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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Blood First Edition Paper, prepublished online March 24, 2014; DOI 10.1182/blood-2013-11-533406 Brief Report: Microvascular Oxygen Consumption During Sickle Cell Pain Crisis Carol A. Rowley,1 Allison K. Ikeda,1 Miles Seidel,2 Tiffany C. Anaebere,1 Matthew D. Antalek,2 Catherine Seamon,3 Anna K. Conrey,3 Laurel Mendelsohn,3 James Nichols,3 Alexander M. Gorbach,2 Gregory J. Kato3 and Hans Ackerman1,* 1 The Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, Maryland 2 The Infrared Imaging & Thermometry Unit, National Institute of Biomedical Imaging and Bioengineering, Bethesda, Maryland 3 The Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland * To whom correspondence should be addressed: Hans Ackerman, MD DPhil Laboratory of Malaria and Vector Research National Institute of Allergy and Infectious Diseases 12735 Twinbrook Parkway, Room 3E-28 Rockville, Maryland 20852 [email protected] Running Title: Oxygen Consumption Increases During Pain Crisis 1 Copyright © 2014 American Society of Hematology From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Key Points: • Patients with sickle cell disease have greater microvascular oxygen consumption rates than healthy individuals. • During sickle cell pain crisis, microvascular oxygen consumption increases further. ABSTRACT Sickle cell disease is an inherited blood disorder characterized by chronic hemolytic anemia and episodic vaso-occlusive pain crises. Vaso-occlusion occurs when deoxygenated hemoglobin-S polymerizes and erythrocytes sickle and adhere in the microvasculature, a process dependent on the concentration of hemoglobin-S and the rate of deoxygenation, among other factors. We measured oxygen consumption in the thenar eminence during brachial artery occlusion in sickle cell patients and healthy individuals (#NCT01568710). Microvascular oxygen consumption was greater in sickle cell patients compared to healthy individuals (median[IQR]; sickle cell: 0.91 [0.75-1.07] vs. healthy: 0.75 [0.62-0.94] -ΔHbO2/min, p<0.05) and was elevated further during acute pain crisis (crisis: 1.10 [0.78-1.30] vs. recovered: 0.88 [0.76-1.03] -ΔHbO2/min, p<0.05). Increased microvascular oxygen consumption during pain crisis could affect the local oxygen saturation of hemoglobin when oxygen delivery is limiting. Identifying the mechanisms of elevated oxygen consumption during pain crisis might lead to the development of new therapeutic interventions. This study was registered at ClinicalTrials.gov, Study ID Number: NCT01568710 (http://www.clinicaltrials.gov/ct2/show/NCT01568710). 2 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. INTRODUCTION Sickle cell disease is a blood disorder caused by homozygous or compound heterozygous inheritance of abnormal hemoglobin-β chains that form hemoglobin-S. Patients with sickle cell disease endure pain crises that may last days and occur multiple times each year.1,2 The etiology of painful crises is unknown, but may involve blockage of vessels by sickled and adherent blood cells3, followed by ischemia reperfusion injury4 and local inflammatory responses.5 Inflammation, in addition to increasing pain, can increase oxygen consumption6,7 and might have adverse effects on hemoglobin oxygenation and sickling when oxygen delivery is limiting. We hypothesized that sickle cell patients would have increased rates of oxygen consumption during acute pain crisis. We measured microvascular oxygen consumption and systemic biomarkers of inflammation in healthy African American volunteers, patients with sickle cell disease in clinical steady state, and in patients both during pain crisis and after recovery. METHODS Patients The Institutional Review Board of the National Heart, Lung and Blood Institute approved clinical protocol 12-H-0101 specifically for this study. All participants provided written informed consent in accordance with the Declaration of Helsinki. See http://www.clinicaltrials.gov/ct2/show/NCT01568710 and Supplemental Table 1 for enrollment criteria. Pain crisis was defined as acute pain occurring in a typical distribution requiring hospital admission and parenteral analgesia. Acute crisis studies were performed within 36 hours of admission, after patients had received intravenous fluids and pain medications. Followup studies were performed more than 3 weeks after resolution of acute pain symptoms. 3 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Near-Infrared Spectroscopy and Oxygen Consumption Calculation Near-infrared spectroscopy has been validated against magnetic resonance spectroscopy as a measure of local oxygen consumption in muscle.8 We used the Inspectra 650 (Hutchinson Technology, Hutchinson, MN) to record tissue hemoglobin oxygen saturation9 StO2 and tissue hemoglobin index10 THI (a measure of hemoglobin signal strength) every two seconds during a 5-minute brachial artery occlusion. Oxygen consumption VO2 was calculated as the sum of each change in StO2 over each 2-second interval, weighted by the THI, all divided by the duration of occlusion t. 1) ሺௌ௧ைమሻశభିሺௌ௧ைమሻ ሺ்ுூ ା்ுூశభሻ ଶ ଵ௧ ∑ ୀଵ ଶ ଵ Our approach is similar to existing methods11 but does not assume a linear decline in hemoglobin saturation. Raw data were processed and analyzed with custom scripts in R.12 Statistics Unpaired t-tests, Mann-Whitney tests, paired t-tests, or Wilcoxon matched-pairs signed-rank tests were performed where appropriate using GraphPad Prism 6 (Graphpad Software, San Diego, CA). Data are presented as median [interquartile range]. 4 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. RESULTS/DISCUSSION Microvascular Oxygen Consumption We estimated microvascular oxygen consumption by monitoring the decline in hemoglobin oxygen saturation in the thenar eminence while preventing arterial inflow from the brachial artery. Microvascular oxygen consumption was greater among sickle cell patients in steady state (0.91[0.75-1.07] -ΔHbO2/min) compared to healthy individuals (0.75[0.62-0.94] -ΔHbO2/min, p<0.05). Oxygen consumption was greater during acute pain crisis (1.10[0.78-1.30] ΔHbO2/min) compared to either steady state (0.91[0.75-1.07] -ΔHbO2/min, p<0.05) or after recovery from pain crisis using paired analysis (recovered: 0.88[0.76-1.03] -ΔHbO2/min, p<0.05), shown in Figure 1. Taken together, these results suggest that oxygen consumption is chronically elevated in sickle cell patients in steady state, increases acutely during pain crisis and then returns to a steady-state baseline after recovery from crisis. Inflammatory Biomarkers We assessed each patient’s inflammatory state by neutrophil count and C-Reactive Protein (CRP) concentration. Absolute neutrophil count was elevated during acute pain crisis compared to steady state (crisis: 5.7[3.3-7.2] vs. steady state: 3.4[2.1-5.2] K/uL, p<0.01) but remained unchanged after recovery from crisis (crisis: 5.7[3.3-7.2] to recovery: 3.6[2.7-6.6], p=0.33). CRP was acutely elevated during pain crisis compared to steady state (crisis: 12[2.4-66] vs. steady state: 3.3[1.3-4.8] mg/L, p<0.01) and decreased after resolution of crisis to 6.0[2.0-8.7] mg/L (p<0.05). Our findings of elevated inflammatory biomarkers during sickle cell pain crisis are consistent with previous studies showing elevated neutrophil count and CRP in steady state with further elevation during pain crisis, though we did not observe an elevated neutrophil count 5 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. in steady state compared to healthy individuals as previously reported.13,14 The observational nature of this study does not allow us to causally link inflammation with increased oxygen consumption; however, these data emphasize the relevance of inflammation to the pathophysiology of sickle cell disease, especially during pain crisis. Possible Causes of Elevated Oxygen Consumption Several factors might elevate oxygen consumption in sickle cell disease and in pain crisis specifically. In steady state, patients with sickle cell disease experience elevated resting energy expenditure (REE), requiring greater systemic oxygen consumption.15,16 This has been attributed to an increased rate of protein synthesis at sites of erythropoiesis. Although oxygen consumption was greater in steady state compared to healthy individuals (0.91[0.75-1.07] vs. 0.75[0.62-0.94] ΔHbO2/min, p<0.05), our measurements are more likely to reflect the local density and activity of intravascular blood cells and myocytes rather than the metabolic demands of erythropoiesis at distant sites. Oxygen consumption by inflammatory cells in the blood contributes measurably to both local and systemic oxygen consumption: stimulation of phagocytes by phorbol myristate acetate (PMA) elevated total body oxygen consumption by 18% in guinea pigs and was prevented by co-administration of an NADPH oxidase inhibitor, indicating that the respiratory burst of phagocytes was responsible for systemic changes in oxygen consumption.17 Similarly, controlled exposure to endotoxin, a potent inducer of inflammation, increased total body oxygen consumption by 39% in human volunteers.7 Our observations that sickle cell patients in pain crisis have local oxygen consumption rates that are 24% greater than steady state (p<0.01) and 46% greater than healthy volunteers (p<0.0001) are similar in magnitude to the changes induced by acute inflammatory stimuli such as PMA and endotoxin. 6 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. In addition to the cellular activity of NADPH oxidase, the process of hemoglobin autoxidation may contribute importantly to microvascular oxygen consumption. Previous studies have attributed the enhanced production of reactive oxygen species in sickled erythrocytes to reactions catalyzed by hemoglobin,18 possibly augmented by increases in membrane-bound heme iron and free iron.19 In this study, we found that sickle cell patients in crisis had greater concentrations of methemoglobin in venous blood than did patients in steady state or healthy individuals (crisis: 1.70[1.43-2.00] %; steady state: 1.40[1.13-1.65] %, p<0.01; healthy: 0.70[0.60-0.80] %, p<0.0001). This suggests an increased rate of hemoglobin autoxidation during sickle cell pain crisis, though impaired reduction of methemoglobin may also play a role.20 Mitochondrial activity could also potentially contribute to increased oxygen consumption during crisis through altered cellular respiration or generation of reactive oxygen species.21 While oxygen consumption at the thenar eminence was elevated among patients experiencing sickle cell pain crisis, it is conceivable that oxygen consumption would be greater still at sites of pain where activated inflammatory cells would be more concentrated.22 Imaging modalities such as computed tomography and magnetic resonance imaging combined with markers of oxygen consumption might better elucidate the changes in oxygen consumption that occur at sites of pain. Nevertheless, the simplicity and safety of near-infrared spectroscopy combined with controlled brachial artery occlusion facilitated the first measurements of microvascular oxygen consumption during sickle cell pain crisis. The discovery of elevated oxygen consumption during crisis identifies a potential new target for the treatment of acute pain crisis. 7 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. ACKNOWLEDGMENTS The authors acknowledge the technical contributions of Ken Chang and Stephen Yoon. Research nursing care was provided by Linda Tondreau, RN, BSN, Bisi Dada, RN, BSN, Mijung Kim, RN, BSN, IckHo Kim, RN, BSN, Elizabeth Witter, RN, BSN, Wendy Holt, RN, BSN, Mashood Esfanaji, RN, Grace Kim, RN, BSN, Miwha Yi, RN, BSN, Elmer Amparo, RN, BSN, and Stella Woo, RN, BSN. Professional protocol management was provided by Stephanie Housel, MS, CIP and Mary Hall, CIP. We thank BethAnn Guthmueller, CCRP and Hutchinson Technology, Inc. for providing a NIRS monitor and sensors for use in this study. Junfeng Sun, PhD, provided statistical advice on the study design. We also acknowledge the contributions of the physicians, nurse practitioners, and nurses who provided care for the patients in this study, and we thank the patients for participating. This study was supported by the Intramural Research Program of the National Institutes of Health, USA at the National Heart, Lung and Blood Institute, the National Institute of Allergy and Infectious Diseases and the National Institute of Biomedical Imaging and Bioengineering. 8 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CURRENT AFFILIATIONS Allison K. Ikeda: The School of Medicine & Health Sciences, The George Washington University, Washington, D.C. Tiffany C. Anaebere: Department of Emergency Medicine, Alameda Health Systems-Highland Hospital, Oakland, CA Gregory J. Kato: Department of Medicine, Division of Hematology; Heart, Lung, Blood, and Vascular Medicine Institute; University of Pittsburgh, Pittsburgh, PA AUTHORSHIP CONTRIBUTIONS/ DISCLOSURE OF CONFLICTS OF INTEREST Study Design: HA, AI, AG, GK Protocol Development: HA, AI, TA, JN Scheduling, Consent and Evaluation: CS, HA, TA, AC Data Collection: AI, MS, TA, MA, HA, AG Blood Sample Processing: AI, LM, CR, HA Data Analysis and Interpretation: CR, HA Manuscript Writing: CR, HA The authors have no conflicts of interest to declare. 9 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. REFERENCES 1. Platt OS, Thorington BD, Brambilla DJ, et al. Pain in Sickle Cell Disease. N Engl J Med. 1991;325(1):11–16. 2. Steiner CA, Miller JL. Sickle Cell Disease Patients in U.S. Hospitals, 2004: Statistical Brief #21. Healthcare Cost and Utilization Project (HCUP) Statistical Briefs. 2006. 3. Steinberg MH. Management of Sickle Cell Disease. N Engl J Med. 1999;340(13):1021–1030. 4. Osarogiagbon UR, Choong S, Belcher JD, et al. Reperfusion injury pathophysiology in sickle transgenic mice. Blood. 2000;96(1):314–320. 5. Hebbel RP, Osarogiagbon R, Kaul D. The endothelial biology of sickle cell disease: inflammation and a chronic vasculopathy. Microcirculation. 2004;11(2):129–151. 6. Vlessis AA, Bartos D, Muller P, Trunkey DD. Role of reactive O2 in phagocyte-induced hypermetabolism and pulmonary injury. J Appl Physiol. 1995;78(1):112–116. 7. Suffredini AF, Shelhamer JH, Neumann RD, et al. Pulmonary and oxygen transport effects of intravenously administered endotoxin in normal humans. Am J Respir Crit Care Med. 1992;145(6):1398–1403. 8. Hamaoka T, Iwane H, Shimomitsu T, et al. Noninvasive measures of oxidative metabolism on working human muscles by near-infrared spectroscopy. J Appl Physiol. 1996;81(3):1410– 1417. 10 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 9. Myers DE, Anderson LD, Seifert RP, et al. Noninvasive method for measuring local hemoglobin oxygen saturation in tissue using wide gap second derivative near-infrared spectroscopy. J Biomed Opt. 2005;10(3):034017–03401718. 10. Myers D, McGraw M, George M, Mulier K, Beilman G. Tissue hemoglobin index: a noninvasive optical measure of total tissue hemoglobin. Crit Care. 2009;13(Suppl 5):S2. 11. Skarda DE, Mulier KE, Myers DE, Taylor JH, Beilman GJ. Dynamic near-infrared spectroscopy measurements in patients with severe sepsis. Shock. 2007;27(4):348-353. 12. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2013. 13. Schimmel M, Nur E, Biemond BJ, et al. Nucleosomes and neutrophil activation in sickle cell disease painful crisis. Haematologica. Prepublished on August 2, 2013, as DOI 10.3324/haematol.2013.088021. 14. Pathare A, Al Kindi S, Alnaqdy AA, et al. Cytokine profile of sickle cell disease in Oman. Am J Hematol. 2004;77(4):323–328. 15. Badaloo A, Jackson AA, Jahoor F. Whole body protein turnover and resting metabolic rate in homozygous sickle cell disease. Clin Sci. 1989;77(1):93–97. 16. Borel MJ, Buchowski MS, Turner EA, et al. Alterations in basal nutrient metabolism increase resting energy expenditure in sickle cell disease. Am J Physiol - Endocrinol Metab. 1998;274(2):E357–E364. 11 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 17. Rossi F. The O2-forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim. Biophys. Acta BBA - Rev Bioenerg. 1986;853(1):65–89. 18. Hebbel RP, Eaton JW, Balasingam M, Steinberg MH. Spontaneous oxygen radical generation by sickle erythrocytes. J Clin Invest. 1982;70(6):1253. 19. Sugihara T, Repka T, Hebbel RP. Detection, characterization, and bioavailability of membrane-associated iron in the intact sickle red cell. J Clin Invest. 1992;90(6):2327. 20. Zerez CR, Lachant NA, Tanaka KR. Impaired erythrocyte methemoglobin reduction in sickle cell disease: dependence of methemoglobin reduction on reduced nicotinamide adenine dinucleotide content. Blood. 1990;76(5):1008-1014. 21. Wood KC, Granger DN. Sickle cell disease: role of reactive oxygen and nitrogen metabolites. Clin Exp Pharmacol Physiol. 2007;34(9):926–932. 22. Hermreck AS, Thal AP. Mechanisms for the high circulatory requirements in sepsis and septic shock. Ann Surg. 1969;170(4):677. 12 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Table 1. Participant characteristics, hematological parameters and inflammatory markers. Beta-globin Genotype Healthy Sickle Cell Sickle Cell Recovered from Volunteer Steady State Pain Crisis Pain Crisis (n = 38) (n = 29) (n = 20) (n = 16) 30 AA, 7 AS, 1 AC 26 SS, 3 SC 18 SS, 2 SC 14 SS, 2 SC Age (yrs) 31 (26-43) 35 (28-43) 36 (25-45) 36 (27-45) Female (%) 24/38 (63%) 17/29 (59%) 13/20 (65%) 11/16 (69%) BMI (kg/m2) 28 (24-32) 24 (21-26)* 23 (21-27) 24 (22-28) Hydroxyurea Use (%) 0/38 (0%) 20/29 (69%) 17/20 (85%) 14/16 (88%) Hemoglobin (g/dL) 13 (12-14) 9.1 (8.1-9.7)* 7.7 (7.1-8.8)† 8.5 (7.4-9.3) Fetal hemoglobin (%) 0.0 9.5 (5.0-18)* 12 (4.0-16) 13 (2.9-16) Hemoglobin S (%) 0.0 76 (61-81)* 81 (77-83) 78 (57-83) Hematocrit (%) 38 (36-41) 25 (22-27)* 21 (20-24)† 24 (21-27) MCV (fL) 85 (79-90) 93 (80-110)* 94 (86-100) 90 (77-100) Reticulocyte (%) 1.0 (0.8-1.5) 8.7 (4.3-11)* 8.1 (5.7-12) 9.0 (6.2-14) Retic Count (K/uL) 50 (36-72) 190 (110-280)* 170 (150-300) 240 (140-320) LDH (U/L) 180 (160-200) 330 (280-470)* 480 (330-660) 390 (350-440) Platelet Count (K/uL) 240 (220-280) 300 (230-370)* 280 (210-380) 310 (230-490) WBC Count (K/uL) 5.4 (4.6-6.9) 7.1 (5.9-9.5)* 9.2 (7.6-13)† 8.9 (7.1-14) Neutrophil Count (K/uL) 3.0 (2.1-3.8) 3.4 (2.1-5.2) 5.7 (3.3-7.2)† 3.6 (2.7-6.6) CRP (mg/L) 1.2 (0.4-2.9) 3.3 (1.3-4.8)* 12 (2.4-66)† 6.0 (2.0-8.7) VO2 (-ΔHbO2/min) 0.75 (0.62-0.94) 0.91 (0.75-1.07)* 1.10 (0.78-1.30)† 0.88 (0.76-1.03) ‡ ‡ ‡ Data are presented as median (interquartile range). BMI = body mass index, RBC = red blood cell, MCV = mean corpuscular volume, Retic = reticulocyte, LDH = lactate dehydrogenase, Abs = absolute, CRP = C-reactive protein. Statistics performed using unpaired t-tests, Mann-Whitney tests, paired t-tests, or Wilcoxon matched-pairs signed rank tests, where appropriate. p < 0.05 for Steady State compared to Healthy Volunteer (*), Acute Crisis compared to Steady State (†), and Recovered from Crisis compared to Acute Crisis (‡). 13 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Figure 1. Elevated oxygen consumption, neutrophil count and C-reactive protein during sickle cell pain crisis. Thenar eminence microvascular oxygen consumption (VO2), absolute neutrophil count and C-reactive protein levels were greater among sickle cell disease (SCD) patients in pain crisis than among patients in steady state or healthy individuals. Horizontal lines indicate the median for each group. Significance levels are indicated by *p < 0.05, **p < 0.01 and ns (not significant). 14 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Prepublished online March 24, 2014; doi:10.1182/blood-2013-11-533406 Microvascular oxygen consumption during sickle cell pain crisis Carol A. Rowley, Allison K. Ikeda, Miles Seidel, Tiffany C. Anaebere, Matthew D. Antalek, Catherine Seamon, Anna K. Conrey, Laurel Mendelsohn, James Nichols, Alexander M. Gorbach, Gregory J. 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