If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, ItalyUOC Patologia Clinica, Azienda Ospedaliera Senese, Siena, Italy
Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, ItalyUOC Medicina Molecolare e Genetica, Azienda Ospedaliera Senese, Siena, Italy
Department of Musculoskeletal Biology, University of Liverpool, Liverpool, UKDepartment of Clinical Biochemistry and Metabolism, Royal Liverpool University Hospital, Liverpool, UK
Address correspondence and reprint requests to: A. Santucci, Università degli Studi di Siena, Dipartimento di Biotecnologie, Chimica e Farmacia, via Aldo Moro 2, 53100 Siena, Italy. Tel: 39-0577-234958; Fax: 39-0577-234254.
The aim of this work was to assess baseline serum levels of established biomarkers related to inflammation and oxidative stress in samples from alkaptonuric subjects enrolled in SONIA1 (n = 40) and SONIA2 (n = 138) clinical trials (DevelopAKUre project).
Methods
Baseline serum levels of Serum Amyloid A (SAA), IL-6, IL-1β, TNFα, CRP, cathepsin D (CATD), IL-1ra, and MMP-3 were determined through commercial ELISA assays. Chitotriosidase activity was assessed through a fluorimetric method. Advanced Oxidation Protein Products (AOPP) were determined by spectrophotometry. Thiols, S-thiolated proteins and Protein Thiolation Index (PTI) were determined by spectrophotometry and HPLC. Patients' quality of life was assessed through validated questionnaires.
Results
We found that SAA serum levels were significantly increased compared to reference threshold in 57.5% and 86% of SONIA1 and SONIA2 samples, respectively. Similarly, chitotriosidase activity was above the reference threshold in half of SONIA2 samples, whereas CRP levels were increased only in a minority of samples. CATD, IL-1β, IL-6, TNFα, MMP-3, AOPP, thiols, S-thiolated protein and PTI showed no statistically significant differences from control population. We provided evidence that alkaptonuric patients presenting with significantly higher SAA, chitotriosidase activity and PTI reported more often a decreased quality of life. This suggests that worsening of symptoms in alkaptonuria (AKU) is paralleled by increased inflammation and oxidative stress, which might play a role in disease progression.
Conclusions
Monitoring of SAA may be suggested in AKU to evaluate inflammation. Though further evidence is needed, SAA, chitotriosidase activity and PTI might be proposed as disease activity markers in AKU.
Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder (MIM 203500) causing an early onset, chronically debilitating spondylo-arthropathy due to high circulating homogentisic acid (HGA, 2,5-dihydroxyphenylacetic acid)
. Accumulation of HGA is due to mutations of the HGD gene causing the production of a defective HGD enzyme in tyrosine and phenylalanine catabolic pathways
. Excess HGA is partly eliminated in the urine, partly contributes to the production of an ochronotic pigment deposited in cartilaginous tissues, which leads to a range of clinical manifestations. AKU causes considerable morbidity in adulthood, and cases of acute fatal metabolic complications (oxidative haemolysis and/or methaemoglobinaemia) were reported
. The use of nitisinone (NTBC) was suggested to lower circulating HGA levels, and clinical trials were undertaken in Europe (DevelopAKUre – Clinical Development of Nitisinone for Alkaptonuria)
Suitability of Nitisinone in Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.
. Recent evidence pointed out also that AKU is a multisystem disease involving amyloid A (AA) amyloidosis due to high circulating Serum Amyloid A (SAA) promoting inflammation, oxidative stress and amyloidosis
In this framework, we undertook the present work to monitor the presence of established biomarkers related to inflammation and oxidative stress in serum of AKU subjects who were/are enrolled in DevelopAKUre clinical trials.
Material and methods
Samples
This study was carried out within the DevelopAKUre project
Suitability of Nitisinone in Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.
for SONIA1 (Suitability of Nitisinone in Alkaptonuria 1), and SONIA2 (Suitability of Nitisinone in Alkaptonuria 2) clinical studies. In SONIA1, samples were collected from 40 AKU subjects at the investigative sites of Liverpool (UK) and Piešťany (SK). In SONIA2, samples were collected from 138 AKU subjects at the investigative sites of Liverpool (UK), Piešťany (SK) and Paris (F). Serum samples were collected under fasting conditions at baseline (i.e., when patients first entered the study before randomisation). Details on inclusion/exclusion criteria can be found in Ranganath et al.
Suitability of Nitisinone in Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.
The same procedures were in place at each clinical site involved in SONIA1 and SONIA2 for blood collection, serum preparation, sample storage and shipment; these procedures were planned and monitored by the Contract Research Organization PSR Group (Amsterdam, the Netherlands). All the serum samples were kept at −80°C before analysis.
Since control populations were not planned for SONIA1 and SONIA2 studies, serum samples from age-matched healthy volunteers (with no concomitant known oxidative stress/inflammatory conditions) were collected at Siena University Hospital (n = 12 for SONIA1; n = 26 for SONIA2). All the available demographic information for both AKU and control subjects is reported in Tables 1S and 2S.
The studies were conducted following the approval of local Ethics Committee. All procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Written informed consent was obtained from all patients before any study procedures at each clinical site
Suitability of Nitisinone in Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.
Assays for pro-inflammatory markers were carried out by means of commercial ELISA kits according to manufacturer's instruction, as follows: SAA (KHA0012), IL-1β (KHC0011), IL-6 (KHC0062); TNFα (KHC3013), CRP (KHA0031), metalloproteinase 3 (MMP-3) (KAC1541) (all from Invitrogen-Life Technologies), cathepsin D (CATD) (ab119586, abcam), IL-1ra (KAC1181, BioSource Europe). Plates were read on a VersaMax microplate reader (Molecular Devices) using Ascent software (Thermo Scientific). Quantification of analytes was obtained against polynomial standard curves generated with appropriate standards.
Laboratory tests
Cholesterol and triglycerides were determined through an enzymatic colorimetric method, and high density lipoprotein (HDL)-cholesterol and low density lipoprotein (LDL)-cholesterol were determined through a homogeneous enzymatic colorimetric method on a Cobas® 6000, Roche/Hitachi cobas c system. Serum HGA levels were previously determined in Ranganath et al.
Suitability of Nitisinone in Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.
Briefly, 2.5 μL of serum were incubated with 50 μL of 22 μM 4-methylumbelliferyl-l-β-D-N,N′,N″-triacetylchitotriose (Sigma) in McIlvaine phosphate-citrate buffer (pH 5.2) for 1 h at 37°C. Reactions were terminated by adding 1.4 mL of 0.2 M glycine buffer (pH 10.8); fluorescence of 4-methylumbelliferone was read in a fluorimeter (Perkin Elmer; excitation 365 nm, emission 435 nm).
Advanced Oxidation Protein Products (AOPP)
AOPP were measured according to Witko-Sarsat et al.
Serum samples were diluted 1:10 or 1:20 in PBS and 200 μL applied to a 96-well microplate. Standards of chloramine T (200 μL; 0–100 μM) were added to the plate. KI (1.16 M, 10 μL) was added to all wells, followed 2 min later by bolus addition of 20 μL glacial acetic acid. The optical density was then read immediately at 340 nm on a microplate reader (VersaMax, Molecular Devices) using Softmax Pro software (Molecular Devices).
Thiols, S-thiolated proteins and Protein Thiolation Index (PTI)
Quantitative determination of free thiols and S-thiolated proteins was carried out according to Giustarini et al.
Briefly, one aliquot of serum (0.03 mL) was used to measure thiol levels by colorimetric reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) by spectrophotometer
. One additional aliquot of serum (0.1 mL) was treated with 0.1 mL of 2 mM N-ethylmaleimide (NEM) for 2 min and then deproteinized by addition of 18 μL of 60% (w/v) trichloroacetic acid (TCA). This second aliquot of serum was used to measure the level of S-thiolated proteins by HPLC. The protein pellets were treated with dithiothreitol (DTT) and the released protein-bound thiols were detected after their labelling with monobromobimane (mBrB)
. HPLC separation was performed on a C18 column (Zorbax Eclipse XDB-C18, 4.6 mm 150 mm, 5 mm, Agilent Technologies). Measurements were carried out with an Agilent series 1100 HPLC.
PTI was calculated as the molar ratio between total S-thiolated proteins (RSSP, where RS is cysteine, cysteinylglycine, homocysteine, γ-glutamylcysteine and glutathione) and the concentration of free, DTNB-titrable protein thiol groups
In SONIA2, quality of life of AKU patients was assessed through the following validated questionnaires:
•
Knee injury and Osteoarthritis Outcome Score (KOOS), evaluating both short- and long-term consequences of knee injury. It holds 42 items in five separately scored subscales [pain, other symptoms, function in daily living, function in sport and recreation, and knee-related quality of life (QoL)]. Scores are normalized to a “0–100” scale, with “0” representing extreme knee problems and “100” representing no knee problems.
•
Health Assessment Questionnaire (HAQ), including a disability index (haqDI) and a global pain visual analog scale (hapVAS). Eight categories are assessed: dressing and grooming, arising, eating, walking, hygiene, reach, grip, common daily activities. Results are scored from 0 (no difficulties) to 3 (unable to do).
•
Short Form-36 (SF-36), a multi-purpose short-form with 36 questions addressing both physical and mental status that measures patients' quality of life across eight domains: vitality, physical functioning, bodily pain, general health perception, physical role functioning, social functioning, emotional role functioning, mental health. A score of “0” indicates maximum disability, while a score of “100” indicates no disability.
•
AKUSSI, which incorporates multiple, clinically meaningful AKU outcomes combined with medical photography imaging investigations, and detailed questionnaires into a single score
. In this work, we limited to non-spine rheumatology (pain in 14 joints) and spine rheumatology (pain in four clinical spine regions) scores, expressed as percentages.
These scores were used to undertake correlation analyses with the measured markers, as detailed below.
Statistical analysis
Results were processed through Excel and GraphPad 6.0. Normal distribution was analysed with D'Agostino-Pearson or Shapiro Wilk test depending on sample size, and descriptive statistics was obtained for each analysed dataset.
When reference thresholds were available (SAA, CRP, AOPP, and chitotriosidase), the number and percentage of values above references were obtained. For IL-1β, IL-6, TNFα, MMP-3, CATD, protein thiols, S-thiolated proteins and PTI, comparisons were made between AKU and controls. For statistical comparisons, non-normally distributed data were previously log-transformed. Then unpaired t-test or ordinary one-way ANOVA followed by Tukey's multiple comparison test were carried out as appropriate. Statistically significant differences between groups were reported as difference between means and 95% confidence intervals of difference. Spearman's rank correlation analysis was also run on non-normally distributed data to investigate possible correlations between the tested markers and AKU patients' data [age, body mass index (BMI), haematological parameters and output of health questionnaires].
Results
The overall aim of this work was to assess baseline levels of established biomarkers related to inflammation and oxidative stress in serum from alkaptonuric patients who were/are enrolled in DevelopAKUre clinical trials. The tested biomarkers included well-known mediators of inflammatory responses (IL-6, IL-1β, TNFα and CRP) and SAA, which play a role in inflammation, oxidative stress, and AA amyloidosis. Serum levels of the following biomarkers were also tested: CATD, a lysosomal aspartic protease taking part in intracellular digestion of proteoglycan in the initial stages of osteoarticular inflammation
; IL-1 receptor antagonist (IL-1ra), which is specific for preventing the activity of IL-1α and IL-1β by competing with them for binding to the ligand-binding chain, termed type I (IL-1RI); MMP-3, which is involved in extracellular matrix remodelling and whose serum levels are increased in inflammatory rheumatic diseases
AOPP were tested as oxidative stress and potential inflammatory mediators, as they are found in several human diseases where these events are involved, such as chronic renal failure and
Advanced oxidation protein products promote inflammation in diabetic kidney through activation of renal nicotinamide adenine dinucleotide phosphate oxidase.
; conversely, only a minority (7/40; 17.5%) had CRP levels above the reference threshold of 5 mg/L. For those inflammatory markers with no established reference thresholds, values were compared between AKU and a control healthy population. Such an analysis revealed no statistically significant differences excepting for IL-1ra, slightly lower in AKU (Table I). Routinely assessed haematological parameters such as: glucose, cystatin C, alkaline phosphatase (data not shown), cholesterol, triglycerides and LDL-cholesterol were generally in range, whereas HDL-cholesterol scored below the reference range in several AKU subjects (96% males, 77% females) (Table 3S).
Table ISONIA1 inflammatory markers: descriptive statistics and comparisons between AKU and controls
SONIA1
AKU
CTR
Difference between means [95% CI interval of difference]
The possible dependence of the tested inflammatory biomarkers from age, BMI, smoking and drinking habits, gender and clinical site was evaluated. No statistically significant differences according to gender or cigarette smoking habits could be highlighted (data not shown). Conversely, we found that CATD serum levels were significantly increased in subjects drinking alcohol (Fig. 1) and that IL-1ra, TNFα and CRP serum levels were significantly higher in overweight/obese subjects (Fig. 1). Interestingly, CATD, IL-1β and MMP-3 showed also significantly different levels according to the clinical site (Fig. 1), like found previously in the same AKU population for extracellular matrix remodelling biomarkers
. Concomitant medications were not found to alter significantly the levels of the tested markers (data not shown). A positive and significant correlation was found for SAA and CRP, and several inflammatory biomarkers were positively correlated to BMI (SAA, IL-6, IL-1ra, TNFα and CRP) (Table 4S). Conversely, none of the tested biomarkers was statistically correlated to serum HGA levels (Table 4S).
Fig. 1Inflammatory markers tested in AKU subjects enrolled in SONIA1. Unpaired t-test (for alcohol drinking habits and clinical site) or one-way ANOVA with Tukey's multiple comparisons test (for BMI) were carried out on log-transformed values. Graphs report mean values ± stdev; tables include differences between means and 95% confidence intervals of differences (within square brackets). Statistically significant differences are highlighted in bold and indicated in graphs for BMI classification as follows: a) compared to normal subjects; b) compared to overweight subjects. Abbreviations: N (normal); OW (overweight); O (obese).
On the basis of SONIA1 results, SAA was the only marker among those tested in SONIA1 that was measured also in SONIA2. Chitotriosidase activity was included in this set of analysis as an additional marker of inflammation
were reported previously in smaller cohorts of AKU patients, AOPP, thiols, S-thiolated proteins and PTI were investigated in SONIA2 as well.
Descriptive statistics of AOPP, SAA and chitotriosidase activity is reported in Fig. 2. Interestingly, only six out of the 138 analysed samples (4%) showed AOPP levels above the reference value (set at 30 μmol/dL). Conversely, SAA serum levels ranged between 3 and 10 mg/L in 18 subjects (13%) and were above the threshold of 10 mg/L
in 119 out of the 138 analysed samples (86%). Chitotriosidase activity was above the reference value (set at 51 nmoL/mL/h) in 72 out of the 138 tested samples (52%) (Fig. 2). As for serum thiols, S-thiolated proteins and PTI, values in AKU were generally comparable to those of the control healthy population (Fig. 1S). Chitotriosidase and SAA showed also statistically significant differences according to age and BMI, respectively (Fig. 3 and Fig. 2S). Concomitant medications were not found to alter significantly the levels of the tested markers (data not shown).
Fig. 2Inflammatory and oxidative stress markers tested in AKU subjects enrolled in SONIA2. AOPP (μM chloramine T equivalents), SAA (mg/L), and chitotriosidase activity (nmoL/mL/h) values are reported in graphs. Grey lines indicate mean values ± stdev; red lines indicate reference limits (300 μM for AOPP, 10 mg/L for SAA and 51 nmoL/mL/h for chitotriosidase activity). Descriptive statistics summarised in the Table.
Fig. 3Variations in the levels of inflammatory and oxidative stress markers tested in AKU subjects enrolled in SONIA2 according to age group (upper panels) or BMI classification (lower panels). Ordinary one-way ANOVA and Tukey's multiple comparisons test were carried out. Statistically significant differences are indicated with letters as follows: a) compared to ≥30 y; b) compared to 31–40 y; c) compared to 41–50 y; d) compared to 51–60 y; f) compared to normal subjects. Differences between means and 95% confidence intervals of differences are reported in Fig. 1S. Abbreviations: U (underweight); N (normal); OW (overweight); O (obese); PTI: Protein Thiolation Index.
No statistically significant differences were found between male and female AKU subjects for all the markers tested in SONIA2 (data not shown). However, chitotriosidase activity and PTI (but not AOPP nor SAA) were found at statistically higher levels in older AKU subjects; furthermore, SAA levels were significantly higher in AKU obese subjects compared to those with a normal BMI (Fig. 3). Significant and positive correlations with age were found in AKU subjects for SAA levels (r = 0.3556, P < 0.0001), chitotriosidase activity (r = 0.4886, P < 0.0001) and PTI (r = 0.4954, P < 0.0001); SAA levels were also positively correlated to BMI (r = 0.3556, P < 0.0001).
When inflammatory and oxidative marker levels were correlated to the outcomes of health questionnaires, we found weak but statistically significant correlations indicating that high levels of SAA were more frequently associated both to a higher degree of difficulties in sport activities as well as to a reduced perceived knee-related quality of life (KOOS questionnaire). Similarly, patients with high PTI and chitotriosidase activity reported more frequently an increased severity of pain and symptoms, difficulties in daily activities and sport, and a reduced perceived knee-related quality of life (KOOS questionnaire) (Table II). We also found that high serum levels of SAA, PTI and chitotriosidase activity were more frequently associated to an increased perception of disability (haqDI, HAQ questionnaire) and to a reduced perceived physical health (i.e., lower levels of functioning according to SF-36) (Table II). Higher PTI values were positively associated to pain in multiple spine regions, and higher chitotriosidase activity was positively associated to joint and spinal pain (AKUSSI questionnaire) (Table II). Positive correlations were also found between PTI and SAA (r = 0.187, P = 0.032), and PTI and chitotriosidase (r = 0.392, P < 0.0001).
Table IICorrelation matrix between markers measured in SONIA2 study and output of patients' questionnaires. Spearman's r and P values are reported; statistically significant correlations highlighted in bold
. Though AKU represents the iconic prototype “inborn error of metabolism” and shares features with other more common rheumatic diseases also at the molecular level
A new light on alkaptonuria: a Fourier-transform infrared microscopy (FTIRM) and low energy X-ray fluorescence (LEXRF) microscopy correlative study on a rare disease.
, SAA was increased in the vast majority of samples and seemed the most promising biomarker to be assessed in AKU to monitor inflammation. Although in fewer AKU subjects, a similar trend was observed for chitotriosidase, a marker related to macrophage activation during inflammation
. Conversely, CRP levels were increased only in a minority of cases. Thus, superiority of SAA and chitotriosidase compared to CRP might be suggested in AKU similarly to what observed for SAA in patients suffering from Familial Mediterranean Fever (FMF)
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
Overall, our findings suggest that sub-clinical inflammation may be relevant in AKU and connected with the development of disease-related complications, similarly to other rheumatic conditions characterized by increased SAA levels, such as: osteoarthritis (OA)
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
Thematic review series: the immune system and atherogenesis. Lipoprotein-associated inflammatory proteins: markers or mediators of cardiovascular disease?.
, the chronic injury sustained by HGA in AKU may potentially deregulate the inflammatory process leading to aggressive resolution mechanisms, i.e., amyloidosis or persistent tissue destruction. In such a scenario, SAA may be considered a marker to monitor progression of both AKU and amyloidosis. Persistently elevated SAA levels represent a risk factor for the development of AA amyloidosis
. However, physiological and pathological functions of SAA are still partly unclear and differences between recombinant and endogenous SAA have been highlighted in in vitro assays, probably due to a difference in association to lipids
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
Endogenous acute phase serum amyloid A lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors.
. Unfortunately, no conclusions can be drawn on the presence of AA amyloidosis in SONIA1 and SONIA2 subjects, as this analysis was not included in the original study design.
We found that SAA serum levels were falling within a wide range in the tested AKU subjects. This becomes particularly relevant in the light of a recent work where HGA was found to act as an amyloid aggregation enhancer in vitro (in a time- and dose-dependent fashion) for amyloidogenic proteins and peptides
, pharmacological control of SAA circulating levels in AKU seems appropriate to be suggested, although such previous results were obtained with a recombinant protein and need further validation with wild-type SAA.
Unfortunately, direct measurement of SAA in AKU synovial fluid has not been reported yet, but since plasma SAA levels correlate with SAA levels in synovial fluid, passive diffusion of SAA from systemic circulation to synovial joint may be speculated
. This is particularly relevant due to the role that SAA might play in joint destruction through induction of metalloproteinases and collagen although different functions have been suggested for systemic and locally produced SAA isoforms, as well as for acute and constitutive SAA
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
A new light on alkaptonuria: a Fourier-transform infrared microscopy (FTIRM) and low energy X-ray fluorescence (LEXRF) microscopy correlative study on a rare disease.
. Mechanisms of ochronosis and pigment toxicity in AKU are not fully clear, but possible factors include the co-presence of amyloidosis and angiogenesis in tissues
In this study, SAA serum levels were increased regardless of AKU subjects' age (Fig. 3), although these parameters were positively correlated. Ageing is an important factor in OA progression
. AKU patients are symptom-free in the first decades of life, but our data suggest that a subclinical inflammation might be present even in young asymptomatic AKU subjects, indicating a possible role of SAA as an early biomarker of disease progression
. Cartilage degeneration (with alterations in collagen composition and depletion of proteoglycans), synovia inflammation and presence of amyloid in labial salivary gland can be detected even in young, asymptomatic AKU subjects
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
, positive association were found between SAA and BMI, since in obesity (where low-grade inflammation is found), adipose tissue is the major source of SAA, which can be considered an obesity-related inflammatory protein
Endogenous acute phase serum amyloid A lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors.
. Interestingly, we found that 90% of the tested AKU subjects enrolled in SONIA1 had lower levels of HDL than what established by reference guidelines. Chronic inflammation, as outlined in FMF, RA and SLE
Are familial Mediterranean fever (FMF) patients at increased risk for atherosclerosis? Impaired endothelial function and increased intima media thickness are found in FMF.
might alter the structure and functions of HDL, overall impairing HDL properties. In particular, a decreased antioxidant activity of HDL might follow displacement of ApoA-I from HDL due to high SAA. Since altered profiles in apolipoproteins were documented by comparative proteomics of AKU serum
, this topic deserves further investigations in AKU.
No statistically significant correlations were found, conversely, between the tested biomarkers and serum HGA (Table 4S). This could be explained by the fact that serum HGA in AKU depends on residual HGD enzyme activity (inter-subject variability) and on tyrosine intake with the diet (inter- and intra-subject variability)
Serum matrix metalloproteinase-3 in comparison with acute phase proteins as a marker of disease activity and radiographic damage in early rheumatoid arthritis.
are markers of disease activity and severity in several rheumatic conditions. Here we provided evidence that AKU patients presenting with significantly higher SAA and chitotriosidase activity (enhanced inflammation) and higher PTI (enhanced oxidative stress) reported more often a decreased quality of life (as assessed through patients' health questionnaires) and scored higher in the AKUSSI scale for joint and spinal pain. This suggests that worsening of symptoms in AKU is paralleled by increased inflammation and oxidative stress, which might play a role in AKU progression. Consequently, SAA, chitotriosidase activity and PTI might be proposed as disease activity and severity markers in AKU, although further evidence is needed.
Conclusions
Due to the ultra-rarity of the disease (affecting 1:250,000–1,000,000
), we were given an invaluable opportunity, as we were able to test for the very first time a high number of alkaptonuric serum specimens collected and stored under standardised procedures. This was also the first time that several inflammatory and oxidative stress biomarkers could be investigated in vivo in AKU.
We found increased SAA and chitotriosidase activity in the vast majority of AKU samples, indicating increased systemic inflammation. Conversely, oxidative stress biomarkers were not significantly different when compared to a normal population. SAA, but especially PTI and chitotriosidase activity were correlated to AKU severity, as assessed through validated health questionnaires, and AKUSSI, indicating a role for both oxidative stress and inflammation in AKU progression and severity. Prospectively, routine assessment of SAA should be recommended in AKU so that proper interventions could be put in place to address the inflammatory-pro-amyloidogenic component of the disease. For instance, low dose methotrexate (MTX) can down-regulate inflammation
. This is also relevant in view of the recent in vitro reports indicating that even nearly physiological HGA concentrations might enhance the aggregation of SAA
Importantly, all the data obtained within this work could be used to populate an AKU database integrating biomarker levels, demographics, patient's quality of life, environmental and life-style data, and clinical outcomes
. Such a database could represent an optimal tool with potential relapses for the study of AKU mechanisms and the development of a precision medicine approach for AKU and other more common rheumatic disorders.
Authors' contribution
All authors contributed to the conception and design of the study, acquisition, analysis or interpretation of the data. All authors were also involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version. Annalisa Santucci ([email protected]) as the corresponding author takes responsibility of the integrity of the work as a whole, from inception to finished article.
Competing interest statement
The authors have no conflicts of interest to declare.
Role of the funding source
This work was supported by European Commission Seventh Framework Programme funding granted in 2012 (DevelopAKUre, project number: 304985). The funding source was not involved in the study design, collection, analysis and interpretation of data, the writing of the manuscript, or in the decision to submit the manuscript for publication.
Acknowledgements
The authors thank aim AKU, Associazione Italiana Malati di Alcaptonuria (ORPHA263402), and Prof. Gabriele Cevenini for assistance with statistical analyses. This work is in memory of Dr Duccio Calamandrei.
Appendix A. Supplementary data
The following is the supplementary data related to this article:
Suitability of Nitisinone in Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.
Advanced oxidation protein products promote inflammation in diabetic kidney through activation of renal nicotinamide adenine dinucleotide phosphate oxidase.
A new light on alkaptonuria: a Fourier-transform infrared microscopy (FTIRM) and low energy X-ray fluorescence (LEXRF) microscopy correlative study on a rare disease.
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
Thematic review series: the immune system and atherogenesis. Lipoprotein-associated inflammatory proteins: markers or mediators of cardiovascular disease?.
Endogenous acute phase serum amyloid A lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors.
Are familial Mediterranean fever (FMF) patients at increased risk for atherosclerosis? Impaired endothelial function and increased intima media thickness are found in FMF.
Serum matrix metalloproteinase-3 in comparison with acute phase proteins as a marker of disease activity and radiographic damage in early rheumatoid arthritis.
31293-7&id=gr1.jpg){kind=link}
31293-7&id=gr2.jpg){kind=link}
31293-7&id=gr3.jpg){kind=link}