Are advanced glycation end-product-modified proteins of pathogenetic importance in fibromyalgia?

G. Hein and S. Franke

Department of Internal Medicine IV, Rheumatology and Osteology, Friedrich-Schiller-University of Jena, 07740 Jena, Germany


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Pentosidine
 Results
 Discussion
 References
 
Objective. To quantify the serum levels of the advanced glycation end-product (AGE) pentosidine in 41 patients with fibromyalgia (FM) and 46 healthy controls. The formation of pentosidine is closely related to oxidative stress.

Methods. Pentosidine was measured by reverse-phased high-performance liquid chromatography with gradient separation on a RP-18 column.

Results. Patients with FM have significantly higher pentosidine serum levels than healthy subjects.

Conclusion. AGE modification of proteins leads to reduced solubility and high resistance to proteolytic digestion of such altered proteins (e.g. AGE-modified collagens). AGEs are also able to stimulate different kinds of cells via activation of the NF{kappa}B, mediated by specific receptors of AGEs (e.g. RAGE) on the cell surface. Both mechanisms may contribute to the development, perpetuation and spreading of pain phenomena in FM patients.

KEY WORDS: Fibromyalgia, Advanced glycation end-product, Pentosidine, Collagen cross-linking, Pain.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Pentosidine
 Results
 Discussion
 References
 
Although fibromyalgia (FM) is a frequent rheumatological disorder, its aetiology and pathogenesis are still unknown.

There are two different models for the pathogenesis of FM. Contrary to the situation a few years ago, the most widely accepted hypothesis now evokes CNS mechanisms like nociception and allodynia rather than pathogenetically painful muscles [1].

On the other hand, there are many findings supporting the hypothesis of primary damage of the muscle tissue. It is also possible that different endogenic and exogenic factors (e.g. psychogenic factors [2], muscle overload, disturbed posture of spine [3], disturbed sleep [4], etc.) lead to chronic local hypoxia [5, 6] in muscle tissue, resulting in structural and functional disturbances of muscles and secondary alteration of nociception.

As early as 1973, Fassbender et al. [5] hypothesized ‘degenerative’ changes in the muscles of FM patients, based on morphological studies. Besides decreased levels of adenosine diphosphate and phosphoryl creatine, they described slight, but frequent histopathological and also histochemical changes as compared with normal muscle tissue.

In particular, they reported the step-wise destruction of myofilaments and swollen endothelial cells. These changes may be associated with the relative hypoxia of the muscle cells in FM patients.

It is known that oxidative stress accelerates the generation of advanced glycation end-products (AGEs).

AGEs are formed by non-enzymatic reactions between sugar-derived aldehyde groups and protein amino groups, known as the Maillard reaction. They constitute a heterogeneous class of structures, mainly characterized by a brown colour, fluorescence, a tendency to polymerization and biological recognition through AGE-specific receptors (e.g. RAGE). A remarkable feature of such AGE-mediated cross-linked proteins is a decrease in solubility and a high resistance to proteolytic digestion [79].

The formation of the AGE pentosidine, a fluorescent cross-link structure of lysine and arginine, requires both glycation and oxidation and is closely related to oxidative processes [10, 11].

AGEs accumulated in vivo are implicated in the process of ageing as well as in the pathogenesis of several diseases, including: diabetes, atherosclerosis, Alzheimer's disease, and renal failure. They are also seen in patients undergoing renal replacement therapy with maintenance haemodialysis [1215].

Also, in chronic inflammatory rheumatic diseases such as rheumatoid arthritis, elevated levels of pentosidine are found. Recent studies have demonstrated increased pentosidine concentrations in the serum, urine and cartilage of patients with rheumatoid arthritis [1618]. Moreover, significant correlations have been found between serum pentosidine and disease activity. These results may indicate an enhanced formation of AGEs in rheumatoid arthritis caused by increased oxidative stress, and are possibly of pathogenic importance in this disease.

It can be hypothesized that oxidative stress in muscle tissue, as well as in the enthesis region, accelerates the formation and accumulation of AGEs; resulting in a disturbed remodelling of the tissue (particularly of the connective tissue structures such as collagen), and an increased synthesis of pro-inflammatory cytokines, probably affecting pain perception.

The aim of this study was to measure the pentosidine levels in the serum of patients with FM and to compare the results with those obtained in age- and sex-related controls. A significant difference in pentosidine serum levels could be a pathogenetically relevant finding.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Pentosidine
 Results
 Discussion
 References
 
The study included 41 patients with FM and 56 healthy subjects (HS). All patients were recruited consecutively in the outpatient department of a rheumatological division (Department of Internal Medicine IV, Friedrich-Schiller-University of Jena) and fulfilled the ACR criteria for diagnosis of FM before the beginning of the therapeutic procedures. The overall pain was measured by the visual analogue scale (VAS). The demographic data of the patients and controls are given in Table 1Go. No significant difference existed between the patients' age (ranging from 15 to 65 yr) and the age of HS (ranging from 21 to 75 yr). In both groups the age levels were normally distributed. No subjects suffered from diabetes mellitus, renal diseases or any other known conditions influencing the AGE status.


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TABLE 1. Epidemiological data of FM patients and HS

 
Thirty-eight FM patients, but no HS, were taking analgesics [NSAID n=20, paracetamol n=8, katadolon® (flupirtine) n=10]; additionally, 10 patients were taking myotonolytic/psychotropic drugs [valium® (diazepam), musaril® (tetrazepam)]. A washout of 3 days was done on all patients.

Non-heparinized blood was collected, centrifuged at 3800g for 5 min and stored at -80°C until testing.


    Pentosidine
 Top
 Abstract
 Introduction
 Patients and methods
 Pentosidine
 Results
 Discussion
 References
 
Pentosidine measurement was performed using the HPLC assay described by Miyata et al. [19].

Serum samples were hydrolysed with 5 N HCl at 110°C under a nitrogen atmosphere for 16 h, subsequently neutralized with 5 N NaOH and 0.5 M phosphate buffer (pH 7.4), filtered through a 0.45 µm-pore filter, and diluted with phosphate-buffered saline (PBS).

Pentosidine was analysed by reverse-phased HPLC with gradient separation on a RP-18 column (Merck, Germany) under fluorescence detection (excitation/emission wavelength: 335/385 nm). Synthetic pentosidine was used to obtain a standard curve (kindly provided by T. Miyata, Tokai University, Japan).

The intra- and inter-assay coefficients of variation were <3% and <6%, respectively.

Measurement of all samples was done in one batch.

Statistics
The results are given as means with standard deviations (mean±S.D.) and medians. Statistics were performed using the Mann–Whitney U-test for comparison of unpaired samples and the Spearman correlation test; a P-value <0.05 was considered to be significant.


    Results
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 Abstract
 Introduction
 Patients and methods
 Pentosidine
 Results
 Discussion
 References
 
The serum concentrations of pentosidine were significantly elevated in patients with FM as compared with HS (Fig. 1Go).



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FIG. 1. Serum levels of pentosidine in patients with FM and HS.

 
No significant differences existed between males and females in the patient group nor in the HS (Table 2Go). In the healthy subjects but not in the FM patients, pentosidine was significantly age-related (r=0.440, P=0.001) (Figs 2 and 3GoGo).


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TABLE 2. Pentosidine serum levels in male and female patients (FM) and healthy subjects (HS)

 


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FIG. 2. Absence of correlation between the serum pentosidine levels and age in patients with FM.

 


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FIG. 3. Positive correlation between the serum pentosidine levels and age in HS.

 

We were not able to show any correlation between the severity of fibromyalgic pain syndrome (number of painful tender points as well as quantification of overall pain by VAS, data not shown) and the serum levels of pentosidine.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Pentosidine
 Results
 Discussion
 References
 
FM is one of the most frequent rheumatological disorders [1, 20]. However, there are different points of view concerning the classification of this disorder and the acceptance of FM as a disease entity [1]. Moreover, there are different opinions concerning the pathogenetic process underlying this disease.

A number of studies of muscle histology and energy metabolism suggest a possible pathological and pathobiochemical basis in the muscle tissue [2123]. An abnormal microcirculation leading to local hypoxia of muscle tissue [5], in the skin surface above the trigger points [6] as well as skin above the tender points [24], is described.

The common finding seems to be a tissue hypoxia of muscles and the related enthesis region.

However, it is also obvious that tissue hypoxia is one of the causes for an accelerated generation of AGEs.

The generation of AGEs is an inevitable process in vivo. They are formed by non-enzymatic reactions of sugar-derived aldehyde groups with protein amino groups (the Maillard reaction). The AGEs induce an increased cross-linking of proteins, resulting in a decrease of solubility and a high resistance to proteolytic digestion [79].

Furthermore, AGEs bind to cell surface receptors and AGE-binding proteins (e.g. RAGE, AGE-R1, AGE-R2, AGE-R3, macrophages scavanger receptor). AGE receptors are found in monocytes/macrophages, endothelial cells, T lymphocytes, fibroblasts and others [2527]. The AGE–RAGE interaction induces activation of NF{kappa}B, resulting in increased expressions of, for example, cytokines, growth factors and adhesion molecules.

Such AGE-induced and RAGE-mediated NF{kappa}B activation has been demonstrable in neurons, endothelial cells, mesangial cells, smooth muscle cells and monocytes/macrophages and is much more prolonged than the activation of NF{kappa}B by cytokines [28].

Although it cannot be excluded that our results only reveal an ‘epiphenomenon’, the increased pentosidine levels in FM patients may provide evidence that AGE-modified proteins are involved in the pathogenesis of FM as well.

Sprott et al. [29] has pointed out that evidence of a disturbed local collagen metabolism could be found in FM. They showed that there are visible collagen cuff sheaths around pre-terminal nerve fibres in the subepidermal connective tissue of FM skin taken from tender points of the trapezius region. No such changes were observed in any control samples [29]. They also found significant alterations in the excretion of collagen cross-links (pyridinoline, deoxypyridinoline) and hydroxyproline in FM patients as compared with the controls, and a tendency toward normalizing, especially of the pyridinoline/deoxypyridinoline ratio, after effective therapy with acupuncture [30].

AGE modification, particularly of long-lived proteins like collagens, leads to an alteration of the tissue protein structure and function.

The more intensive cross-linking of collagen fibrils caused by these modifications result in a proteolytic resistance of collagenous structures, which may contribute to the process of increased collagen deposition around the nerve fibres.

It seems that this local alteration of collagen metabolism resulting in ‘collagen cuff sheaths' around sensitive neurons (as described by Sprott et al. [29, 30]) may be involved in the development of inflammatory pain, hypothesized by Weihe et al. [31] as ‘neurogenic inflammation’. It is known that afferent sensitive neurons act as pain receptors [32]. So, it may be that collagen deposits around the neurons could act as compressive lesions or, on the other hand, may disturb the diffusion, traffic and binding of neuropeptides/monoamines thereby contributing to the pain syndrome, as experienced by patients with FM.

On the other hand the AGE modification of proteins, accelerated by a hypothesized tissue hypoxia in the muscles, tendons and related skin tissues of FM patients, may also induce cell activation of, for example, macrophages, fibroblasts or periphereal nerve cells via the binding of AGE structures to their cellular receptor RAGE, followed by a prolonged activation of NF{kappa}B. Unlike unmodified proteins, AGE-modified proteins are able to stimulate the secretion of pro-inflammatory cytokines in RAGE-bearing cells.

Pro-inflammatory cytokines in turn may contribute to pain generation and perpetuation.

Both mechanisms, caused by the AGE modification of proteins, ultimately interact with one another, and in a possible circulus vitiosus, the clinical symptomatology swells leading to the spreading and increased levels of pain as often seen in patients with FM.


    Notes
 
Correspondence to: G. Hein. Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Pentosidine
 Results
 Discussion
 References
 

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Submitted 20 March 2002; Accepted 25 March 2002