Complex regional pain syndrome (CRPS) is a posttraumatic disorder, which occurs in about 2% of patients after a distal radial fracture. Epidemiologic data suggest that taking angiotensin-converting enzyme (ACE) inhibitors, which are indicated to treat hypertension, increases the odds ratio to develop CRPS after a radial fracture by a factor of 3.10 Angiotensin-converting enzyme is a membrane-bound zinc metalloproteinase of the gluzincin family extracellularly degrading angiotensin 1 to angiotensin 2 (ANG2), which is a potent vasoconstrictor. It also degrades inflammatory mediators such as bradykinin (BK) and substance P (SP) (for review, see Ref. 29). Indeed, the serum concentrations of the ACE substrates BK4 and SP38 as well as the activity of skin SP are increased in both CRPS patients25 and CRPS rodent models.45 Ultimately, a sufficient function of ACE could be important to prevent the development of CRPS after fracture. It is even more likely that, beyond ACE, a sufficient function of a whole peptidase network36,40 including peptidases degrading, eg, pronociceptive cytokines or the vasoconstrictive endothelin in primary cold CRPS, is important to prevent CRPS after fracture.
One method to assess the serum peptidase activity in individual patients is to quantify the degradation of substrates by standardized serum aliquots (serum degradation capacity). In recent years, this approach has been increasingly used in conjunction with mass spectrometry–based profiling experiments, in particular, in cancer.11,32 For the detection of ACE specifically, activity fluorescence assays are used, which are based on short substrates (tripeptides).21 However, using a full-length bioactive peptide such as BK (nonapeptide) or SP (undecapeptide) as a substrate in such assays provides additional information about related peptidase pathways because longer peptides have more cleavage sites. Thus, degradation of full-length peptides provides information about protease network activity rather than that of a single protease.
The clinical symptoms of CRPS, either exaggerated inflammation in the primary warm subtype or impaired vasomotor activity in the primary cold subtype,7 together with the biomarker findings mentioned above, suggest a decreased peptidase activity in these patients. Variability of proteinase capacity, as a phenotypic trait or occurring in response to the trauma, might be one factor that contributes to or predisposes for posttraumatic CRPS. To verify this hypothesis, we first developed a novel dabsyl-peptide protease activity assay based on the dabsylation of BK and thin-layer chromatography (TLC) for the separation of the cleavage products.2 We then used this assay to investigate the serum proteinase activity in patients with CRPS as well as in healthy and pain controls.
2.1. Patients and controls
The study was approved by the IRB of the Rhineland–Palatinate Medical Association (registration number 4208); the recruitment and clinical investigation of the patients was registered at the German Clinical Trials Register (registration number DRKS00008964). Fifty-two patients with CRPS who met the inclusion/exclusion criteria were recruited consecutively from the CRPS outpatient clinic in Mainz, Germany. Nineteen painful diabetic neuropathy (dPNP) patients were selected from the “ncRNAPain” study cohort (http://www.ncrna-pain.eu/)35 and served as pain controls; 31 healthy controls (HCs) were recruited from the staff of the University Medical Centre Mainz (Mainz, Germany).
The exclusion criteria for both patients and controls were the presence of neoplasms, alcohol consumption, infections, major affective disorders, chronic pain other than the diseases of interest, and unrelated surgery during the past 4 weeks; they were assessed by a structured interview. This was confirmed by investigating the blood cell count, C-reactive protein, liver enzymes, and creatinine serum levels. Another exclusion criterion was the use of ACE inhibitors, eg, for treating hypertension.
Patients with CRPS were included if they fulfilled the research diagnostic criteria for CRPS.16 We did not include multilimb or immobilized patients with CRPS. Healthy controls were included if they reported no relevant disease, in particular, no pain in a structured face-to-face interview before inclusion; dPNP patients were included if they suffered from diabetes and a structured neurological examination, nerve conduction studies, or skin biopsy confirmed the diagnosis of dPNP. The dPNP patients included here are a subgroup of the dPNP cohort that was published recently,35 selected to match the demographic characteristics of the patients with CRPS as closely as possible and to meet the comprehensive exclusion criteria, particularly the absence of hypertension and the treatment with antihypertensive drugs potentially affecting BK degradation. However, dPNP patients took diabetes-related drugs as medically needed.
2.2. Clinical investigation
The patients with CRPS were systematically examined by experienced neurologists (M.H., F.E.-L., and F.B.) adhering to a standard protocol that is in place in the Mainz CRPS clinic.6 All signs that are necessary to prove the CRPS diagnosis and to determine the CRPS severity score (CSS)17 were assessed. The patients with CRPS also completed the German versions of the trait part of the State–Trait Anxiety Inventory (STAI-T),24 the Beck Depression Inventory,23 the Neuropathic Pain Symptom Inventory,5 and the skin temperature on either the hands or feet.
2.3. Quantitative sensory testing
Quantitative sensory testing (QST) was performed according to the guidelines of the German Research Network on Neuropathic Pain (DFNS).37 It was performed on the back of the hand or top of the foot on both the affected and contralateral unaffected side. Thermal parameters included warm detection thresholds, cold detection thresholds, thermal sensory limen (TSL), heat pain thresholds, and cold pain thresholds. Thermal thresholds were assessed using a Thermal Sensory AnalyzerII (Medoc, Ramat Yishai, Israel). Mechanical sensation was assessed as detection thresholds by calibrated von Frey filaments (MARSTOCK nervtest, Schriesheim, Germany); pain thresholds, mechanical pain sensitivity, and the wind-up ratio were determined by calibrated pinprick stimulators (MRC Systems GmbH, Heidelberg, Germany), and the pressure pain threshold by a pressure gauge device (FDN200; Wagner Greenwich Instruments, Greenwich, CT). Log-transformed (when appropriate) QST values were used for analysis. Z transformation was performed for comparison with multicenter age- and sex-adjusted normative values of the DFNS.
2.4. Proteinase activity assay
Detection was based on the chromophore label dabsyl chloride (4-(4-dimethyl aminophenylazo)benzenesulfonyl chloride), which reacts with free amino groups. In BK (sequence RPPGFSPFR), the label is located at the N-terminus, which potentially allows for monitoring the known enzyme cleavage products of aminopeptidase P (APP; DBK1), ACE (DBK1-5), and carboxypeptidase N (CPN, DBK1-8) following incubation of dabsyl-BK (DBK) with serum. Cleavage specificity of ACE to BK and DBK was determined during assay development; the known ACE cleavage product of BK, BK1-7, was only transient under these conditions.2 The assay workflow is visualized in Figure 1. Synthesis and purification of the labeled substrate as well as TLC separation of the cleavage products were performed as described earlier2 with slight modifications. The TLC running buffer consisted of chloroform, methanol, water, and acetic acid (11:4:0.6:0.09). Serum (3 µL) was incubated for 60 minutes with dabsylated BK (DBK; 527.2 pmol), yielding the fragments DBK1-8 and DBK1-5 before stopping the reaction by adding 18-µL ice-cold acetone. The time point 60 minutes was chosen as measurement value because DBK1-9 degradation was nearly complete (better than 90% in HCs) at this time under our experimental conditions (Fig. 1). Thin-layer chromatography plates were scanned using a conventional flatbed scanner. The images were converted to 16-bit black and white tiff using the Photoshop plug-in silver efex pro 2 and analyzed with JustTLC (Sweday; Sodra Sandby, Sweden). Samples were analyzed in triplicate as described.2 Briefly, the staining intensity of each spot was determined. Plate-to-plate variations were accounted for by normalizing the intensity of each spot to the sum of the intensities of DBK1-9, DBK1-8, and DBK1-5 for this time point, providing relative values for DBK and its fragments. It is important to note that this assay does not rely on TLC retention times as analytical parameter, as they may slightly vary depending on the laboratory temperature or miniscule variations in solvent composition.
Statistical analysis was conducted using SPSS (Version 23.0, SPSS, Inc, Chicago, IL). Quantitative sensory testing data were transformed into standard normal distributions corrected for body region, sex, and age (Z values). Z transformation allows for the comparison of values independent of their physical dimensions. Increased sensitivity results in positive Z scores, whereas decreased sensitivity results in negative Z scores.
The analysis was exploratory. The a priori determined primary hypothesis is the difference of DBK serum degradation between patients with CRPS and healthy and pain controls. Under the assumption that in a limited-sized sample only strong effects (f = 0.4) could demonstrate significant differences (alpha P < 0.05) between groups, a priori sample size calculation revealed that in total 102 subjects had to be included.
All data were normally distributed. Therefore, we used parametric tests: the chi-squared test for categorical variables; Student t tests to compare QST parameters between the affected and unaffected sides; analysis of variance to compare clinical parameters, DBK1-8, DBK1-5, and the DBK1-8/DBK1-5 ratio between groups; Pearson correlation coefficients to compare relations between 2 parameters; and a forward stepwise multiple regression (probability for F inclusion P < 0.05 and exclusion P < 0.1) fixing the influence of all continuous measures on DBK1-8, DBK1-5, and the DBK1-8/1-5 ratio.
The true positive rate (sensitivity) as a function of the false positive rate (1−specificity) was plotted to produce a receiver operating characteristic (ROC) curve. For the estimation of the discriminatory accuracy of DBK1-8 and DBK1-5, the area under the ROC curve (AUC) was calculated.
Data are presented as mean ± SD throughout the article. Statistical significance was considered if P < 0.05.
2.6. Data availability
Anonymized data not published within the article will be shared by the request from any qualified investigator.
The biographic data of patients and controls are reported in Table 1. Pain intensity was at a median level and did not differ between patients with CRPS and pain controls (dPNP). Due to the natural course of the diseases, pain duration was shorter in CRPS, which was often subacute when the patients came to our clinic. By contrast, dPNP is a stable chronic condition. Healthy controls were slightly younger than both patient groups, and women were predominant in the CRPS group. However, neither age nor sex had a significant influence on the BK degradation products DBK1-8 and DBK1-5 in HCs (see Supplementary Table 1, available at http://links.lww.com/PAIN/A745).
The majority of the patients suffered from CRPS type I; most patients belonged to the primarily warm subtype, and the most affected extremity was the upper limb. Absolute skin temperature was not different at the time of investigation after acclimatization, but the temperature difference between the affected and the unaffected limb varied between −6°C and +4°C. For further details and CRPS characteristics, see Table 2.
The QST profile was typical for CRPS.13 It is characterized by sensory deficits and different hyperalgesia phenomena when the affected limb is compared with the unaffected one. Loss of nonpainful sensations was more pronounced than hyperalgesia. For details, see Figure 2.
As is exemplarily visualized in Figure 3, sera of HCs could be distinguished from sera of patients with CRPS by the levels of the degradation products DBK1-8 and DBK1-5.
The assay was analyzed in all subjects. In HCs, we found that the amounts of DBK1-8 and DBK1-5 were not related to age or sex (Supplementary Table 1, available at http://links.lww.com/PAIN/A745). In all groups, DBK1-8 and DBK1-5 were negatively correlated as expected (HC: r = −0.49, P < 0.01; dPNP: r = −0.84, P < 0.001; CRPS: r = −0.66, P < 0.001; Supplementary Fig. 1, available at http://links.lww.com/PAIN/A745).
DBK1-8 (F = 17.43, P < 0.01) and DBK1-5 (F = 10.47, P < 0.01) differed between groups. Post hoc tests with Bonferroni correction revealed higher values for DBK1-8 in CRPS vs HC (P < 0.001), but also in dPNP vs HC (P < 0.01). For DBK1-5, post hoc tests revealed lower values in CRPS vs HC (P < 0.01) and vs dPNP (P < 0.001).
Because DBK1-8 and DBK1-5 were linearly correlated in all groups (see Supplementary Fig. 1, available at http://links.lww.com/PAIN/A745), we additionally calculated the ratio DBK1-8/DBK1-5. This ratio differed between groups (F = 10.86, P < 0.001); post hoc tests with Bonferroni correction revealed higher ratios in CRPS vs HC (P < 0.001) and vs dPNP (P < 0.01). There was no difference between HC and dPNP. For details, see Figure 4.
3.2. Serum bradykinin degradation capacity and complex regional pain syndrome phenotype
We analyzed the influence of independent clinical variables on the DBK degradation capacity (DBK1-8, DBK1-5, and the DBK1-8/DBK1-5 ratio) using 2 methods: Clinical signs (presence vs absence) were analyzed by calculating one ANOVA analyzing the main effects; continuous variables were investigated by calculating a stepwise multiple regression. Neither DBK1-8 nor DBK1-5 nor the DBK1-8/1-5 ratio was influenced by the basic parameters and the clinical signs, which are depicted in Table 1, including CRPS type I or II. Stepwise multiple regression with DBK1-5 or the DBK1-8/DBK1-5 ratio as the dependent variable did not include any of the following independent variables: current, mean, or maximum pain; duration of CRPS; STAI-T; Beck Depression Inventory; temperature difference between the affected and unaffected side; CSS sum score; or any of the QST findings on the affected side. However, stepwise multiple regression with DBK1-8 included TSL and current pain (r2 = 0.23; F = 5.0; P < 0.02) indicating that the higher DBK1-8, the higher TSL and the more severe the actual pain.
3.3. Receiver operating characteristics
To determine the diagnostic value of the DBK degradation serum capacity for CRPS, we conducted a ROC analysis against HCs. For DBK1-8, the AUC was 0.83 (95% confidence interval [CI] 0.75-0.92) indicating a good diagnostic value (P < 0.001). DBK1-8 values of 0.64 or higher separated CRPS from HC with a sensitivity of 0.73 and a specificity of 0.81. For DBK1-5, the AUC was 0.71 (95% CI 0.56-0.84) indicating only a fair diagnostic value (P < 0.001). DBK1-5 values of 0.28 or lower separated CRPS from HC with a sensitivity of 0.79 and a specificity of 0.68. Receiver operating characteristic analysis for DBK1-8/DBK1-5 revealed a moderate diagnostic value (AUC 0.75; CI 0.64-0.87; P < 0.001); a DBK1-8/DBK1-5 ratio of 2.30 distinguished CRPS from HC with a sensitivity of 0.79 and a specificity of 0.65. For details, see Figure 5.
In a second ROC analysis, we also calculated the diagnostic sensitivity of the different parameters for the separation of CRPS vs the pain controls (dPNP). Although DBK1-5 distinguished CRPS from dPNP (AUC 0.80; 95% CI 0.68-0.91; P < 0.001), DBK1-8 did not (P > 0.05). However, DBK1-8/DBK1-5 separated CRPS from dPNP with an AUC of 0.76 indicating a moderate diagnostic quality for this biomarker (P < 0.01; CI 0.63-0.88).
This study demonstrates that patients with CRPS differ from both HCs and pain controls (dPNP) regarding their degradation pathways of proalgesic inflammatory mediators in the serum. After inventing a simple and resource-effective assay, we have been able to show that BK degradation pathways are affected in CRPS. Sera in HCs and pain controls degrade the labeled nonapeptide BK to the main fragments DBK1-5 and DBK1-8 after 1 hour. In comparison, in patients with CRPS, the measured concentration of DBK1-8 is increased and that of DBK1-5 is reduced. DBK degradation to DBK1-8 is known to be due to the activity of carboxypeptidase N (CPN), but it is possibly also due to other undescribed serum peptidases. Degradation to DBK1-5 further includes ACE activity. Our results indicate that the resolving anti-inflammatory protease network in patients with CRPS is different. Taking into account the clinical signs of CRPS, which resemble inflammation, this network could be less effective.
Complex regional pain syndrome is a clinically heterogeneous entity. The most important signs are sensory symptoms such as pain and hyperalgesia and “visible” signs of inflammation (“warm” CRPS) or presumed sympathetic dysfunction (“cold” CRPS). In addition, these signs may change during the CRPS course, either spontaneously or due to treatment, in a patient-specific manner.7,8 The clinical diagnostic criteria, however, do not reflect this heterogeneity,16 although it is clearly seen in our patient group as indicated in Table 2. This means that the pathophysiology of CRPS may not be singular and thus that an “all-explaining” pathomechanism for CRPS may never be found.3 Supporting this hypothesis, a plethora of single inflammatory mediators in different tissues and body fluids, including serum markers, of patients with CRPS differ from controls, with variable effect sizes in different studies.33 At the first glance, it seems puzzling that serum markers contribute to a focal disease such as CRPS. However, serum transfer (IgG) from CRPS to mice only works pronociceptive and proinflammatory if a trauma is prevalent beforehand, and this trauma is focal.41 This might explain the regional symptoms.
In the blood and skin, these “dysregulated” inflammatory mediators include (neuro-) peptides such as kinins, inflammatory or anti-inflammatory cytokines, and their soluble receptors.4,14,18,19,26,42 There are only 2 principle mechanisms how this could occur: increased (or decreased in case of anti-inflammatory mediators) biosynthesis or hampered (or enhanced) inactivation, or both. In this study, we concentrated on the inactivation, namely the degradation capacity of CRPS serum, ie, of the serum protease network. The diversity of clinical signs, the multiplicity and interaction of mediators, and the lack of comprehensive knowledge about the bioactivity of the different degradation products, which might be agonistic or antagonistic,15 probably render analyzing single proteases and mediators useless. It seemed more promising to concentrate on “networks,” ie, the proteolytic capacity of the serum as a whole. Therefore, we abstain from interpreting our results as an indication for a reduced activity of single proteases such as ACE or CPN, for which BK is a known and well-described substrate. We regard such a simplification as not possible because the network of proteases, which degrade these inflammatory mediators and their fragments, and their endogenous inhibitors are complex. It includes endopeptidases (eg, ACE, neutral endopeptidase, and endothelin-converting enzyme), aminopeptidases (eg, APP and dipeptidyl peptidase IV), and carboxypeptidases (eg, CPN). It is the orchestra of these enzymes that contribute to the termination of symptoms, which are mediated by cytokines (pain and hyperalgesia39 and peptides [calcitonin gene-related peptide and SP for neurogenic inflammation,44 endothelin for cold skin,14 and BK for pain and hyperalgesia34]). For summary, see Ref. 43. If we had a similar test for SP, calcitonin gene-related peptide, endothelin, or cytokine degradation, we could speculate that CRPS also differed from the controls. We acknowledge that this assumption might lose some information by failing to detect changes in individual enzymes. However, it may not only be valid for CRPS because dPNP patients are also slightly different from HCs. It may be of interest to compare painful with painless dPNP cohorts in future trials.
In this study, we have chosen BK as an index mediator in our assay for several reasons: (1) BK has been shown to be increased in CRPS serum samples.4 (2) Angiotensin-converting enzyme contributes to the degradation of BK, and taking an antihypertensive ACE inhibitor significantly increases the odds to develop CRPS after fracture, probably by inhibition of degradation of the substrates BK and SP.9 (3) The analysis of BK degradation provided robust data in our ex vivo assay. We first tried mass spectroscopy for the detection of BK (and of other peptides) and found it very sensitive and specific, but in particular with nanobore chromatography technologies coupled to mass spectrometers, we observed little technical robustness towards complex biological matrices such as serum so that it rendered it impossible to investigate suitable cohort sizes reproducibly. We thus developed this low-tech, low-cost approach requiring only a drop of serum. Looking at enzyme activity by supplying the substrate rather than trying to detect the endogenous compound itself in the complex biological background simplifies the analytical task. Moreover, especially considering the likely interference by endogenous BK and biological processes such as the activation of the kallikrein–kinin cascade during sampling and probe handling, the assay is independent from these variations, as DBK is preferentially degraded.2
The assay is robust and reliable, and the data were independent from age and sex. Taking ACE inhibitors could have influenced the results, but we strictly excluded patients and controls with such a medication. This was particularly a challenge for recruiting the dPNP pain control group.35 The negative correlation of the degradation products DBK1-8 and DBK1-5 separately in all subject groups was properly evidenced in the assay. It also exhibits the sensitivity and specificity to potentially tag CRPS. Receiver operating characteristic analysis allows for a moderate to good differentiation between CRPS, HC, and dPNP. Sensitivity and specificity is comparable to the diagnostic criteria for CRPS, which were evaluated against different neuropathic pain conditions including dPNP,16 or to technical investigations such as 3-phase bone scintigraphy46 or serum osteoprotegerin analysis.22 On the other hand, the results are variable, and there is a considerable overlap between cases and controls. This variability mirrors the clinical and pathophysiological variability of CRPS. We found only a very moderate correlation of DBK1-8 with clinical symptoms, mainly pain, which should, however, not be overrated because of the complex network of inflammatory mediators and proteases in human tissue.
Our study has limitations. The cohorts were not perfectly matched. However, neither age nor sex had a significant influence on the BK degradation products DBK1-8 and DBK1-5 in HCs. Another limitation is that we could not differentiate whether our results are the consequence of, or the prerequisite to get, CRPS. Although the finding that the duration of CRPS had no effect on the results favors a “prerequisite” assumption, definite answers can only come from longitudinal investigations. Such investigations might include patients with normal fracture healing as another control group. Alternatively, tissue from the affected vs the unaffected limb could be investigated for degradation capacity. If the “prerequisite” assumption is right, both sides should differ from controls; if the “consequence” assumption is right, it might be mainly the affected limb. The third limitation is that we can only provide information about the black box “serum degradation capacity.” We cannot comment on the bioactivity of the dabyslated BK (DBK) degradation products. However, the natural BK1-8 is the major agonist on the BK B1 receptor and becomes functional in inflammation and contributes to pain and hyperalgesia.1 BK1-5 has strong anti-inflammatory (in sepsis)28 and antithrombotic actions, eg, via antagonism on different proteinase-activated receptors.30 As in our previous study,42 this could be an indication for increased inflammatory responses in CRPS. However, to gain more specific information would require analyzing many more mediators including peptides and cytokines alone and in combination, with serum and purified proteases. This is not easily done and can only be achieved by consortia focusing on this topic. But even such a complex approach would neglect the bioactivity of the fragments in vivo. Nevertheless, the present results show that a comprehensive analysis might be rewarding. One possible approach would be an in silico bioinformatics network analysis including all available data regarding inflammatory mediators in CRPS and then looking for master switches, which control the activity of, eg, several proteases. Possible candidates would be small noncoding RNAs. First results in CRPS have been promising.27,31
Conflict of interest statement
The authors have no conflict of interest to declare.
Supported by DFG Bi579/8, KO1694/13-1, and the EU FP7 program ncRNAPain under the agreement 602133 to F.B., E.V., J.B., and H.R. The authors thank Ms Cheryl Ernest for her help improving the language.
Supplemental digital content
Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/A745.
Supplemental video content
Video content associated with this article can be found at http://links.lww.com/PAIN/A746.
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