Sensitive fluorimetric quantitation of pyridinium and pentosidine crosslinks in biological samples in a single high-performance liquid chromatographic run

Bank, Ruud A.; Beekman, B.; Verzijl, Nicole; Roos, Jeroen A.D.M. de; Sakkee, A.N.; TeKoppele, J.M.

doi:10.1016/s0378-4347(97)00391-5

Cited by 163 publications

(138 citation statements)

References 21 publications

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“…Within 1 hour after dissection, full-thickness cartilage slices (excluding the subchondral bone) were cut into square pieces (3-10 mg) and processed for histologic and biochemical analyses (40). Tissue pentosidine levels (2 cartilage samples per dog per joint) were determined by reverse-phase highperformance liquid chromatography after acid hydrolysis, as previously described (21,42). In addition, pentosidine levels were determined in stored cartilage samples (Ϫ20°C; remnants from unrelated studies) of young (mean Ϯ SEM 1.5 Ϯ 0.2 years; n ϭ 20) and old (4.0 Ϯ 0.9 years; n ϭ 8) dogs.…”

Section: Methodsmentioning

confidence: 99%

Accumulation of advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis

DeGroot¹,

Verzijl²,

Wijk

et al. 2004

Arthritis & Rheumatism

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177

109

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Objective. Osteoarthritis (OA) is one of the most prevalent and disabling chronic conditions affecting the elderly. Its etiology is largely unknown, but age is the most prominent risk factor. The current study was designed to test whether accumulation of advanced glycation end products (AGEs), which are known to adversely affect cartilage turnover and mechanical properties, provides a molecular mechanism by which aging contributes to the development of OA.Methods. The hypothesis that elevated AGE levels predispose to the development of OA was tested in the canine anterior cruciate ligament transection (ACLT) model of experimental OA. Cartilage AGE levels were enhanced in young dogs by intraarticular injections of ribose. This mimics the accumulation of AGEs without the interference of other age-related changes. The severity of OA was then assessed 7 weeks after ACLT surgery in dogs with normal versus enhanced AGE levels.Results. Intraarticular injections of ribose enhanced cartilage AGE levels ϳ5-fold, which is similar to the normal increase that is observed in old dogs. ACLT surgery resulted in more-pronounced OA in dogs with enhanced AGE levels. This was observed as increased collagen damage and enhanced release of proteoglycans. The attempt to repair the matrix damage was impaired; proteoglycan synthesis and retention were decreased at enhanced AGE levels. Mankin grading of histology sections also revealed more-severe OA in animals with enhanced AGE levels.Conclusion. These findings demonstrate increased severity of OA at higher cartilage AGE levels and provide the first in vivo experimental evidence for a molecular mechanism by which aging may predispose to the development of OA.The population of Western society is aging rapidly. Consequently, age-related diseases will increase greatly over the coming decades and will have a great impact on the quality of life of the elderly. Osteoarthritis (OA) is one of the most prevalent and disabling chronic conditions affecting the elderly and poses a significant public health problem (1). The most prominent feature of OA is the progressive destruction of articular cartilage, resulting in impaired joint motion, severe pain, and, ultimately, disability (2). As yet, the etiology of OA remains largely unknown. The incidence of OA increases with age: Ͼ50% of the population over 60 years of age is affected (3,4). Although age is identified as the main risk factor for the development of OA, the mechanism by which aging is involved remains unclear. Age-related changes in the articular cartilage are expected to play an important role in the susceptibility of cartilage to OA.Articular cartilage derives its mechanical properties from its extracellular matrix. This matrix is composed of type II collagen, which forms a 3-dimensional network that provides the cartilage with resistance to tensile forces (5).

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Section: Methodsmentioning

confidence: 99%

Accumulation of advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis

DeGroot¹,

Verzijl²,

Wijk

et al. 2004

Arthritis & Rheumatism

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177

109

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“…The crosslinks were analyzed by reverse-phase high-performance liquid chromatography (23). The amounts of cross-links were calculated based on an internal pyridoxine standard and a cross-link standard containing pyridoxine, HP, and LP.…”

Section: Construction Of the Targeting Vector And Gene Targeting Inmentioning

confidence: 99%

Tissue-specific Changes in the Hydroxylysine Content and Cross-links of Collagens and Alterations in Fibril Morphology in Lysyl Hydroxylase 1 Knock-out Mice

Takaluoma

Hyry

Lantto

et al. 2007

Journal of Biological Chemistry

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We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1 ؊/؊ mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1 ؊/؊ aortas showed degenerative changes. Collagen fibrils in the Plod1 ؊/؊ aorta and skin had an abnormal morphology. The LH activity level in the Plod1 ؊/؊ skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1 ؊/؊ tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1 ؊/؊ tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1 ؊/؊ collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1 ؊/؊ mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.Lysyl hydroxylase (LH, 2 EC 1.14.11.4) catalyzes the hydroxylation of lysine residues mainly but not exclusively in -X-LysGly-triplets in collagens and proteins with collagen-like sequences (1). The enzyme resides within the endoplasmic reticulum and has three human and mouse isoenzymes, LHs 1-3 (1-7). The hydroxylysine residues formed have two important functions: they are essential for the stability of the intermolecular cross-links that provide the collagen fibrils with their tensile strength and mechanical stability, and they serve as attachment sites for carbohydrate units, either the monosaccharide galactose or the disaccharide glucosylgalactose (1). Collagen cross-link formation occurs in the extracellular matrix and is initiated by the conversion of specific lysine or hydroxylysine residues in the telopeptides, i.e. the short non-triple helical ends of collagen molecules, into the aldehydes allysine or hydroxyallysine, respectively, catalyzed by lysyl oxidase (8). The telopeptides are connected with the triple helical part of an adjacent molecule by difunctional immature cross-links in the characteristic staggered array of collagen molecules in a fibril. The three main fibril-forming collagens, types I, II, and III, have four cross-linking sites, one in each telopeptide and two in the triple helical region, close to its N-and C-terminal ends (9). If the residue in the telopeptide is hydroxyallysine, the difunctional cross-links can mature into trifunctional non-reducible cross-links comprising lysylpyri...

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“…In short, dried hydrolysates were dissolved in internal standard solution containing 10 µM pyridoxine (Sigma) and 2.4 mM homoarginine (Sigma). For pentosidine analysis, samples were diluted fivefold with 0.5 % (v\v) heptafluorobutyric acid (Fluka) in 10 % (v\v) acetonitrile (Rathburn, Walkerburn, Scotland, U.K.) and analysed by HPLC [25]. Pentosidine was generously given by Professor V. M. Monnier (Case Western Reserve University, Cleveland, OH, U.S.A.), and calibrated against our pentosidine standard [10].…”

Section: Analytical Proceduresmentioning

confidence: 99%

“…The FL, CML and CEL content of the collagen samples is expressed as mmol per mol of lysine residues. Historical data for lens protein and skin collagen have been determined using the same methodology as described above [1,2,9,21].Pentosidine content and amino acid composition were determined by HPLC as described previously [24,25]. In short, dried hydrolysates were dissolved in internal standard solution containing 10 µM pyridoxine (Sigma) and 2.4 mM homoarginine (Sigma).…”

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confidence: 99%

“…The quantities of arginine and the sum of the primary amines hydroxylysine and lysine, i.e. (hydroxy-)lysine, are expressed as mol per mol of collagen, assuming 300 hydroxyproline residues per triple-helical collagen molecule [25].Collagen-linked fluorescence and browning were measured in cartilage and skin collagen (1-5 mg) digested for 2 h at 65 mC with 2.5 units\ml of papain (from Papaya latex, Sigma) in 300 µl of papain buffer [50 mM phosphate buffer (pH 6.5), 2 mM -cysteine and 2 mM EDTA] [26]. Fluorescence of the papain digests was measured at λ ex l 360 nm and λ em l 460 nm in a Cytofluor II Multi-Well Plate Reader (PerSeptive Biosystems), because those filters were available for the plate reader.…”

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confidence: 99%

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Age-related accumulation of Maillard reaction products in human articular cartilage collagen

Verzijl

DeGroot

Oldehinkel³

et al. 2000

Biochemical Journal

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235

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Non-enzymic modification of tissue proteins by reducing sugars, the so-called Maillard reaction, is a prominent feature of aging. In articular cartilage, relatively high levels of the advanced glycation end product (AGE) pentosidine accumulate with age. Higher pentosidine levels have been associated with a stiffer collagen network in cartilage. However, even in cartilage, pentosidine levels themselves represent 1 cross-link per 20 collagen molecules, and as such cannot be expected to contribute substantially to the increase in collagen network stiffness. In the present study, we investigated a broad range of Maillard reaction products in cartilage collagen in order to determine whether pentosidine serves as an adequate marker for AGE levels. Not only did the well-characterized AGEs pentosidine, N ε -(carboxymethyl)lysine, and N ε -(carboxyethyl)lysine increase with age in cartilage collagen (all P 0.0001), but also general measures of AGE cross-linking, such as browning and fluorescence (both P 0.0001), increased. The levels of these AGEs are all higher in cartilage collagen than in skin collagen. INTRODUCTIONDuring aging, long-lived proteins such as collagen and eye lens proteins are non-enzymically modified by reducing sugars. The major initial product is fructose-lysine (FL) [1,2], which results from glycation of ε-amino groups on lysine residues. In subsequent Maillard or browning reactions, products known as advanced glycation end products (AGEs) are formed from FL [3,4], and accumulate with age in long-lived proteins [1,2,[5][6][7][8]. These AGEs include structurally characterized adducts, such as N ε -(carboxymethyl)lysine (CML) [1][2][3] and N ε -(carboxyethyl)-lysine (CEL) [9], fluorescent cross-links, such as pentosidine formed between lysine and arginine residues [5,10], as well as chemically unidentified compounds which result in proteinbound browning or fluorescence, and cross-linking [11][12][13][14].In comparison with other collagen-rich tissues such as skin, cartilage contains relatively large amounts of pentosidine [5,15]. Pentosidine levels in articular cartilage increase linearly with age [6-8], as was previously described for skin collagen [16] and lens proteins [10]. Pentosidine is also present in the proteoglycans in articular cartilage [17], but appears to be localized predominantly in the collagen component of the tissue [7]. Age-related accumulation of AGE cross-links in articular cartilage collagen may result in increased stiffening of the collagen network, as was shown after in itro ribosylation of cartilage [7]. Thus AGE cross-linking in i o may contribute to the age-related impairment of the ability of articular collagen to resist mechanically induced damage and eventually cartilage degeneration [18][19][20].Abbreviations used : AGE, advanced glycation end product ; CEL, N ε -(carboxyethyl)lysine ; CML, N ε -(carboxymethyl)lysine ; FL, fructose-lysine. 1 To whom correspondence should be addressed (e-mail JM.TeKoppele!pg.tno.nl).As a functional measure of glycation the digestibility o...

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Sensitive fluorimetric quantitation of pyridinium and pentosidine crosslinks in biological samples in a single high-performance liquid chromatographic run

Cited by 163 publications

References 21 publications

Accumulation of advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis

Accumulation of advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis

Tissue-specific Changes in the Hydroxylysine Content and Cross-links of Collagens and Alterations in Fibril Morphology in Lysyl Hydroxylase 1 Knock-out Mice

Age-related accumulation of Maillard reaction products in human articular cartilage collagen

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