Differential Acetone Extraction of Total and Hemoprotein-Unbound Heme to Quantify Heme Binding Capacity of Plasma in Patients with Sickle Cell Disease: The Role of Heme Scavengers
Authors: Vissa Madhav , Soupene Eric , Larkin Sandra K. , Neumayr Lynne D. , Vichinsky Elliott P. , Kuypers Frans A. 
Published: November 2020

Introduction:
Heme, an iron-containing protoporphyrin, is an essential component of hemoglobin that binds oxygen for delivery to tissues. In sickle cell disease, intravascular hemolysis leads to the presence of cell-free hemoglobin and heme, which may contribute to oxidative damage and activation of inflammatory pathways. Hemoproteins such as haptoglobin and hemopexin provide pathways to remove hemoglobin and heme, respectively, from circulation. Due to its hydrophobic nature, heme also intercalates in cell membranes and binds to plasma components such as albumin and lipoproteins, though with varying affinity. Hemopexin has high affinity for heme and removes heme from other heme pools in blood to counter the highly toxic properties of heme unbound to hemoproteins. Due to chronic hemolysis, hemopexin is depleted in individuals with sickle cell disease. We hypothesize that the reduction in heme binding capacity leads to increased unbound heme in blood and contributes to the pathogenesis of sickle cell disease. To define the different heme binding pools in patients with sickle cell disease, we developed a method requiring small amounts of plasma which allows measurement of total and hemoprotein-unbound heme. With this method, we can quantify the binding capacity of plasma for heme and correlate that with measurement of heme scavenging proteins.
Methods:
Blood from healthy individuals and sickle cell patients was collected in EDTA as anticoagulant under IRB approval. Plasma was separated by centrifugation from whole blood, and either processed fresh or after freezing at -80°C. Plasma protein was precipitated with a 4-fold volume of acetone at neutral pH (NA) or acidic pH (AA). Under acidic condition, heme is released from all heme binding pools, including hemoglobin, and provides detection of the total heme present in plasma. Under neutral pH condition, only heme unbound to plasma proteins is extracted. Once extracted, samples were dried and resuspended in DMSO. Heme concentration was spectrophotometrically determined at 400nm using standard curves prepared from hemin added in AA or NA. To determine heme binding capacity, hemin was added to serial dilutions of plasma and extracted in NA and AA as above. The appearance of heme in NA relative to AA represents the point at which heme binding capacity of plasma was saturated. This was compared to measurement of hemopexin and haptoglobin using commercially available ELISA measurements. Hemopexin and albumin were added to samples to modulate heme binding capacity.
Results:
Heme concentration closely correlates with spectroscopic measurement of heme in DMSO confirming reliable quantification of total and unbound heme in acidic and neutral acetone extractions as low as 2.5µM. We next show that heme binding capacity can be determined. Heme added to plasma was effectively recovered in AA extracts and begins to appear in the NA extract when binding sites start to become saturated. We note that not all sites appear to be fully saturated before heme is detected in NA extract. Addition of hemopexin to plasma increased the binding capacity on an equimolar basis, indicating that hemopexin effectively binds heme present in plasma. In samples from patients with sickle cell disease, concentration of total and unbound heme varied widely, and did not necessarily correlate with degree of intravascular hemolysis, estimated based on the measurement of cell free hemoglobin. Both the capacity of plasma to bind heme and levels of hemopexin indicated that, in a number of patients, the amount of heme present was greater than the ability of hemopexin to bind cell free heme.
Discussion:
We present a novel method to quantitatively differentiate hemoprotein-bound and unbound heme in plasma, the latter of which is pathologically relevant in sickle cell disease. Our data show significant variation in the concentration of total and unbound heme in sickle cell patient samples, and that the binding capacity in sickle cell plasma only partially correlates to the degree of hemolysis measured based on cell free hemoglobin. Patients are currently enrolled in a clinical study to measure intra-patient differences in heme and heme-binding capacity during steady state and during acute sickle cell-related illness. Understanding the clinical implications of heme and heme scavengers may provide insights into diagnostic and therapeutic targets for patients with sickle cell disease.

Disclosures
Neumayr: Emmaus: Consultancy; Bayer: Consultancy; CTD Holdings: Consultancy; Pfizer: Consultancy; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Micelle: Other: Site principal investigator; GBT: Other: Site principal investigator; PCORI: Other: site principal investigator; Novartis: Other: co-investigator; Bluebird Bio: Other: co-investigator; Sangamo Therapeutics: Other; Silarus: Other; Celgene: Other; La Jolla Pharmaceuticals: Other; Forma: Other; Centers for Disease Control and Prevention: Other; Seattle Children's Research: Other; Imara: Other; National Heart, Lung, and Blood Institute: Other; Health Resources and Services Administration: Other. Vichinsky:Bluebird Bio: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; GBT: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Kuypers:Forma Therapeutics, Inc.: Research Funding.