Urinary markers of bone resorption, pyridinoline and deoxypyridinoline, are increased in sickle cell patients with further increments during painful crisis
Authors: Erfan Nur, Willem Mairuhu, Bart J. Biemond, Anton P. van Zanten, John‐John B. Schnog, Dees P. Brandjes, Hans‐Martin Otten
Journal: American Journal of Hematology (2010)

The painful crisis is the hallmark of sickle-cell disease (SCD). Bone resorption, as part of physiological bone turnover, results in release into the circulation with subsequent urinary excretion of the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD). Urinary PYD and DPD concentrations could reflect the extent of bone infarction during painful sickle-cell crisis. Urinary concentrations of PYD and DPD, adjusted for urine creatinine, were measured in sickle-cell patients (38 clinically asymptomatics and 27 during painful crisis) and healthy controls (n = 25) using high-performance liquid chromatography (HPLC). PYD and DPD concentrations were higher in asymptomatic HbSS/HbSβ0-thalassemia patients compared to controls (P < 0.05) with further increments during painful crisis in both HbSS/HbSβ0-thalassemia and HbSC/HbSβ+-thalassemia patients (P < 0.05). In the asymptomatic HbSS/HbSβ0-thalassemia patients, there was a statistically significant positive correlation between DPD and hemolytic rate. Based on urinary PYD and DPD concentrations, bone degradation is increased in asymptomatic sickle-cell patients, with further increments during painful crisis. Urinary PYD and DPD concentrations are potentially diagnostic and prognostic tools in SCD. Sickle-cell disease (SCD) affects millions worldwide. One of the most frequently occurring complications is the painful crisis [1]. The painful crisis manifests as acute musculo-skeletal (usually juxta-articular) and/or visceral pain mostly associated with mild pyrexia, which often necessitates treatment with parenteral opiates and thus hospital-based medical care [2]. The pathophysiology involves multiple mechanisms ultimately leading to the obstruction of microvasculature with subsequent tissue ischemia and infarction as result [3-5]. Even though mostly self-limiting, painful crises are associated with severe complications such as the acute chest syndrome, stroke, multiorgan failure, and sudden death [6-8]. Furthermore, patients experiencing three or more painful crises per year requiring medical attention carry a higher risk of early death [1, 9, 10]. One of the most challenging aspects in the management of patients with acute painful crises is the lack of objective laboratory tools to confirm the diagnosis and to estimate its severity. Next to parameters such as reticulocyte and leukocyte counts and lactate dehydrogenase (LDH) levels, studies have focused on laboratory markers involved in the pathophysiological processes of SCD such as markers of endothelial activation, cytokine profiles, and coagulation proteins [11-18], none of which have made the transition to the clinic. The lack of diagnostic tools to diagnose vaso-occlusion can contribute to misinterpretation of symptoms, unjust withholding of adequate analgesia, distorted communication, and doctor–patient relationships with unjust stigmatization of patients as drug addicts. Therefore, objective laboratory tools accurately reflecting the vaso-occlusive process would be of great value to those caring for patients with SCD. As marrow ischemia and infarction potentially leads to bone degradation, laboratory markers of bone resorption may be of value in monitoring vaso-occlusion in SCD. Pyridinoline (PYD) and deoxypyridinoline (DPD) are collagen cross-links, and their urinary concentrations have been proven accurate markers of bone resorption [19-22]. After bone degradation, PYD and DPD are released in the circulation and excreted directly into urine without further systemic metabolism. Pyridinium-based cross-links are an important part of the extracellular collagen fibrils in most connective tissue types. However, unlike other connective tissue types, bone is continuously remodeled and therefore forms the main source of urinary cross-links. Furthermore, the ratio of PYD to DPD in urine is approximately the same as in adult human bone (3.5:1), further supporting bones as the predominant source of urinary PYD and DPD [23]. Given the above, we set out to determine profiles of urinary PYD and DPD in SCD in both the clinically asymptomatic state as well as during painful crisis. Twenty-seven HbSS/HbSβ0-thal [median age (interquartile range [IQR]) 26 (18-30) years; 23 HbSS and 4 HbSβ0-thalassemia] and 11 HbSC/HbSβ+-thal [age 29 (21-30); all HbSC] sickle-cell patients during clinically asymptomatic state and 21 HbSS/HbSβ0-thal [age 25 (19-30); all HbSS] and 6 HbSC/HbSβ+-thal [age 25 (21-30); 1 HbSβ+-thalassemia and 5 HbSC] during painful crisis were included. Twenty-five race- and age-matched healthy HbAA [age 28 (18-30)] volunteers were included as controls. See Supporting Information 1 for detailed baseline characteristics. Although urinary PYD and DPD to creatinine ratios were comparable between healthy controls and HbSC/HbSβ+-thal patients in steady state, they were significantly higher in asymptomatic state HbSS/HbSβ0-thal patients (Fig. 1A,B). Further increments were observed during painful crisis, although the differences did not reach statistical significance for PYD in both groups and for DPD in HbSC/HbSβ+-thal patients. In a paired analysis of 19 patients who were included both during asymptomatic state and painful crisis, the urinary cross-links increased during painful crisis, although the difference was only statistically significant for DPD (see Fig. 2). Urinary concentrations of pyridinoline (PYD) and deoxypyridinoline (DPD), adjusted for urine creatinine, in 25 healthy controls with normal HbAA hemoglobin (CTRL), 27 HbSS/HbSβ0-thalassemia (SS), and 11 HbSC/HbSβ+-thalassemia (SC) sickle-cell patients during asymptomatic state (A) and 21 HbSS/HbSβ0-thal and 6 HbSC/HbSβ+-thal patients during painful crisis (C). A: PYD concentrations. B: DPD concentrations. *Significantly different when compared with healthy controls (P < 0.05). †Significantly different when compared with asymptomatic state (P < 0.05). Bars indicate means ± SEM. Deoxypyridinoline (DPD) in 19 patients included both during asymptomatic state (ASST) and painful crisis (CRIS). Urinary concentrations of DPD are higher during painful crisis as compared to the asymptomatic state. Urinary concentrations of PYD and DPD were significantly related to hemolytic rate in asymptomatic state HbSS/HbSβ0-thal patients but not HbSC/HbSβ+-thal patients (Supporting Information 2). No correlations between the cross-links and the hemolytic rate were observed during painful crisis. Gender was not related to the degree of urinary excretion of PYD and DPD (data not shown). PYD and DPD after the first night of hospital admission were not related to the duration of hospital stay (days) for the treatment of painful crisis (data not shown). The findings in this study indicate a higher degree of chronic bone degradation in SCD with exacerbations during painful crisis. Increased urinary excretion of PYD and DPD during painful crisis primarily suggests that bone ischemia and subsequent necrosis due to microvascular occlusion induces bone degradation. However, increased metabolic bone turnover during painful crisis could also contribute. The degree of increments in PYD and DPD concentrations during painful crisis in most patients of the paired analysis was comparable. Because of interpatient variation in baseline concentrations during asymptomatic state, it was not possible to determine a normal cut-off value. Baseline PYD and DPD values were elevated in HbSS/HbSβ0-thal patients when compared with healthy controls. Given the fact that sickle-cell patients are characterized by an increased renal creatinine excretion [24], the expressed cross-links to creatinine ratios are likely to be underestimated in these patients. PYD and DPD concentrations were highest in patients with the greatest hemolytic rate, and it may well be possible that hemolytic anemia-induced bone marrow expansion contributes to bone degradation. SCD is characterized by a continuous state of inflammation, which could also be a significant contributory factor to increased bone resorption [25]. Ongoing clinically silent vaso-occlusion is another potential explanation of increased bone ischemic damage and degradation [26]. Hemolysis-induced vasculopathy in SCD, mediated by oxidative stress, reduced nitric oxide bioavailability, hemostatic activation, adhesion of activated leukocytes, and platelets to endothelial cells might result in ischemia-reperfusion injury and thus bone degradation, especially in genotypes characterized by severe hemolysis (HbSS/HbSβo-thalassemia) [27, 28]. It is very likely that a combination of these factors in addition to bone volume and bone metabolism related factors such as calcium, phosphate, vitamin D, parathyroid hormone, and age ultimately determines (the variation in) base-line PYD and DPD values in the clinically asymptomatic state. Associations with biomarkers of inflammation and bone metabolism were out of the scope of this study. Currently, it is unknown whether each individual patient has a stable base-line value over time. In interpreting these data, some pitfalls should be considered. As there is no gold standard for diagnosing a sickle-cell crisis (let alone its severity), one should be cautious to relate the urinary concentrations of PYD and DPD to the severity of a vaso-occlusive crisis. We could not find a correlation between the urinary PYD and DPD concentrations and crisis duration. The number of patients included was low, and these preliminary findings need confirmation in a large, prospective study where serial measurements of PYD and DPD during painful crisis are studied in relation to clinical outcomes such as pain score. In the paired analysis, DPD, but not PYD, increased significantly during painful crisis. This could be explained by the greater bone specificity of DPD, as PYD is also a major component of collagen fibrils in other tissue types [23]. In conclusion, the findings of increased PYD and DPD in asymptomatic state sickle-cell patients with further increments during painful crisis seem not only of potential diagnostic importance but of pathophysiological importance as well, since sickle-cell patients may have an increased risk of osteoporosis due to continuously increased bone degradation, with further increments during each painful crisis [3, 29]. Urinary PYD and DPD concentrations can be determined rapidly with a widely available technique, and their value as potential diagnostic tools of the painful sickle cell crisis is now subject of further study. Consecutive clinically asymptomatic adult (≥18 years old) patients with SCD [HbSS, HbSβ0-thalassemia, HbSβ+-thalassemia, and HbSC confirmed by high-performance liquid chromatography (HPLC)], visiting the outpatient clinic and patients admitted with a painful crisis at the Academic Medical Centre and Slotervaart Hospital, Amsterdam, the Netherlands, were eligible for the study. A painful crisis was defined as musculo-skeletal pain not otherwise explained and recognized as such by the patient. The clinically asymptomatic state was defined as being free of SCD-related acute events, such as painful crises, priapism, acute chest syndromes, and strokes during at least 4 months before study participation. Exclusion criteria were autoimmune inflammatory diseases, active infection, pregnancy, and women in (post-) menopausal stage. Healthy race and age-matched HbAA volunteers served as controls. All participants gave written informed consent. The protocol was reviewed and approved by the local medical ethical committee and conducted in agreement with the Helsinki declaration of 2000. Blood samples were drawn via venipuncture. Standard blood counts were performed in EDTA-anticoagulated blood (Cell-Dyn 4000, Abbott, IL). LDH and total bilirubin levels were measured in heparinized plasma with spectrophotometry (P800 Modular, Roche, Basel, Switzerland). Urine samples were collected in the morning between 8 and 10 a.m. after overnight fasting of at least 8 hr and stored at −80○C until measurements. Cross-links are determined by HPLC using commercial reagents (ChromSystems, München, Germany). To adjust for the degree of urine concentration, urinary creatinine is measured, and the cross-links are expressed as PYD to creatinine and DPD to creatinine ratios. For data analysis, patients were divided in two groups with patients with the relatively severe genotypes HbSS and HbSß0-thalassemia grouped in one group (HbSS/HbSβ0-thal) and patients with the relatively milder HbSC and HbSß+-thalassemia genotypes collected in the other group (HbSC/HbSβ+-thal) [30]. As data were not normally distributed, statistical tests for nonparametric data were used. For multiple-group comparisons of continuous variables, the Kruskal–Wallis test was used. The Mann–Whitney U-test was used for comparison between two groups. The Wilcoxon Signed Rank Test was used for a paired analysis of patients included both during asymptomatic state and painful crisis. For correlation studies, the Spearman Rank correlation coefficient (rS) was determined. Continuous data are presented as medians with corresponding IQR, unless stated otherwise. P < 0.05 was considered statistically significant (SPSS 16.0, SPSS, Chicago, IL). 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