Professional Documents
Culture Documents
Information about reproducing this article in parts or in its entirety may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Advance online articles have been peer reviewed and accepted for publication but have not yet
appeared in the paper journal (edited, typeset versions may be posted when available prior to
final publication). Advance online articles are citable and establish publication priority; they are
indexed by PubMed from initial publication. Citations to Advance online articles must include
digital object identifier (DOIs) and date of initial publication.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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
*
1
Copyright 2014 American Society of Hematology
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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] -HbO2/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] -HbO2/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).
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. For personal use only.
INTRODUCTION
Sickle cell disease is a blood disorder caused by homozygous or compound heterozygous
inheritance of abnormal hemoglobin- 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.
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. For personal use only.
1)
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].
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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] -HbO2/min) compared to healthy individuals (0.75[0.62-0.94] -HbO2/min,
p<0.05). Oxygen consumption was greater during acute pain crisis (1.10[0.78-1.30] HbO2/min) compared to either steady state (0.91[0.75-1.07] -HbO2/min, p<0.05) or after
recovery from pain crisis using paired analysis (recovered: 0.88[0.76-1.03] -HbO2/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 patients 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
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. For personal use only.
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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.
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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.
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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):1116.
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.
4. Osarogiagbon UR, Choong S, Belcher JD, et al. Reperfusion injury pathophysiology in sickle
transgenic mice. Blood. 2000;96(1):314320.
5. Hebbel RP, Osarogiagbon R, Kaul D. The endothelial biology of sickle cell disease:
inflammation and a chronic vasculopathy. Microcirculation. 2004;11(2):129151.
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):13981403.
10
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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):03401703401718.
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):323328.
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):9397.
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):E357E364.
11
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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):6589.
18. Hebbel RP, Eaton JW, Balasingam M, Steinberg MH. Spontaneous oxygen radical
generation by sickle erythrocytes. J Clin Invest. 1982;70(6):1253.
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):926932.
22. Hermreck AS, Thal AP. Mechanisms for the high circulatory requirements in sepsis and
septic shock. Ann Surg. 1969;170(4):677.
12
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. For personal use only.
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)
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)
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)
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)
240 (220-280)
300 (230-370)*
280 (210-380)
310 (230-490)
5.4 (4.6-6.9)
7.1 (5.9-9.5)*
9.2 (7.6-13)
8.9 (7.1-14)
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 (-HbO2/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)
From bloodjournal.hematologylibrary.org at SAMSUNG MEDICAL CENTER on April 2, 2014. 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