Microvascular Oxygen Consumption During Sickle Cell Pain Crisis 2014

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doi:10.1182/blood-2013-11-533406Prepublished online March 24, 2014;

and Hans AckermanSeamon, Anna K. Conrey, Laurel Mendelsohn, James Nichols, Alexander M. Gorbach, Gregory J. KatoCarol A. Rowley, Allison K. Ikeda, Miles Seidel, Tiffany C. Anaebere, Matthew D. Antalek, Catherine Microvascular oxygen consumption during sickle cell pain crisis

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1

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. Kato

3 and Hans Ackerman

1,*

1 The Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious

Diseases, Rockville, Maryland2 The Infrared Imaging & Thermometry Unit, National Institute of Biomedical Imaging and

Bioengineering, Bethesda, Maryland3 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 Diseases12735 Twinbrook Parkway, Room 3E-28

Rockville, Maryland 20852

[email protected]

Running Title: Oxygen Consumption Increases During Pain Crisis

Blood First Edition Paper, prepublished online March 24, 2014; DOI 10.1182/blood-2013-11-533406

Copyright © 2014 American Society of Hematology

For personal use only.on April 2, 2014. at SAMSUNG MEDICAL CENTERbloodjournal.hematologylibrary.orgFrom








2

Key Points:

• Patients with sickle cell disease have greater microvascular oxygen consumption ratesthan 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).









3

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 injury

4 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. Follow-

up studies were performed more than 3 weeks after resolution of acute pain symptoms.









4

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)

∑

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].









5

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 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









6

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] -

∆HbO2 /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.









7

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.









8

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.









9

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, CAGregory 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, JNScheduling, Consent and Evaluation: CS, HA, TA, AC

Data Collection: AI, MS, TA, MA, HA, AG

Blood Sample Processing: AI, LM, CR, HAData Analysis and Interpretation: CR, HA

Manuscript Writing: CR, HA

The authors have no conflicts of interest to declare.









10

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.









11

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 non-

invasive 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.









12

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.









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Table 1. Participant characteristics, hematological parameters and inflammatory

markers.

Healthy

Volunteer

(n = 38)

Sickle Cell

Steady State

(n = 29)

Sickle Cell

Pain Crisis

(n = 20)

Recovered from

Pain Crisis

(n = 16)

Beta-globin Genotype 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 (- 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)

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 Wilcoxonmatched-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 (

‡).









14

Figure 1. Elevated oxygen consumption, neutrophil count and C-reactive protein during

sickle cell pain crisis. Thenar eminence microvascular oxygen consumption (VO2), absoluteneutrophil 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).







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