RESUMO
Intermolecular cross-linking of bone collagen is intimately related to the way collagen molecules are arranged in a fibril, imparts certain mechanical properties to the fibril, and may be involved in the initiation of mineralization. Raman microspectroscopy allows the analysis of minimally processed bone blocks and provides simultaneous information on both the mineral and organic matrix (mainly type I collagen) components, with a spatial resolution of ~1 µm. The aim of the present study was to validate Raman spectroscopic parameters describing one of the major mineralizing type I trivalent cross-links, namely pyridinoline (PYD). To achieve this, a series of collagen cross-linked peptides with known PYD content (as determined by HPLC analysis), human bone, porcine skin, predentin and dentin animal model tissues were analyzed by Raman microspectroscopy. The results of the present study confirm that it is feasible to monitor PYD trivalent collagen cross-links by Raman spectroscopic analysis in mineralized tissues, exclusively through a Raman band ~1660 wavenumbers. This allows determination of the relative PYD content in undecalcified bone tissues with a spatial resolution of ~1 µm, thus enabling correlations with histologic and histomorphometric parameters.
Assuntos
Aminoácidos/metabolismo , Osso e Ossos/metabolismo , Colágeno/metabolismo , Análise Espectral Raman , Reagentes de Ligações Cruzadas , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Análise Espectral Raman/métodos , Dente/patologiaRESUMO
Lysyl hydroxylase 3 (LH3, encoded by PLOD3) is a multifunctional enzyme capable of catalyzing hydroxylation of lysyl residues and O-glycosylation of hydroxylysyl residues producing either monosaccharide (Gal) or disaccharide (Glc-Gal) derivatives, reactions that form part of the many posttranslational modifications required during collagen biosynthesis. Animal studies have confirmed the importance of LH3, particularly in biosynthesis of the highly glycosylated type IV and VI collagens, but to date, the functional significance in vivo of this enzyme in man is predominantly unknown. We report here a human disorder of LH3 presenting as a compound heterozygote with recessive inheritance. One mutation dramatically reduced the sugar-transfer activity of LH3, whereas another abrogated lysyl hydroxylase activity; these changes were accompanied by reduced LH3 protein levels in cells. The disorder has a unique phenotype causing severe morbidity as a result of features that overlap with a number of known collagen disorders.
Assuntos
Doenças do Tecido Conjuntivo/genética , Mutação , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Adolescente , Sequência de Bases , Colágeno/metabolismo , Saúde da Família , Feminino , Glicosilação , Heterozigoto , Humanos , Masculino , Modelos Biológicos , Fenótipo , Análise de Sequência de DNARESUMO
Protein-bound pyrroles are a sign of oxidative damage. Here we report a specific method for detecting pyrrole-containing proteins using biotin-labeled Ehrlich's reagent (ER-B). After treatment of either human serum or isolated human serum proteins with various oxidizing agents, damaged, biotin-labeled components could be detected by blotting. Combining the use of ER-B with proteomic techniques allowed human serum proteins susceptible to oxidative damage to be detected and then identified by LC/MS/MS. Identification of such proteins in different human conditions such as obesity, diabetes, and cardiovascular disease should lead to the discovery of new biomarkers and the development of specific assays to monitor health status.
Assuntos
Benzaldeídos/química , Biotina/análogos & derivados , Proteínas Sanguíneas/química , Proteômica/métodos , Pirróis/química , Biotina/química , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Western Blotting , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Humanos , Indicadores e Reagentes/química , Estresse Oxidativo , Pirróis/análise , Espectrometria de Massas em TandemRESUMO
Although median levels of bone turnover are increased in postmenopausal women, it is unclear whether the low circulating levels of endogenous estrogen exert a regulatory role on these levels. This issue was evaluated by assessing the effect of a blockade of estrogen synthesis on bone turnover markers in 42 normal women (mean age +/- SD, 69 +/- 5 years) randomly assigned to groups receiving the potent aromatase inhibitor letrozole or placebo for 6 months. Letrozole treatment reduced serum estrone (E1) and estradiol (E2) to near undetectable levels (p < 0.0001). This treatment did not affect bone formation markers but, as compared with the placebo group, increased bone resorption markers (urine 24-h pyridinoline [PYD] by 13.3% [p < 0.05] and 24-h urine deoxypyridinoline [DPD] by 14.2% [p < 0.05]) and decreased serum parathyroid hormone (PTH) by 22% (p = 0.002). These data indicate that in late postmenopausal women even the low serum estrogen levels present exert a restraining effect on bone turnover and support the concept that variations in these low levels may contribute to differences in their rate of bone loss.
Assuntos
Reabsorção Óssea/sangue , Estrogênios/sangue , Idoso , Inibidores da Aromatase , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/fisiologia , Reabsorção Óssea/induzido quimicamente , Método Duplo-Cego , Inibidores Enzimáticos/farmacologia , Estradiol/sangue , Estrona/sangue , Feminino , Humanos , Letrozol , Menopausa/sangue , Pessoa de Meia-Idade , Nitrilas/farmacologia , Osteoporose Pós-Menopausa/sangue , Estudos Prospectivos , Triazóis/farmacologiaRESUMO
In an attempt to identify potential staging markers of effective healing, changes in connective tissue properties were measured in a human skin excisional wound healing model in which tissue was re-excised at intervals up to 6 months after injury. The proportion of collagen III relative to collagen I increased significantly (p<0.001) up to 6 weeks after initial injury and remained elevated up to 6 months, at which time the proportion of collagen III was 70% above baseline values. Extractability of biopsy tissue collagen by pepsin increased significantly throughout the study (baseline, 32.8+/-6.8%; 6 months, 89.1+/-8.9%), with inverse changes in the mature skin cross-link, histidinohydroxylysinonorleucine (baseline, 1.18+/-0.11 mol/mol collagen; 6 months, 0.27+/-0.09 mol/mol collagen). Pyridinoline content increased over the period of the study, although remaining at relatively low concentrations (baseline, 0.037+/-0.011; 6 months, 0.063+/-0.014 mol/mol collagen), and the pyridinoline/deoxypyridinoline ratio was significantly higher (baseline, 3.5+/-0.6; 6 months, 10.3+/-2.2). Elastin content, measured as desmosine cross-links, decreased significantly in the first 3 weeks and continued to decline over the period of study. Overall, the data suggest that remodeling of the wound tissue continues at least up to 6 months after injury. The close inverse correlation between histidinohydroxylysinonorleucine concentrations and extractability by pepsin (r2=0.89, p<0.0001) suggests a causal relationship, consistent with the likely effects of a substantial network of mature, inter-helical bonds in collagen.
Assuntos
Colágeno Tipo III/metabolismo , Colágeno/isolamento & purificação , Histidina/análogos & derivados , Pele/química , Pele/lesões , Cicatrização , Ferimentos Penetrantes/fisiopatologia , Adulto , Aminoácidos/metabolismo , Colágeno Tipo I/metabolismo , Dipeptídeos/metabolismo , Elastina/metabolismo , Histidina/metabolismo , Humanos , Masculino , Fatores de TempoRESUMO
BACKGROUND: The link between acid-base homeostasis and skeletal integrity has gained increasing prominence in the literature. Estimation of the net rate of endogenous non-carbonic acid production (NEAP) from dietary protein and potassium content enables exploration of the effects of dietary acidity or alkalinity on bone. OBJECTIVE: The study aimed to ascertain whether lower dietary acidity (lower dietary protein intake but higher potassium intake-ie, low estimate of NEAP) was associated with greater axial and peripheral bone mass and less bone turnover, independent of key confounding factors. DESIGN: Baseline (cross-sectional) results of a population-based study were examined further. The database includes spine and hip bone mineral density (BMD) in 1056 premenopausal or perimenopausal women aged 45-54 y and forearm bone mass and the urinary markers of bone resorption in 62 women. A validated food-frequency questionnaire was used to measure dietary intakes. RESULTS: Lower estimates of energy-adjusted NEAP were correlated with greater spine and hip BMD and greater forearm bone mass (P < 0.02 to P < 0.05). Hip and forearm bone mass decreased significantly across increasing quartiles of energy-adjusted NEAP (P < 0.02 to P < 0.03), and trends at the spine were similar (P < 0.09). Differences remained significant after adjustment for age, weight, height, and menstrual status. Lower estimates of energy-adjusted NEAP were also correlated with lower excretion of deoxypyridinoline and were significant predictors of spine and forearm bone mass. CONCLUSIONS: These novel findings provide evidence of a positive link between a ratio of lower protein to higher potassium dietary intake (ie, less dietary acid) and skeletal integrity.
Assuntos
Osso e Ossos/metabolismo , Climatério/metabolismo , Proteínas Alimentares/metabolismo , Homeostase/fisiologia , Potássio/metabolismo , Pré-Menopausa/metabolismo , Densidade Óssea , Feminino , Humanos , Estudos Longitudinais , Pessoa de Meia-IdadeRESUMO
In bone matrix, type I collagen is stabilised by covalent cross-links formed between adjacent collagen molecules; the majority of which is believed to be immature, divalent bonds. For studying these immature forms in detail, we have developed an immunoassay for a synthetic peptide SP 4 that is analogous with and detects a linear epitope within the C-telopeptide of alpha1-chain of type I collagen. The SP 4 assay, together with the ICTP assay, which is specific for the trivalently cross-linked C-telopeptide, was used for the isolation of the differently cross-linked C-telopeptide structures of the alpha1-chain of type I collagen present in mineralised human bone. Amino acid analysis, peptide sequencing and MALDI-TOF mass spectrometry were used to identify and characterise each of the isolated structures. The cross-link content of each isolated peptide was identified. In the trivalent ICTP peptide, only 40% was cross-linked with pyridinoline, the remainder of the cross-links being currently uncharacterized. The divalent peptides contained only previously characterised cross-linking structures. Most of the divalent cross-links were dihydroxylysinonorleucine (DHLNL), with minor amounts of hydroxylysinonorleucine (HLNL). The relative proportion of the HLNL cross-link was slightly higher in the divalent alpha1Calpha2H peptide. A substantial amount of uncross-linked telopeptide structures was also found. Previous studies, where direct chemical cross-link analyses have been used to assess the maturity of cross-linking, have inferred that bone contains more divalently than trivalently cross-linked C-telopeptides. The immunochemical peptide approach used in this study may help to detect presently uncharacterized, trivalent cross-links, the presence of which is strongly suggested in this study. It also provides additional information regarding the extent and maturity of tissue type I collagen cross-linking in health and disease.
Assuntos
Calcificação Fisiológica/fisiologia , Colágeno Tipo I/metabolismo , Colágeno/metabolismo , Peptídeos/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Colágeno/química , Colágeno/isolamento & purificação , Colágeno Tipo I/química , Feminino , Glicosilação , Humanos , Imunoensaio , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Phosphatases are essential for the mineralization of the extracellular matrix within the skeleton. Their precise identities and functions however remain unclear. PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase involved in the generation of inorganic phosphate for bone mineralization. It is highly expressed at sites of mineralization in bone and cartilage. The bones of Phospho1(-/-) mice are hypomineralized, bowed and present with spontaneous greenstick fractures at birth. In this study we show that PHOSPHO1 is essential for mechanically competent mineralization that is able to withstand habitual load. Long bones from Phospho1(-/-) mice did not fracture during 3-point bending but deformed plastically. With dynamic loading nanoindentation the elastic modulus and hardness of Phospho1(-/-) tibiae were significantly lower than wild-type tibia. Raman microscopy revealed significantly lower mineral:matrix ratios and lower carbonate substitutions in Phospho1(-/-) tibia. The altered dihydroxylysinonorleucine/hydroxylysinonorleucine and pyridinoline/deoxypyridinoline collagen crosslink ratios indicated possible changes in lysyl hydroxylase-1 activity and/or bone mineralization status. The bone formation and resorption markers, N-terminal propeptide and C-terminal telopeptide of Type I collagen, were both increased in Phospho1(-/-) mice and this we associated with increased bone remodeling during fracture repair or an attempt to remodel a mechanically competent bone capable of withstanding physiological load. In summary these data indicate that Phospho1(-/-) bones are hypomineralized and, consequently, are softer and more flexible. An inability to withstand physiological loading may explain the deformations noted. We hypothesize that this phenotype is due to the reduced availability of inorganic phosphate to form hydroxyapatite during mineralization, creating an undermineralized yet active bone.
Assuntos
Calcificação Fisiológica/fisiologia , Fraturas Espontâneas/prevenção & controle , Fraturas Espontâneas/fisiopatologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos/fisiologia , Remodelação Óssea/fisiologia , Colágeno/metabolismo , Elasticidade , Fêmur/patologia , Fêmur/fisiopatologia , Fraturas Espontâneas/enzimologia , Dureza , Camundongos , Monoéster Fosfórico Hidrolases/deficiência , Tíbia/patologia , Tíbia/fisiopatologia , ViscosidadeRESUMO
While there is an appreciable understanding of the importance of collagen breakdown in contributing to atherosclerotic plaque vulnerability and rupture, little is known about changes in collagen maturation in the atherosclerotic plaque. This is achieved through the formation of the covalent intermolecular cross-links pyridinoline (Pyd) and deoxypyridinoline (Dpd). In this study we collected carotid endarterectomy specimens from patients and undertook (i) histological assessment of collagen and inflammatory cell distribution and (ii) biochemical analysis of total collagen and cross-link content. Greater collagen deposition, increased presence of CD68 positive cells and an increased Pyd:Dpd ratio (an indicator of lysyl hydroxylase (LH-1) activity) were found in plaque versus normal vascular tissue. These findings are the first measurements of Pyd and Dpd cross-links in normal and atherosclerotic vascular tissue. The observed differences in cross-links in the plaque may adversely affect tensile strength and may have relevance to the mechanisms underlying rupture of vulnerable plaques.
Assuntos
Estenose das Carótidas/diagnóstico , Colágeno/química , Reagentes de Ligações Cruzadas/farmacologia , Idoso , Aminoácidos/farmacologia , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Aterosclerose/patologia , Cardiologia/métodos , Estenose das Carótidas/patologia , Endarterectomia das Carótidas/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/biossínteseRESUMO
Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase (LH), hydroxylysyl galactosyltransferase (GT) and galactosylhydroxylysyl glucosyltransferase (GGT) activities in vitro. To investigate the in vivo importance of LH3-catalyzed lysine hydroxylation and hydroxylysine-linked glycosylations, three different LH3-manipulated mouse lines were generated. Mice with a mutation that blocked only the LH activity of LH3 developed normally, but showed defects in the structure of the basement membrane and in collagen fibril organization in newborn skin and lung. Analysis of a hypomorphic LH3 mouse line with the same mutation, however, demonstrated that the reduction of the GGT activity of LH3 disrupts the localization of type IV collagen, and thus the formation of basement membranes during mouse embryogenesis leading to lethality at embryonic day (E) 9.5-14.5. Strikingly, survival of hypomorphic embryos and the formation of the basement membrane were directly correlated with the level of GGT activity. In addition, an LH3-knockout mouse lacked GGT activity leading to lethality at E9.5. The results confirm that LH3 has LH and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity and that the GGT activity, not the LH activity of LH3, is essential for the formation of the basement membrane. Together our results demonstrate for the first time the importance of hydroxylysine-linked glycosylation for collagens.
Assuntos
Membrana Basal/enzimologia , Colágeno/metabolismo , Hidroxilisina/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Animais , Catálise , Colágeno/química , Galactosiltransferases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Glucosiltransferases/metabolismo , Glicosilação , Camundongos , Camundongos Knockout , Mutação , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Especificidade por SubstratoRESUMO
PURPOSE OF REVIEW: The primary aim of this review is to highlight recent advances in techniques available for assessing collagen turnover, particularly in relation to the diagnosis and management of bone disorders. As collagen is the major protein constituent of bone, its metabolites form the basis of most of the biochemical markers, but their efficacy needs to be viewed in the context of other non-collagenous markers, for which methodology is also advancing rapidly. RECENT FINDINGS: New markers of bone metabolism have been developed utilising the age-dependent isomerisation and racemisation of aspartyl residues at the C-terminal end of collagen. These methods allow measurement of the ratio between newly synthesised and mature collagen: this ratio appears to provide a novel indicator of the fracture risk for osteoporosis. Other studies have led to an improved understanding of biological variability, particularly in relation to the effects of feeding. Bone resorption assays have been applied to a wide range of diseases and have been especially useful in monitoring the efficacy of novel therapies. SUMMARY: New assays have been developed to facilitate better monitoring of collagen metabolism in bone diseases. A more complete understanding of biological variability, particularly the effects of feeding, have led to improved clinical applicability of these assays in detecting disease and monitoring therapy. Part of the future challenge, however, is to ensure that commercial assay developments keep pace with clinical expectations.
Assuntos
Doenças Ósseas/metabolismo , Colágeno/metabolismo , Biomarcadores , Doenças Ósseas/diagnóstico , Reabsorção Óssea/diagnóstico , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Humanos , Osteoporose/diagnóstico , Osteoporose/metabolismoRESUMO
These guidelines review the relevant literature on the way plant phyto-oestrogens act on bone and the responsiveness of different bone cell systems to phyto-oestrogenic compounds. The primary emphasis is on the experimental conditions used, the markers available for assessing osteoblast and osteoclast function, and their expected sensitivity. Finally, we assess the published results to derive some general recommendations for in vitro experiments in this area of research.
Assuntos
Osso e Ossos/efeitos dos fármacos , Estrogênios não Esteroides/farmacologia , Isoflavonas , Técnicas de Cultura de Células/métodos , Genômica , Guias como Assunto , Humanos , Técnicas de Cultura de Órgãos/métodos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fitoestrógenos , Preparações de PlantasRESUMO
Lysyl hydroxylases (LH) (procollagen-lysine 2-oxoglutarate 5-dioxygenase; PLOD) catalyse the hydroxylation of lysine residues during the post-translational modification of collagenous proteins. In this paper, we describe the first identification and cloning of LH isoforms 2 and 3 from the rat, including both LH2 splice variants (LH2a and LH2b). The rat LHs are expressed in almost all tissue and cell types examined, indicating a probable lack of tissue specificity for LH function. All LH isoforms were stably transfected into CHO-K1 cells and this represents the first example of recombinant LH production in a eukaryotic cell line. Expression and production of all LH isoforms led to an increase in total collagen synthesis. LH1 and LH2a expression and production led to an increase in total pyridinium cross-link production. Evidence that LH2a possesses telopeptide lysyl hydroxylase activity, previously thought to be a novel enzyme, is presented.
Assuntos
Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Clonagem Molecular , Colágeno/biossíntese , Colágeno/metabolismo , Colágeno Tipo I , Cricetinae , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Peptídeos/metabolismo , Splicing de RNA , Ratos , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Distribuição Tecidual , Transcrição GênicaRESUMO
Intervention with selective endothelin (ET)A receptor antagonists within 24h after myocardial infarction (MI) in rats has been reported to aggravate left ventricular (LV) remodeling. In contrast, beneficial effects are reported when initiation of treatment is delayed 7 days or more after MI. However, bosentan, a mixed ET(A)/ET(B) receptor antagonist with low affinity for the ET receptors, has been shown to exert beneficial effects independent of the time point of initiation of treatment after MI. The aim of the present study was to investigate to what extent early intervention with a mixed ET(A)/ET(B) receptor antagonist with higher affinity at the ET receptors (SB 209670) would also exert beneficial effects on postinfarction LV remodeling. After ligation of the left coronary artery, rats were randomized to treatment with SB 209670 (6.25 mg x kg(-1) SC b.i.d., n = 10) or vehicle (n = 12) for 26 days, starting 48h after MI. Treatment with SB 209670 adversely affected the postinfarction remodeling process causing further dilatation of the LV (LV end-diastolic diameter: 10.4+/-0.5 vs 9.1+/-0.2 mm; LV end-systolic diameter: 8.5+/-0.4 vs 7.2+/-0.2 mm, P < 0.05). However, SB 209670 did not significantly affect infarct size, compensatory cardiac hypertrophy, nor the myocardial mRNA levels of procollagen type I and III, and prolyl 4-hydroxylase and lysyl oxidase, 2 important enzymes affecting collagen secretion, stability and functionality. In addition, SB 209670 had no significant effects on LV collagen cross-linking or extent of fibrosis. Thus, our data demonstrate that early intervention with a potent, mixed ET(A)/ET(B) receptor antagonist after MI may promote dilatation of the LV without significant alterations of infarct size and extracellular matrix composition. Our data support the notion that the timing of initiation of ET receptor antagonism after MI is critical and that potent ET receptor antagonists may be harmful during the first few days after MI.
Assuntos
Antagonistas dos Receptores de Endotelina , Indanos/farmacologia , Infarto do Miocárdio/fisiopatologia , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Cardiomegalia/diagnóstico por imagem , Colágeno/química , Colágeno/metabolismo , Ecocardiografia , Endotelina-1/sangue , Matriz Extracelular/genética , Fibrose , Expressão Gênica , Coração/fisiopatologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Receptor de Endotelina A , Receptor de Endotelina B , Fatores de TempoRESUMO
Research on the bone effects of natural phyto-oestrogens after menopause is at a relatively early stage. Published studies are few, difficult to compare and often inconclusive, due in part to design weaknesses. Currently, many questions remain to be answered including to what extent a safe daily intake may prevent postmenopausal bone loss. These questions can only be addressed by conducting well-planned, randomised clinical trials that take into consideration present knowledge in the oestrogen, phyto-oestrogen and bone fields. This review is intended to provide hints for critical decision-making about the selection of subjects, type of intervention, suitable outcome measures and variables that need to be controlled.
Assuntos
Dieta , Estrogênios não Esteroides/uso terapêutico , Isoflavonas , Osteoporose Pós-Menopausa/prevenção & controle , Feminino , Humanos , Pessoa de Meia-Idade , Fitoestrógenos , Preparações de Plantas , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Projetos de Pesquisa , Glycine max/química , Resultado do TratamentoRESUMO
BACKGROUND: Pyridinoline (PYD) and deoxypyridinoline (DPD) are two of the most extensively characterized biochemical bone markers, but the interpretation of results is hampered by biologic and other preanalytical variability. We reviewed factors contributing to preanalytical variation of pyridinium cross-links in urine. METHODS: We searched four databases for English-language reports on PYD and/or DPD in urine. Searches were restricted to humans, except for studies of stability, when the search was expanded to other species. The 599 identified articles were supplemented with references from those articles and with articles known to the authors. RESULTS: The mean reported within-day variability was 71% for PYD (range, 57-78%) and 67% for DPD (range, 53-75%). The mean interday variability was 16% for both DPD and PYD (range for PYD, 12-21%; range for DPD, 5-24%). The mean intersubject variabilities across studies were 26% for PYD (range, 12-63%) and 34% for DPD (range, 8-98%) for healthy premenopausal women and 36% (range, 22-61%) and 40%, (range, 27-54%) for postmenopausal women, respectively. Specimen instability and errors in creatinine measurements were additional sources of variability. CONCLUSIONS: Intra- and intersubject variability can be reduced by collecting specimens at a specific time of the day and by maintaining similar patient status at each specimen collection regarding factors such as medications and dietary supplements.