Download Microvascular Oxygen Consumption During Sickle Cell Pain Crisis

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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
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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
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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.
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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.
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ଶ ଵ௧ ∑௠
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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].
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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 (‡).
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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
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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. Kato
and Hans Ackerman
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