Carbon Monoxide Levels and Sickle Cell Disease Severity

Sickle cell disease is an autosomal recessive disorder and the most common genetic disease
affecting African-Americans. Approximately 0.15% of African-Americans are homozygous for
sickle cell disease, and 8% have sickle cell trait. Hemoglobin S polymerization leads to red
cell rigidity, microvascular obstruction, inflammation, and end-organ ischemic injury. Our
published data indicate that up to 50% of sickle cell patients have vascular dysfunction due
to impaired bioavailability of endogenous nitric oxide, due in large part to scavenging of
nitric oxide by cell-free hemoglobin. In previous studies we have demonstrated that
steady-state serum LDH is strongly associated with 1) other markers of intravascular
hemolysis including plasma cell-free hemoglobin and arginase levels, 2) levels of soluble
endothelial adhesion molecules, and 3) an impaired vasodilatory response to an NO donor.
Further, significant steady-state LDH elevation identified a subset of patients in our cohort
as well as the CSSCD cohort at increased risk for developing pulmonary hypertension,
cutaneous leg ulceration, priapism, and early death. Previous biochemical studies have
demonstrated significant transient increases in serum LDH and plasma hemoglobin levels during
VOC, and this presumed hyperhemolysis has been confirmed by 51Cr labeled RBC studies that
revealed further decreases in RBC survival during VOC. However, serum LDH levels are not a
specific biomarker of hemolysis, and furthermore these observations on RBC survival have not
been correlated with markers of intravascular hemolysis at baseline in patients with sickle
cell disease in order to confirm the presence of chronic hyperhemolysis subphenotypes in
sickle cell disease as posited in our previous work.

The current gold standards of random and cohort labeling of RBCs used to quantitate RBC
survival suffer from many technical drawbacks that make them impractical for routine clinical
use. The production rate of expired CO has previously been used to assess RBC survival, based
upon the principal that virtually all CO produced in human beings results from cleavage of
the ?-methene bond of heme and is completely excreted via the lungs. Because RBC destruction
accounts for approximately 80% of heme turnover in the body, endogenous CO production can be
used as a quantitative indicator of RBC life span. Furne et al have previously reported on
the development of a simple, rapid, and noninvasive method for determining RBC life span
based on measurement of exhaled alveolar CO concentration immediately upon awakening
corrected for atmospheric CO, as determined with a device that simulates the body s
equilibration with CO with results comparable to standard labeling techniques. We propose
that this methodology could also provide a quantitative, simple, and noninvasive test to
study the RBC life span and thus rate of hemolysis in those patients with sickle cell
disease.

This trial will aim to 1) establish the use of end-alveolar CO concentration as a
quantitative measure of RBC life span and hemolytic rate in subjects with sickle cell
disease; 2) investigate the association between end-alveolar CO concentration-derived RBC
life span and laboratory measures of hemolytic severity; and 3) investigate the association
between end-alveolar CO concentration-derived RBC life span and the incidence of various
clinical sequelae of sickle cell disease.

- ELIGIBILITY CRITERIA:

All volunteer subjects must be at least 18 years of age and have provided informed, written
consent for participation in this study. Eligibility in the study is determined prior to
enrollment on the basis of the following inclusion and exclusion criteria. Laboratory
values obtained within the preceding 60 days are sufficient for screening purposes.

INCLUSION CRITERIA for SCD Cohort

Males or females 18 years of age or older

Diagnosis of sickle cell disease (any form; electrophoretic or HPLC documentation is
required)

EXCLUSION CRITERIA for SCD Cohort

Chronic scheduled transfusions

Current known pregnancy or lactation

Hemoglobin <5.0 g/dL; however, subjects may return for repeat evaluation at a later date

Currently smoking and unable to refrain from smoking for 24 hours

Subjects previously known to have conditions that may independently affect hemolytic rate:

- Infection or sepsis in the 2 weeks prior to screening

- Autoimmune hemolytic anemia

- Systemic lupus erythematosus (SLE)

- Myelodysplastic disorders, leukemia, or lymphoma

- Hereditary spherocytosis or elliptocytosis

- Severe cardiac valve dysfunction (e.g. AS, MS) or prosthetic heart valve recipients

INCLUSION CRITERIA for Controls

In order to validate the methodology for endogenous CO measurement, initially for each
enrolled study subject with sickle cell disease (up to the first 30 subjects), we will
recruit an African-American healthy control subject of the same gender, within 3 years of
age older or younger than the matched subject with SCD. Additionally, 20 healthy control
subjects will be enrolled for adenosine and any functionally or chemically related
molecules blood testing, and venous blood gas testing only, to compare against subjects
with sickle cell disease. Their participation in this study will consist of one blood draw
of 11 mL for research laboratory testing.

EXCLUSION CRITERIA for Controls

Current pregnancy or lactation

Serum ALT values >80 IU/L

Serum creatinine >2.0 mg/dL

Hemoglobin <11.2 g/dL for females, <13.7 for males; however, subjects may return for repeat
evaluation at a later date

Currently smoking

Subjects with any known form of sickle cell disease (sickle trait will NOT be excluded)

Subjects with any other known forms of hemolytic anemia