mean age, 62.8 ± 5.1 years; 26 males and 34 females) were analyzed. Patients were divided into 2 groups: the NAC group (31 patients receiving 600-mg NAC daily for 3 months) and the control group (29 patients with no prophylactic medication). CRPS-1 diagnosis was based on Budapest criteria during multiple follow-up visits. Serum levels of interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha (TNF-α), total oxidant status (TOS), and total antioxidant status (TAS) were measured at baseline and study end point. CRPS-1 positive patients had significantly higher levels of IL-6, TNF-α, and IL-1 (P < .001 for all), higher TOS (P < .001) and oxidative stress index (P < .001), and lower TAS (P < .001) compared with CRPS-1 negatives. The incidence of CRPS-1 was significantly lower in the NAC group (9.7%) compared with the control group (31.0%; P = .039). Logistic regression indicated a 78% reduction in CRPS-1 odds ratio with NAC treatment (odds ratio, 0.219 [95% confidence interval, 0.053–0.895]; P = .0322). NAC significantly reduced end-point levels and changes in IL-6 (P < .001), TNF-α (P < .001), and IL-1 (P = .038) and improved oxidative stress markers, showing higher TAS (P < .001), lower TOS (P < .001), and oxidative stress index (P < .001) compared with controls. NAC significantly reduced the risk of developing CRPS-1 by decreasing levels of proinflammatory cytokines and oxidative stress. This study highlights NAC’s potential as a preventive treatment for CRPS-1 and emphasizes the importance of early intervention.

complex regional pain syndrome type 1 (CRPS-1)
proinflammatory cytokines
N-acetylcysteine (NAC)
oxidative stress
OPEN-ACCESSTRUE
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pmc1. Introduction

Orthopedic surgeons witness distal radius fractures (DRFs) more often than any other type of fracture in patients with trauma. Up to 18% of all fractures in the elderly are caused by these types of traumas.[1,2] The majority of patients experience uncomplicated healing following these fractures; however, up to 37% of cases may result in the development of complex regional pain syndrome (CRPS), a chronic neurological condition.[1,2]

Sudomotor nerve anomalies, localized edema, alterations in blood flow, and acute pain are hallmarks of CRPS type 1 (CRPS-1), a complex clinical disease. It often occurs in the upper extremities and is frequently observed after fractures or surgery.[3] Consequently, DRFs are among the leading causes of CRPS-1. When CRPS-1 arises, patients’ quality of life and everyday function are substantially hampered because of acute pain, making it difficult for them to continue employment.[4] Therefore, early diagnosis and intensive treatment of CRPS-1 are crucial for the recovery of function.[4,5] Therefore, to recognize and treat CRPS-1 early, it is crucial to identify its risk factors.

The exact causes of CRPS-1 are still not fully understood and involve multiple factors. Nevertheless, clinical observations of the acute phase of CRPS-1, such as pain, swelling, redness with vasomotor instability, increased temperature, and impaired function, suggest that this condition may be linked to an excessive inflammatory response to trauma.[5] The release of proinflammatory cytokines such as interleukin (IL)-1 beta, IL-2, IL-6, and tumor necrosis factor-alpha (TNF-α) occurs as a result of tissue damage.[6,7] Both the local and systemic levels of these cytokines are dramatically increased in patients with CRPS. This heightened inflammatory response to tissue injury, neurogenic inflammation, ischemia, and reperfusion injuries can lead to the overproduction of free radicals and oxidative stress.[8,9] Free radicals have the capacity to increase the permeability of blood vessels, trigger the release of neuropeptides, amplify the process of inflammation, and, consequently, lead to additional tissue damage.[5,7–9]

The interconnection between oxidative stress and inflammation underscores the critical requirement for treatment strategies that can effectively target and alleviate both components of the disease. Several antioxidants, including vitamin C, mannitol, and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), have been studied for their potential advantages in managing CRPS-1. Their ability to neutralize free radicals and reduce oxidative stress is the reason for this. N-acetylcysteine (NAC) has emerged as a particularly promising candidate because of its dual role as an antioxidant and anti-inflammatory agent. NAC is a sulfur-containing amino acid that is a derivative of l-cysteine and has direct antioxidant properties. NAC contains sulfhydryl groups that can interact with free radicals and function as anti-inflammatory agents by reducing proinflammatory cytokines.[10,11]

Most research in the field of literature has focused on exploring the effects of free radical scavengers, particularly vitamin C, on CRPS-1. However, there has been limited exploration of the role of NAC, and existing studies have mainly focused on its clinical effects without thoroughly examining the underlying mechanisms. Consequently, a thorough examination of the role of reactive oxygen species and mediators of inflammation in the development of CRPS-1 was the primary aim of our study. Specifically, we aimed to evaluate the multidirectional impact of NAC on proinflammatory cytokines and oxidative stress markers, including total oxidant status (TOS) and total antioxidant status (TAS), in the plasma. By including baseline measurements and tracking changes from the initial fracture event to the onset of CRPS-1, our study seeks to offer new insights into the potential of NAC as a preventive treatment that addresses both the inflammatory and oxidative pathways in CRPS-1.

2. Material and methods

We conducted a retrospective, single-center study within the orthopedic clinic department of Bursa City Hospital. Good Clinical Practice (International Council for Harmonisation-Good Clinical Practice) guidelines and the guiding principles of the Declaration of Helsinki were followed throughout the study. This study was approved by the Institutional Ethics Committee (date: February 21, 2024; decision number: 2024-2/1).

2.1. Patient selection

Patients over the age of 50 years who underwent DRFs treated with closed reduction and cast application between January 1, 2021, and September 31, 2023, were enrolled and followed up for the next 3 months.

The sample approach was census-based, with patients referred or presented to the hospital’s emergency room with an acute DRF.

The inclusion criteria were patients aged 50 years and above who had closed, unilateral, extra-articular DRFs and had been treated with closed reduction and cast application. Patients who had a history of taking certain medications for CRPS, fractures in the same hand or wrist, articular displacement requiring open reduction, open fractures, neurovascular injury, high-energy mechanism fractures, or multiple trauma injuries were not eligible for inclusion in the study. Each of the 170 patients with fractures was evaluated separately. A total of 60 patients were assessed after data loss and patient exclusions.

2.2. Study protocol

Sample size calculation was conducted using the G*Power 3.1.9.6 program. The power analysis was performed to determine the difference in CRPS-1 incidence between the groups. With an effect size of 0.35, the analysis indicated that a total of 60 participants, with 30 subjects in each group, would be sufficient to achieve 80% power at a significance level of 0.05. The analysis was repeated to ensure the robustness of the sample size determination.

The protocol was initiated in the emergency room after informed consent was obtained. Sixty patients who had DRFs were allocated to receive either NAC or not. Thirty-one patients were found to use NAC 600 mg once daily for 3 months from that moment on the day of the fracture, as the literature shows that this standard dose effectively provides antioxidant and anti-inflammatory benefits across various patient populations without needing weight-based adjustments,[12] and 29 patients in the control group did not take any prophylactic medication. The patients were evaluated clinically at 6, 8, and 12 weeks after management. CRPS-I was diagnosed using the Budapest diagnostic criteria on multiple posttreatment visits.[13] The primary end point of the study was the diagnosis of CRPS-1 within 3 months of treatment.

2.3. Follow-up

Upon arrival at the emergency department, demographic factors such as age, sex, fracture location, hand dominance, and fracture type Arbeitsgemeinschaft für Osteosynthesefragen/Orthopedic Trauma Association classification were documented. Additional data regarding symptoms and indicators of CRPS were documented during the follow-up period. Clinical assessments of the patients were conducted in the sixth week after cast removal, as well as in the eighth and 12th weeks. Laboratory tests were conducted to analyze the cytokines and oxidative state. Blood samples were taken as a baseline before treatment was applied at the end of the study. Clinical assessment of CRPS-1 was conducted using the “Budapest criteria,” which demonstrated satisfactory sensitivity and specificity. If the patient was diagnosed with CRPS, the trial was terminated, and the patient was treated with reassurance, analgesics, and cautious physical therapy to prevent the worsening of pain.

2.4. Biochemical analysis

2.4.1. Serum

Venous blood was obtained from the cubital vein of the upper extremities. Venous blood was withdrawn from the unaffected limb to avoid additional inconvenience to our patients, and previous studies have shown no significant difference between plasma from the involved and uninvolved extremities.[14] For the measurement of inflammatory mediators in serum, 10 mL of blood was drawn into endotoxin-free blood collection tubes (S-Monovette; SARSTEDT AG & Co., Nümbrecht, Germany) and centrifuged for 10 minutes at 20 °C and 3000 U/min. Thereafter, the serum was portioned into 1.5-mL Eppendorf conical polypropylene tubes and stored at −80 °C until analysis.

2.4.2. Cytosine analysis with enzyme-linked immunosorbent assay

Assays were conducted according to the manufacturer’s protocol for the determination of cytokines (PeliKine compact human enzyme-linked immunosorbent assay kits from CLB in Amsterdam, the Netherlands). The standard curve ranges and detection limits, respectively, were given as follows: for IL-1b, 0 to 300 and 0.2 pg/mL; for IL-6, 0 to 450 and 0.3 pg/mL; and for TNF-a, 0 to 1000 and 1 pg/mL.

2.4.3. Determining oxidative stress by measurement of TOS, TAS, and oxidative stress index

TOS and TAS levels were assessed using the Erel methodology.[15] Nearly 5 mL of each peripheral venous blood sample was collected from both patients and controls. The samples were preserved in Becton Dickinson Vacutainer tubes and used for blood collection. The sera were separated by centrifugation and promptly quantified using a commercially available test kit (Rel Assay Diagnostics kit; Mega Tip, Gaziantep, Turkey) according to the manufacturer’s instructions, utilizing their reagents and equipment. The TAS results are reported in millimoles of Trolox equivalent per liter, whereas the TOS results are reported in micromoles of hydrogen peroxide equivalent per liter. The oxidative stress index (OSI) was determined by dividing the TOS by TAS. The formula for calculating the OSI (arbitrary unit) was given as follows: OSI = TOS (μmol H2O2 Eq/L) divided by TAS (μmol Trolox Eq/L), multiplied by 100.

3. Statistical analysis

The data were examined using the Shapiro-Wilk test to determine whether the data presented a normal distribution. The results are presented as mean ± standard deviation, median (minimum-maximum), or frequency and percentage. Normally distributed data were compared using independent sample t tests or Mann-Whitney U tests for nonnormally distributed data. Repeated measurements were compared between groups by calculating the percentage change (percentage change = end-point measurement-baseline measurement/baseline measurement) according to the baseline measurement. Categorical variables were compared between groups using Pearson χ2, Fisher exact, and Fisher-Freeman-Halton tests. A logistic regression model was used to compare the dependent variables to the independent variables. Odds ratios (ORs), including 95% confidence interval (CI), were used to describe the association. Statistical significance was set at α = 0.05. Statistical analyses were performed using IBM SPSS, version 28.0 (IBM Corp, Released 2021; IBM SPSS Statistics for Windows, version 28.0; Armonk, NY: IBM Corp).

4. Results

The mean age was 62.14 ± 5.22 years in the NAC group and 63.03 ± 4.98 years in the control group, with no significant difference between the groups (P = .494). Hand dominance was not significantly different between the groups (P = .264). Similarly, there was no significant difference in the fracture side (P = .828) or type (P = .670) between the NAC and Control groups.

For body mass index (BMI) <25, 38.7% of the NAC group and 58.6% of the control group fell into this category. Conversely, for BMI ≥25, 61.3% of the NAC group and 41.4% of the control group were represented. BMI categories showed no significant difference between the groups for BMI <25 (P = .343) or BMI >25 and above (P = .170).

Table 1 shows the comparison results between the NAC and control groups in terms of demographic characteristics. In summary, the NAC and control groups were comparable in terms of age, hand dominance, fracture side, and fracture type with no significant differences in these parameters, indicating homogeneity between the groups. In addition, while there was a notable difference in BMI distribution, with a higher proportion of individuals in the NAC group having a BMI of ≥25, this difference was not statistically significant.

Table 1 Comparison of demographic characteristics.

	NAC (n = 31)	Control (n = 29)	P	
Age, yr	62.48 ± 4.70	63.66 ± 4.73	.340	
Gender (female)	19 (61.3)	18 (62.1)	.951	
Hand dominance	.264	
 Right	26 (83.9)	27 (93.1)		
 Left	5 (16.1)	2 (6.9)		
Fracture side	.828	
 Right	18 (58.1)	17 (58.6)		
 Left	13 (41.9)	12 (41.4)		
Fracture type	.670	
 AO A2	22 (71.0)	20 (69.0)		
 AO A3	9 (29.0)	9 (31.0)		
BMI <25	12 (38.7)	17 (58.6)	.343	
BMI ≥25	19 (61.3)	12 (41.4)	.170	
The descriptive statistics were presented as mean ± standard deviation, median (minimum-maximum), or n (%).

AO = Arbeitsgemeinschaft für Osteosynthesefragen, BMI = body mass index, NAC = N-acetylcysteine.

Table 2 shows the occurrence of CRPS was compared between the NAC group (31 patients) and the control group (29 patients). In the NAC group, 3 patients (9.7%) developed CRPS, while 9 patients (31.0%) in the control group were CRPS-positive. This difference was statistically significant (P = .039). A logistic regression analysis was conducted to further evaluate the association between group assignment and CRPS status. The results revealed that the NAC group had significantly lower odds of developing CRPS than the control group, with an OR of 0.219 (95% CI, 0.053–0.895; P = .0322). This finding suggests that patients in the NAC group were less likely to develop CRPS than those in the control group. The OR of 0.219 (95% CI, 0.053–0.895) suggests that patients in the NAC group were significantly less likely to develop CRPS than those in the control group.

Table 2 Comparison of CRPS-1 occurrence between NAC and control groups.

	NAC (n = 31)	Control (n = 29)	P	Odds ratio	95% CI	
CRPS-1 (+)	3 (9.7)	9 (31.0)	.039	0.219	0.053–0.895	
CRPS-1 (−)	28 (90.3)	20 (69.0)				
CI = confidence interval, CRPS-1 = complex regional pain syndrome type 1, NAC = N-acetylcysteine.

The results of whether there was a statistically significant difference between cytokine and oxidative stress levels between the NAC and control groups are shown in Table 3. While there was no difference between the NAC and control groups regarding baseline measurements, a significant difference was found between the end point and changes. The comparison of cytokine and oxidative stress levels between the NAC group (n = 31) and the control group (n = 29) revealed significant differences at the end point. For IL-6, the end-point median level was significantly lower in the NAC group (1.4) than in the control group (2.0; P < .001), and the median change (Δ) was also significantly lower (0.36 vs 1.00; P < .001). TNF-α levels at the end point were lower in the NAC group (4.7 vs 4.9; P < .001), with a median change of 0.45 compared with 0.58 in the control group (P = .002). IL-1 end-point levels were lower in the NAC group (2.0 vs 2.1; P = .038), with a median change of 0.25 compared with 0.38 in the control group (P = .035).

Table 3 Comparison of cytokine and oxidative stress levels between NAC and control groups.

		NAC (n = 31)	Control (n = 29)	P	
Median	Minimum	Maximum	Median	Minimum	Maximum	
IL-6, pg/mL	Baseline	1.0	0.8	1.3	1.0	0.8	1.3	.481	
	End point	1.4	1.0	8.5	2.0	1.2	9.1	<.001	
	Δ	0.36	−0.09	9.38	1.00	0.15	9.13	<.001	
TNF-α, pg/mL	Baseline	3.2	2.9	3.5	3.1	2.9	3.5	.339	
	End point	4.7	4.1	9.4	4.9	4.3	9.9	<.001	
	Δ	0.45	0.24	1.82	0.58	0.34	2.30	.002	
IL-1, pg/mL	Baseline	1.6	1.4	1.9	1.6	1.4	1.9	.892	
	End point	2.0	1.7	6.9	2.1	1.8	7.1	.038	
	Δ	0.25	0.0	3.64	0.38	−0.05	3.93	.035	
TAS, μmol Trolox Eq/L	Baseline	1.7	1.5	1.9	1.7	1.5	1.9	.526	
	End point	2.8	0.7	2.9	2.4	0.6	2.8	<.001	
	Δ	0.61	−0.61	0.93	0.39	−0.67	0.87	<.001	
TOS, μmol H2O2 Eq/L	Baseline	3.9	3.5	4.5	3.9	3.5	4.5	.666	
	End point	5.7	4.8	7.7	5.8	5.5	7.9	<.001	
	Δ	0.41	0.20	1.14	0.57	0.30	1.17	.002	
OSI*	Baseline	0.23	0.18	0.29	0.23	0.18	0.29	.355	
	End Point	0.20	0.17	1.07	0.24	0.21	1.30	<.001	
	Δ	−0.15	−0.33	4.36	0.10	−0.26	4.89	<.001	
Bold P values show the statistically significance.

Δ: changes were calculated using a baseline measurement (Δ = percentage change = (end-point measurement−base line measurement)/baseline measurement.

IL = interleukin, NAC = N-acetylcysteine, OSI = oxidative stress index, TAS = total antioxidant status, TNF-α = tumor necrosis factor-alpha, TOS = total oxidant status.

* Measurement unit arbitrary.

Regarding oxidative stress markers, the NAC group had significantly higher end-point TAS levels (2.8 vs 2.4; P < .001) and a greater median change (0.61 vs 0.39; P < .001). TOS levels were lower in the NAC group at the end point (5.7 vs 5.8; P < .001), with a median change of 0.41 compared with 0.57 in the control group (P = .002). The OSI levels were also significantly lower in the NAC group (0.20 vs 0.24; P < .001), with a median change of −0.15 compared with 0.10 in the control group (P < .001).

Table 4 shows the comparison of cytokine and oxidative stress levels between CRPS-positive and CRPS-negative samples. The CRPS-positive group exhibited significantly higher end-point levels of proinflammatory cytokines, IL-6 (8.6 vs 1.5; P < .001), TNF-α (9.6 vs 4.7; P < .001), and IL-1 (6.9 vs 2.0; P < .001), than the CRPS-negative group. In addition, the CRPS-positive group had significantly lower posttreatment TAS levels (0.8 vs 2.7; P < .001) and higher levels of TOS (7.7 vs 5.7; P < .001) and OSI (1.00 vs 0.21; P < .001). The changes (Δ) from baseline to posttreatment also indicated significant differences between the groups, with CRPS-positive patients showing larger increases in IL-6 (7.75 vs 0.45; P < .001), TNF-α (1.87 vs 0.47; P < .001), IL-1 (3.19 vs 0.24; P < .001), TOS (0.95 vs 0.45; P < .001), and OSI (2.97 vs −0.09; P < .001) and a larger decrease in TAS (−0.55 vs 0.54; P < .001).

Table 4 Comparison of cytokine and oxidative stress levels between CRPS-1 positives and negatives.

		CRPS-1	P	
Yes (n = 12)	No (n = 48)	
Median	Minimum	Maximum	Median	Minimum	Maximum	
IL-6, pg/mL	Baseline	1.0	0.8	1.3	1.0	0.8	1.3	.829	
	End point	8.6	8.1	9.1	1.5	1.0	2.5	<.001	
	Δ	7.75	5.54	9.38	0.45	−0.09	2.13	<.001	
TNF-α, pg/mL	Baseline	3.3	2.9	3.5	3.2	2.9	3.5	.266	
	End point	9.6	9.1	9.9	4.7	4.1	5.1	<.001	
	Δ	1.87	1.68	2.30	0.47	0.24	0.72	<.001	
IL-1, pg/mL	Baseline	1.6	1.4	1.9	1.6	1.4	1.9	.529	
	End point	6.9	6.5	7.1	2.0	1.7	2.3	<.001	
	Δ	3.19	2.53	3.93	0.24	−0.05	0.64	<.001	
TAS, μmol Trolox Eq/L	Baseline	1.7	1.5	1.9	1.7	1.5	1.9	.491	
	End point	0.8	0.6	0.9	2.7	0.6	2.9	<.001	
	Δ	−0.55	−0.67	−0.44	0.54	−0.62	0.93	<.001	
TOS, μmol H2O2 Eq/L	Baseline	4.0	3.5	4.5	3.9	3.5	4.5	1.000	
	End point	7.7	7.4	7.9	5.7	4.8	6.0	<.001	
	Δ	0.95	0.68	1.17	0.45	0.20	0.69	<.001	
OSI*	Baseline	0.23	0.18	0.28	0.23	0.18	0.29	0.684	
	End point	1.00	0.84	1.30	0.21	0.17	0.97	<.001	
	Δ	2.97	2.64	4.89	−0.09	−0.33	3.18	<.001	
Bold P values show the statistically significance.

Δ: changes were calculated using a baseline measurement (Δ = percentage change = (end-point measurement−baseline measurement)/baseline measurement.

CRPS-1 = complex regional pain syndrome type 1, IL = interleukin, OSI = oxidative stress index, TAS = total antioxidant status, TNF-α = tumor necrosis factor-alpha, TOS = total oxidant status.

* Measurement unit arbitrary.

5. Discussion

In the present study, our data showed that elevated levels of proinflammatory cytokines (IL-6, TNF-α, and IL-1) and oxidative stress play an important role in the development of postfracture CRPS-1.

Our findings also reveal that NAC significantly reduces the levels of proinflammatory cytokines (IL-6, TNF-α, and IL-1) and improves oxidative stress markers (TAS, TOS, and OSI). This study is notable as it is the first to demonstrate that NAC can decrease proinflammatory cytokines and free radicals in patients with CRPS-1 and is also the first to focus on the acute phase of CRPS-1 while including baseline biomarker measurements.

The relationship between oxidative stress and inflammation is reciprocal and synergistic. Reactive oxygen species (ROS) generated during oxidative stress can activate several signaling pathways, such as the nuclear factor-kappa B pathway. Nuclear factor-kappa B is a transcription factor that plays a central role in the inflammatory response by upregulating the expression of proinflammatory cytokines, chemokines, and adhesion molecules. Oxidative stress can also activate other signaling pathways, such as mitogen-activated protein kinases, which further propagate inflammatory signals. In addition, oxidative damage to cell membranes and other structures can directly trigger inflammatory responses by releasing damage-associated molecular patterns, which are recognized by immune cells and initiate inflammation.[16] During inflammation, activated immune cells, such as macrophages and neutrophils, produce ROS. While this is beneficial in acute inflammation, excessive or chronic ROS production during prolonged inflammation can lead to further oxidative stress. The inflammatory process can deplete antioxidant defenses, either by consuming antioxidants or downregulating their synthesis, thereby exacerbating oxidative stress. Chronic inflammation, through the sustained release of inflammatory cytokines, can perpetuate oxidative stress, leading to a continuous cycle of damage.[17]

Our results are in line with the fact that CRPS-1’s pathogenesis is still not fully understood. Recent data have pointed to exaggerated inflammation after trauma as an igniting factor. Elevated levels of inflammatory cytokines have been well documented in patients with CRPS-1. For instance, Munnikes et al[18] reported higher levels of IL-6 and TNF-α from blister fluid in the involved extremities of patients during the intermediate stages of CRPS-1. Likewise, according to Huygen et al,[7] a local inflammatory process is suggested by an increase in IL-6 and TNF-α levels in the blister fluid of the affected limb compared with the uninvolved limb during the acute phase. Üçeyler et al[19] observed differential expression patterns of cytokines in patients with CRPS, with elevated proinflammatory cytokines (TNF and IL-2) and reduced anti-inflammatory cytokines (IL-4 and IL-10) in serum examination. Regardless of the duration or severity of CRPS, Alexander et al[6] discovered that compared with healthy controls, patients with CRPS had significantly higher levels of plasma cytokines, chemokines, and their soluble receptors. Huygen et al[14] explored the treatment of CRPS-1 with anti-TNF agents and found significant reductions in inflammatory cytokines (IL-6 and TNF-α) and clinical improvement in patients treated with infliximab, and this study confirms the involvement of cytokines in the inflammatory process of CRPS-1 and suggests that targeting these cytokines can provide therapeutic benefits.

However, some studies showed no effect of proinflammatory cytokines on the disease period. Schinkel et al[20] studied serum cytokine levels in the acute and chronic phases of patients with CRPS-1 and found no significant increase in cytokines among the control group. Huygen et al[7] showed no significant difference between any measured values of IL-1b, IL-6, and TNF-α in the plasma from involved and uninvolved extremities. In a study with a mean disease duration of 10 years, Van de Beek et al[21] discovered no significant variations in several cytokines between control subjects and patients with CRPS-1. The study by Parkitny et al[22] collected cytokine data within 28 days of the fracture and followed the participants for 16 weeks to determine who developed CRPS. They analyzed whether there was a correlation between cytokine levels measured within the first 28 days and the occurrence of CRPS at the 16-week follow-up. There was no significant association between baseline cytokine concentration (measured within 28 days post-injury) and the development of CRPS at 16 weeks. Our study provides a significant advancement over previous research by including both baseline and end-point measurements of cytokine levels in patients with and without CRPS. Unlike the study by Parkitny et al,[22] which only assessed cytokine levels within the first 28 days post-injury, our longitudinal approach allows for a comprehensive analysis of how these biomarkers change over time and their relationship with CRPS development.

Our findings are consistent with those of other studies that investigated the causes of CRPS-1. In our study, elevated levels of inflammatory cytokines were well documented in patients with CRPS-1, particularly during the triggering event of distal radial fractures to the onset of CRPS-1. However, differences in study findings can be attributed to variations in the disease stage, duration of the disease, sample types, measurement methods, patient populations, timing of sample collection, and one-time-point evaluation. In contrast, our study included baseline measurements, which allowed for the tracking of changes in cytokine levels over time. This approach provides a clearer understanding of the progression to CRPS, highlighting the importance of changes in cytokine levels. Most previous studies did not include baseline measurements and instead focused on cytokine levels during the disease period. Our findings suggest that tracking these changes from baseline to disease onset is crucial to understand the role of cytokines in CRPS-1.

Although inflammatory and neural mechanisms have been suggested as potential contributors to type I CRPS, recent studies have suggested that inflammation induces oxidative stress and free radicals in the pathogenesis of CRPS-1. Eisenberg et al[23] identified increased oxidative stress markers in patients with CRPS-1, specifically malondialdehyde (MDA) and F2-isoprostanes, which are indicative of lipid peroxidation and oxidative damage to cell membranes. Baykal et al[24] reported elevated levels of oxidative stress markers in the serum, including MDA, glutathione, and superoxide dismutase in patients with CRPS. Elevated MDA levels indicate increased lipid peroxidation, whereas alterations in glutathione and superoxide dismutase enzyme activities suggest a disrupted antioxidant defense system.[23] Our study findings also confirmed the effect of oxidative stress on CRPS pathophysiology by showing increased levels of TOS and OSI. One key question is whether oxidative changes contribute to CRPS throughout the course of the disease or are primarily relevant during the acute phase when inflammatory features are more pronounced. This distinction is crucial for the development of targeted therapies and understanding the timing of antioxidant interventions. These studies mostly focused on the chronic phase of CRPS and investigated the preventive effects of NAC from the initial fracture event. This preventive approach may address some of the unresolved issues mentioned by providing insights into the role of oxidative stress and inflammation in the earliest stages of CRPS development.

The pathophysiology mentioned above is based on the literature and our findings. Multiple lines of indirect evidence corroborated these findings, indicating that oxidative stress is present in CRPS-1. Furthermore, they provide support for the use of antioxidants and free radical neutralizers in the management and prevention of this condition. A 50-day, double-blind, prospective, multicenter study randomized 416 patients with 427 wrist fractures to receive either placebo or different dosages of vitamin C daily. The incidence of CRPS type I was analyzed by Zollinger et al[25] who concluded that vitamin C reduced the prevalence of CRPS type I after wrist fractures. A study conducted by Laumonerie et al[26] showed that the incidence of CRPS-I in postoperative patients undergoing subacromial shoulder surgery was considerably reduced when vitamin C was administered. According to research by Alimian et al,[27] the risk of CRPS is reduced when 500 mg of vitamin C is added to the local anesthetic in the Bier block. The impact of vitamin C on preventing CRPS type I after foot and ankle surgery was demonstrated by Besse et al.[28] Taking 1 g of vitamin C daily for 40 days following foot or ankle surgery decreases the likelihood of CRPS according to a study by Hernigou et al.[29] The study by Coderre et al[30] explored the development of a novel animal model for CRPS-1, known as chronic postischemia pain, and tested the effects of free radical scavengers NAC and Tempol on mechanical allodynia in the chronic postischemia pain model. Both NAC and Tempol significantly reduced mechanical allodynia, highlighting the role of oxidative stress in the maintenance of neuropathic pain–like symptoms in CRPS-I. Evidence of the antioxidant activity of NAC was established in a study by Koksal et al,[31] who mitigated ischemia/reperfusion injury in rats using a hindlimb model. The researchers observed that the plasma levels of creatine kinase, lactate dehydrogenase, and thiobarbituric acid reactive substances decreased. Although prior research has examined the efficacy of these scavengers, the underlying mechanisms of their actions have received less attention. Our study is the first to objectively demonstrate the effectiveness of NAC in reducing both proinflammatory cytokine and oxidative stress marker levels, highlighting its potential as a comprehensive treatment option for CRPS-1.

Notably, this is the first study to demonstrate that NAC decreases proinflammatory cytokines in patients with CRPS-1. Therefore, our study provides significant insights into the roles of cytokines and oxidative stress in the development of CRPS-1 and demonstrates the potential of NAC as a preventive treatment. One of the major strengths of our research is the detailed examination of proinflammatory cytokines (IL-6, TNF-α, and IL-1) from the initial fracture event to the onset of CRPS-1. By including baseline biomarker measurements, we were able to track temporal changes in cytokine levels, providing a comprehensive understanding of their role in disease progression. This approach contrasts with many previous studies, which only measured cytokine levels during the disease period, thus missing critical early changes.

In addition, our study is the first to show that NAC can significantly reduce both proinflammatory cytokines and oxidative stress markers (TAS, TOS, and OSI) in patients with CRPS-1. This dual effect underscores the potential of NAC not only as an anti-inflammatory agent but also as a powerful antioxidant capable of mitigating oxidative damage that contributes to CRPS pathogenesis. The comprehensive analysis of oxidative stress markers further enhances the robustness of our findings, as it provides a detailed picture of the oxidative stress landscape in patients with CRPS-1.

Moreover, our preventive approach, which focuses on the administration of NAC from the initial fracture event, addresses some unresolved issues in the literature regarding the timing of antioxidant intervention. By demonstrating the efficacy of NAC in the acute phase, our study suggests that early intervention is crucial for preventing the onset of CRPS-1. Overall, the inclusion of baseline measurements, comprehensive analysis of both cytokines and oxidative stress markers, and the novel preventive approach make our study a substantial contribution to the understanding and management of CRPS-1.

One potential limitation of our findings is the lack of generalizability due to the limited sample size. A more robust statistical power and larger investigations are required to validate our findings. Second, although we demonstrated the effectiveness of NAC in reducing cytokine levels and oxidative stress markers, the study design did not allow for the exploration of potential long-term effects of NAC treatment. Further research is required to assess the sustained effect of NAC on clinical outcomes in patients with CRPS-1 over an extended period. In addition, our study did not compare NAC with other standard treatments for CRPS-1, such as physical therapy, nonsteroidal anti-inflammatory drugs, or other antioxidants, which may have provided a broader context for our findings. Finally, the study design did not account for the potential synergistic effects of NAC when used in combination with other therapies. Future studies should investigate these combinations to determine the most effective treatment protocol for CRPS-1.

In conclusion, our study revealed the significant impact of NAC in reducing proinflammatory cytokine and oxidative stress marker levels in patients with CRPS-1 following DRFs. By providing evidence for the effectiveness of NAC during the acute phase and its role in early intervention, we highlight its potential as a preventive treatment for CRPS-1. These findings pave the way for new approaches in managing CRPS-1, emphasizing the importance of addressing both the inflammatory and oxidative pathways from the onset of injury. Future research should build on these results to confirm the broader applicability of NAC in diverse patient populations and investigate its long-term benefits. Our study provides valuable insights into the prevention of CRPS-1, offering hope for improved therapeutic strategies and patient outcomes.

Acknowledgments

The authors would like to acknowledge www.makaletercume.com for their outstanding scientific proofreading and editing services that were provided for this manuscript and DrPH Güven Özkaya for his outstanding statistical analysis.

Author contributions

Conceptualization: Mustafa Dinç, Ömer Cevdet Soydemir.

Data curation: Mustafa Dinç.

Formal analysis: Mustafa Dinç.

Investigation: Mustafa Dinç, Ömer Cevdet Soydemir.

Methodology: Mustafa Dinç, Ömer Cevdet Soydemir.

Resources: Mustafa Dinç.

Software: Mustafa Dinç.

Supervision: Mustafa Dinç.

Validation: Mustafa Dinç.

Writing – original draft: Mustafa Dinç, Ömer Cevdet Soydemir.

Writing – review & editing: Mustafa Dinç.

Abbreviations:

BMI body mass index

CI confidence interval

CRPS complex regional pain syndrome

CRPS-1 complex regional pain syndrome type 1

DRF distal radius fracture

IL interleukin

MDA malondialdehyde

NAC N-acetylcysteine

OR odds ratio

OSI oxidative stress index

ROS reactive oxygen species

TAS total antioxidant status

Tempol 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl

TNF-α tumor necrosis factor-alpha

TOS total oxidant status

This study was approved by the Clinical Research Ethics Committee of Bursa City Hospital (date: February 21, 2024, decision number: 2024-2/1).

The authors have no conflicts of interest to disclose.

The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author upon reasonable request.

How to cite this article: Dinç M, Soydemir ÖC. Efficacy of N-acetylcysteine in reducing inflammation and oxidative stress to prevent complex regional pain syndrome type 1. Medicine 2024;103:38(e39742).
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