Biological response of human aortic endothelial cells exposed to acellular hemoglobin solutions developed as potential blood substitutes

Toussaint-Hacquard, M.; Devaux, Yvan; Longrois, Dan; Faivre-Fiorina, Béatrice; Müller, S.; Stoltz, Jean‐François; Vigneron, C.; Menu, Patrick

doi:10.1016/s0024-3205(02)02368-8

Cited by 11 publications

(8 citation statements)

References 28 publications

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“…This dose is supposed to be efficient for increasing an athlete's performance. Considering a total product dilution in the whole blood volume, a concentration of Oxyglobin in the circulatory system of ∼16 g L -1 is expected . We chose to spike 25 μL of human serum sample aliquots with different amounts of Oxyglobin solution [13 g/dL], resulting in final concentrations from 0 to 50 g L -1 in serum samples.…”

Section: Methodsmentioning

confidence: 99%

N-Terminal Adducts of Bovine Hemoglobin with Glutaraldehyde in a Hemoglobin-Based Oxygen Carrier

2005

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Hemoglobin-based oxygen carriers (HBOCs) are being developed for the medical field, but because they could increase an athlete's performance, they are misapplied for doping purposes. We previously presented a screening method to detect Oxyglobin (Biopure Corp.) and PolyHeme (Northfield Laboratories Inc.) in serum samples using total acid hydrolysis followed by electrospray mass spectrometry analyses. An alternative mass spectrometric method involving enzymatic hydrolysis is here presented. Digestion of Oxyglobin by endoproteinase Glu-C and LC/MS analyses of the mixture allowed the detection of unique peptidic fragments in comparison with a bovine hemoglobin digest. Tandem mass spectrometry experiments of these peptide ions were performed, and two specific species were actually identified as the N-terminal enzymatic fragment of the beta chain carrying two different modifications. Sequential MS3 experiments using an ion trap mass spectrometer permitted us to locate the chemical modification by the glutaraldehyde on the NH2-terminal group and to propose a structure for the modified peptides. In another set of experiments, screening of these two diagnostic ions into Oxyglobin-spiked serums using precursor ion scan mode in a triple quadrupole instrument allowed the detection of this HBOC with a detection limit of 2 g L(-1).

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Section: Methodsmentioning

confidence: 99%

N-Terminal Adducts of Bovine Hemoglobin with Glutaraldehyde in a Hemoglobin-Based Oxygen Carrier

2005

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“…But Oxyglobin-specific tryptic fragments were not detected by this way, probably due to their poor relative abundance in the peptide mixture and the heterogeneity of the adducts. However, even if the αK 99 could be preferentially modified, the reaction should also occur randomly on other sites of the sequence. , For instance, previous experiments performed on trypsin digests showed that the relative intensities of the chromatographic peaks of all the tryptic fragments were lower for Oxyglobin when balanced by the heme moiety chromatographic peak (data not shown). Consequently, chances could be increased to detect modified species by working on amino acids rather than on digest fragments and sensitivity could also be drastically improved.…”

Section: Resultsmentioning

confidence: 94%

“…For therapeutic purposes, a hemodilution of 20% is needed for an adult. Supposing this efficient dose is the same for increasing an athlete's performance and considering a total dilution of the product in the blood volume, the concentration of Oxyglobin in the circulatory system would be ∼16 g·L -1 . To reproduce experimentally these physiological conditions, 50 μL of human serum has to be spiked with 800 μg of Oxyglobin.…”

Section: Methodsmentioning

confidence: 99%

“…Supposing this efficient dose is the same for increasing an athlete's performance and considering a total dilution of the product in the blood volume, the concentration of Oxyglobin in the circulatory system would be ∼16 g‚L -1 . 27 To reproduce experimentally these physiological conditions, 50 µL of human serum has to be spiked with 800 µg of Oxyglobin. To stand below the theoritical detection limit, we deliberately chose to spike 50 µL of human serum samples aliquots with either 3.9 (500 µg, 10 g‚L -1 ) or 1.5 µL (200 µg, 4 g‚L -1 ) of Oxyglobin solution (13 g‚dL -1 ).…”

Section: Acid Hydrolysismentioning

confidence: 99%

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Simple Identification of A Cross-Linked Hemoglobin by Tandem Mass Spectrometry in Human Serum

et al. 2004

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Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1).

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“…Besides, this discovery established the foundation for seeing Hb as a signaling molecule [21,177,229,250]. Presently, a group of investigators from Universite Henri Poincare-Nancy, verified this finding by reporting that in fact Hb-based oxygen carriers induce expression of ICAM-1 that is consistent with the endothelial cell activation/cell signaling mechanism [251].…”

Section: Hemoglobin and Nuclear Factor Kappa Bmentioning

confidence: 87%

Endothelial Cell Response to Hemoglobin Based Oxygen Carriers. Is the Attenuation of Pathological Reactions Possible?

Simoni

Artificial Oxygen Carrier

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There is no doubt that in order to design an effective free hemoglobin (Hb)-based oxygen carrier, a full understanding of the mechanism of Hb toxicity is required. The current knowledge of Hb's overall toxicity however, is very limited. Hb is such an intriguing molecule that even after many decades of researching its physical, biochemical, physiological and pathological profile, many aspects of its intrinsic toxicity are yet to be uncovered [1]. This is probably the main reason for not having a viable substitute of human blood on the market. In fact, no product has yet been approved in the U.S. for any indication [2]. The currently tested, Hb-based oxygen carriers trigger a complex array of reactions as a result of Hb's natural features [3]. A number of unwanted effects have been observed in human clinical trials. Regardless of the type of Hb chemical modification procedure, all the first generation Hb-based oxygen carriers are vasoactive [1][2][3][4][5][6]. Some products have also been linked with oxidative and inflammatory reactions, often described as "flu like" symptoms, gastrointestinal side effects and myocardial lesions [1,3,7,8].The products currently in various phases of clinical trials were developed before the recognition of Hb's intrinsic toxicity problems. These products seem only to address the problems of Hb purity, high oxygen affinity and short circulatory half-life; which were uncovered in the 1970s and had been resolved in various ways. There are, however, newly discovered problems that need resolution. These are vasoactivity, pro-oxidant and pro-inflammatory properties of Hb [1][2][3][4][5][6][7][8]. Therefore, it was not surprising that the commercial

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Biological response of human aortic endothelial cells exposed to acellular hemoglobin solutions developed as potential blood substitutes

Cited by 11 publications

References 28 publications

N-Terminal Adducts of Bovine Hemoglobin with Glutaraldehyde in a Hemoglobin-Based Oxygen Carrier

N-Terminal Adducts of Bovine Hemoglobin with Glutaraldehyde in a Hemoglobin-Based Oxygen Carrier

Simple Identification of A Cross-Linked Hemoglobin by Tandem Mass Spectrometry in Human Serum

Endothelial Cell Response to Hemoglobin Based Oxygen Carriers. Is the Attenuation of Pathological Reactions Possible?

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