Urinary zinc loss in sickle cell disease primarily due to increased bone degradation
Authors: M. Schimmel, E. Nur, W. Mairuhu, D.P.M. Brandjes, R.H.G. Olde Engberink, L. Vogt, B.J. Biemond
Journal: American Journal of Hematology (2016)

To the Editor: Sickle cell disease (SCD) is associated with zinc deficiency, which can be found in a majority of steady state sickle cell patients 1, 2. Zinc is a vital component of many metalloproteinases and is required for cellular metabolism, playing an essential role in normal growth, sexual development, and function of the immune system. Correction of zinc deficiency in children with SCD seems to improve growth and reduces the incidence of infections in these patients 3. Previous studies indicate that zinc deficiency in sickle cell patients is caused by increased urinary zinc loss 1, which seems to increase further during painful crisis 4. The exact pathogenesis of this urinary loss is unclear. Renal tubular injury as a result of chronic oxidative stress and inflammation, especially during painful crisis with increased ischemia-reperfusion damage 5, might limit tubular zinc resorption. Alternatively, increased degradation of bone, the main site of zinc storage, due to recurrent bone ischemia especially during painful crisis 6, may also contribute to increased urinary zinc loss in sickle cell patients. Furthermore, ongoing hemolysis in SCD could also contribute to increased zinc loss in SCD as 75% of the zinc content in the circulation is found in erythrocytes. To investigate whether urinary zinc loss in SCD is related to bone degradation or disturbed renal tubular handling of zinc, we investigated urinary and serum levels of zinc in relation to markers of bone degradation, pyridinoline (PYD) and deoxypyridinoline (DPD) and markers of, respectively, proximal and distal tubular renal injury, Kidney-Injury-Molecule-1 (KIM-1) and Heart-type-fatty-acid-binding protein (H-FABP). Measurements were performed in two cohorts of patients (≥18 years) with sickle cell anemia (HbSS), sickle-β0-thalassemia (HbSβ0-thal), sickle-β+-thalassemia (HbSβ+-thal) or sickle-hemoglobin C (HbSC), in steady state and during painful crisis and in healthy HbAA race-matched controls. Urinary markers, adjusted for urinary creatinine, were measured in samples collected in the morning after overnight fasting, within 24 hr after admission. Zinc levels were determined spectrophotometrically, using the colorimetric method 5-Br-PAPs kit (Instruchemie, Delfzijl, The Netherlands). Total DPD and PYD levels were quantified by high performance liquid chromatography (ChromSystems, Műnchen, Germany). All protocols were approved by the Medical Ethical Committee and conducted in agreement with the Helsinki declaration. The primary cohort consisted of 38 patients (27 HbSS/HbSβ0-thal, 11 HbSC/HbSβ+-thal) in steady state and 27 patients (21 HbSS/HbSβ0-thal, 6 HbSC/HbSβ+-thal) during painful crisis and 25 controls. (see Supporting Information documentation for baseline characteristics in Table 1). Compared with controls [0.84 (IQR 0.72–1.13) µmol/mmol creatinine], urinary zinc levels were higher in patients in steady state [1.80 (IQR 1.49–2.39) µmol/mmol creatinine, P < 0.001] with further increments during painful crisis [3.15 (IQR 1.74–4.14) µmol/mmol creatinine, P = 0.004]. Serum levels of zinc were comparable between controls and steady state patients [17.9 (IQR 17.3–18.6) μmol/l vs. 18.9 μmol/l (IQR 16.6–19.1), P = 0.436]. During painful crisis, serum zinc levels in HbSS/HbSß0-thal patients were significantly lower compared with steady state levels [17.1 μmol/l (IQR 15.6–18.1) vs. 18.9 (17.5–19.5), P = 0.032]. Both fractional excretion of zinc (FEZ) and fractional excretion of sodium (FEN) were higher in patients during painful crisis when compared with steady state (Fig. 1A). In patients during painful crisis a correlation was observed between urinary levels of zinc and PYD (Sr 0.417, P = 0.030), but not DPD. Urinary levels of zinc did not correlate with established plasma markers of hemolysis such as reticulocyte counts, LDH and bilirubin, excluding SCD-related hemolysis as factor of importance in zinc deficiency in SCD. (A,B) Fractional (urinary) excretion of sodium and zinc. (A) Fractional excretion of both zinc and sodium were increased in sickle cell patients during painful crisis when compared with steady state values. Data obtained from the primary cohort. (B) In healthy controls, fractional excretion of sodium was increased four hours after NaCl challenge. In contrast, the fractional excretion of zinc was decreased in healthy controls in the post-challenge sample. (C) Levels of zinc in urinary samples obtained from patients with sickle cell disease on subsequent days of admission for painful crisis show a significant increment during painful crisis. Fasting samples were taken on the first, second, third, and fifth morning of painful crisis. Means with SEM, Kruskal–Wallis statistics. Data obtained from the secondary cohort. In the secondary cohort, an increase in urinary zinc excretion was observed on subsequent days of painful crisis (Fig. 1C) in 32 painful crises (22 HbSS/HbSβ0-thal, 10 HbSC/HbSβ+-thal) in 24 patients. The increase in urinary zinc excretion during painful crisis while serum zinc levels dropped, is suggesting increased renal loss of zinc originating from bone degradation during painful crisis. Indeed, urinary zinc levels correlated significantly to urinary PYD levels in steady state (Sr 0.701, P = 0.008) and both DPD (Sr 0.439, P = 0.012) and PYD (Sr 0.388, P = 0.028) during painful crisis, although the latter just failed to reach statistical significance after applying Bonferroni correction. However, urinary zinc levels were not significantly related to urinary levels of KIM-1 [ELISA (R&D systems, Minneapolis)] or H-FABP [ELISA (Haemoscan, Groningen, The Netherlands)], in patients in steady state or painful crisis, failing to show a significant relationship between renal urinary zinc excretion and renal tubular injury in SCD (see Supporting Information Table 2). To investigate whether renal excretion of zinc during painful crisis is influenced by saline infusion administered to patients during painful crisis, we also determined sodium and zinc levels in urinary samples of 12 healthy male volunteers [age 21 (IQR 19.5–25.5) years] that were loaded with intravenous hypertonic 2.7% (SD 0.2) NaCl solution [5 mmol sodium/liter body water, mean volume of 542 (SD 6) mL] administered in 30 minutes. Urinary samples were obtained 120 and 240 minutes after infusion. We observed a comparable increase in FEN in controls treated with NaCl and patients during painful crisis. However, while the FEZ increased in patients, it decreased in healthy controls (Fig. 1B). Although controls were loaded with a smaller volume of a higher concentration NaCl solution, this observation indicates that the increased zinc excretion is specific for SCD and not the result of iatrogenic sodium load. Previous studies have shown a relation between zinc deficiency and increased urinary zinc excretion in SCD. We demonstrate that the increased urinary zinc excretion may be mediated by increased zinc mobilization from bones due to ischemia-induced degradation as we observe a significant relation between urinary levels of zinc and markers of bone degradation in two cohorts of sickle cell patients, especially during vaso-occlusive crisis. Whether zinc loss can be reduced by prevention of recurrent ischemia or the use of bisphosphonates should be studied in a prospective clinical trial. Life-long urinary wasting of zinc in SCD may justify supplementation of zinc in sickle cell patients. M. Schimmel, E. Nur and B.J. Biemond had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: MS, EN, LV, BB. Acquisition of data: MS, ROE. (Statistical) analysis and interpretation of data: MS, EN, DB, ROE, LV, BB. Drafting of the manuscript: MS, EN and BB. Critical revision of the manuscript for important intellectual content: all named co-authors. The authors would like to thank Huib Bout, Ismael Derraz, Yvonne van der Heide and Natasja Huisman (Slotervaart Hospital, Amsterdam) for their laboratory work and support with collection of samples. We thank Marjolein Spiering (Academic Medical Center, Amsterdam) for study support. Marein Schimmel,1* Erfan Nur,1 Willem Mairuhu,2 Dees P.M. Brandjes,2 R.H.G. Olde Engberink,3 Liffert Vogt,3 and Bart J. Biemond1 1Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands 2Department of Internal Medicine, Slotervaart Hospital, Amsterdam, The Netherlands 3Department of Nephrology, Academic Medical Center, Amsterdam, The Netherlands Additional Supporting Information may be found in the online version of this article Additional Supporting Information may be found in the online version of this article. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.