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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Technion Faculty of Medicine, Technion-Israel
Institute of Technology, Bat Galim, Israel.
Haptoglobin serves as an antioxidant by virtue of its ability to
prevent hemoglobin-driven oxidative tissue damage. It was recently
demonstrated that an allelic polymorphism in the haptoglobin gene is
predictive of the risk for numerous microvascular and macrovascular diabetic complications. Because these complications are
attributed in large part to an increase in oxidative stress, a study
was conducted to determine whether the different protein products of
the 2 haptoglobin alleles differed in the antioxidant protection they
provided. A statistically significant difference was found in the
antioxidant capacity of purified haptoglobin protein produced from the
2 different alleles, consistent with the hypothesis that differences in
genetically determined antioxidant status may explain differential
susceptibility to diabetic vascular complications. These differences
may be amplified in the vessel wall because of differences in the
sieving capacity of the haptoglobin types. Therefore, an attempt was
made to identify the minimal haptoglobin sequences necessary to inhibit
oxidation by hemoglobin in vitro, and 2 independent haptoglobin
peptides that function in this fashion as efficiently as native
haptoglobin were identified. Identification of the biochemical basis
for differences among haptoglobin types may lead to the rational
development of new pharmacologic agents, such as the mini-haptoglobin
described here, to avert the development of diabetic vascular complications.
(Blood. 2001;98:3693-3698) Haptoglobin is a serum protein that functions as an
antioxidant by virtue of its ability to bind to
hemoglobin1 and thereby to prevent the oxidative tissue
damage that may be mediated by free hemoglobin.2 The
importance of this protective mechanism has been demonstrated in
haptoglobin knockout mice in which a marked increase in oxidative
tissue damage develops in response to hemolysis.3 In
humans, 2 alleles (denoted 1 and 2) exist for the haptoglobin
gene.1,2,4 Biophysical and biochemical properties of the
haptoglobin polymeric molecules resulting from the 3 possible
combinations (haptoglobin 1-1, 2-1, or 2-2) of these 2 alleles are
dramatically different.2
We have recently found in multiple independent studies5-8
(and in A.P.L. et al, manuscript submitted for publication) of more
than 1000 persons in Israel, Belgium, and the United States that the
haptoglobin phenotype is a predictor of the risk for microvascular and
macrovascular complications of diabetes. Specifically, diabetic
patients with the haptoglobin 1-1 phenotype were shown to be remarkably
resistant to the development of diabetic retinopathy, diabetic
nephropathy, and cardiovascular disease.5-8 Moreover, we
found that a graded effect was evident with regard to risk and to the
number of haptoglobin 2 alleles.7,8 For example, in a
prospective study of incident cardiovascular disease, we found that
participants homozygous for the haptoglobin 2 allele had a 5-fold
increase in the risk for cardiovascular disease compared with patients
homozygous for the haptoglobin 1 allele, whereas heterozygotes were
found to have an intermediate risk (A.P.L. et al, manuscript submitted
for publication).
It has been proposed that an increase in oxidative stress plays a
crucial role in the development of diabetic vascular
complications.9,10 Accordingly, differences in genetically
endowed antioxidant status may confer increased or decreased
susceptibility to the development of these diabetic vascular
complications. We demonstrate here that purified haptoglobin 1-1 is a
superior antioxidant to purified haptoglobin 2-2 in vitro. A key site
of action of haptoglobin in neutralizing the oxidative capacity of
hemoglobin is the extravascular space, particularly after endothelial
injury. Haptoglobins 1-1 and 2-2 clearly differ in their ability to
sieve into the extravascular compartment across the endothelial cell
barrier.2 Because this difference in sieving is almost
certainly a reflection of the profound differences in the sizes of
haptoglobin 1-1 dimers and haptoglobin 2-2 cyclic polymers, we sought
to identify a minimal haptoglobin peptide with preserved antioxidant
function that would have an improved ability to penetrate the
extravascular space. We describe identification of 2 distinct
haptoglobin peptides, each of which can function as efficiently as
haptoglobin in preventing oxidation by hemoglobin in vitro.
Oxidation of linolenic acid by hemoglobin
The standard reaction (720 µL) consisted of the following reagents,
all incubated at room temperature: 120 µL micelles (final concentration linolenic acid, 0.55 mM), 3 µL of a 10-mg/mL solution of hemoglobin in 1 mL buffer A (final concentration hemoglobin, 0.62 µM), and haptoglobin diluted in buffer A to the desired
concentration. Additional buffer A was added to achieve a final volume
of 720 µL. All components except for hemoglobin were added directly
to a quartz cuvette and mixed by inverting 6 times. Three microliters hemoglobin solution was then added, and the cuvette was inverted to mix
the ingredients. The zero time point was designated as the time at
which hemoglobin was added to the solution. The formation of conjugated
dienes was monitored by the change in absorption of the solution at 232 nm (A232) at room temperature for 60 minutes using a
Lightwave S2000 spectrophotometer (WPA, Cambridge, United Kingdom).
Readings were taken every 10 minutes. For all experiments assessing the
ability to inhibit diene formation with haptoglobin or vitamins, 6 simultaneous reactions were performed to permit direct comparison of
the increase in A232 obtained from the incubation of
hemoglobin alone compared with hemoglobin with the antioxidants to be
tested at the various concentrations. The change in A232 with hemoglobin alone at 60 minutes was taken as the 100% value, and
the change in A232 with each of the antioxidants at the
different concentrations of haptoglobin at 60 minutes was determined
relative to this value and was expressed as a percentage of relative
oxidation. For each concentration of each antioxidant, the reaction was
performed at least 6 separate times, and the mean ± SEM was
determined. P values were determined using the paired
Student t test, with P < .05 considered
statistically significant.
Oxidation of low-density lipoprotein by
hemoglobin.
Low-density lipoprotein (LDL) was isolated from human plasma by
sequential ultracentrifugation as previously
described.11,12 Oxy-Hb was obtained by chromatography
methods, verified spectrophotometrically, and converted to met-Hb as
previously described.13 LDL (200 µg/mL) was incubated
for 4 hours at 37°C with met-Hb (10 µM) in the presence of
H2O2 (20 µM). To this standard assay were
added various concentrations of haptoglobin 1-1 or 2-2. Oxidation of LDL lipids was determined using the thiobarbituric reactive substances (TBARS) assay14 using a WPA Lightwave
spectrophotometer. All experiments were performed at least 3 times, and
the data are presented as mean inhibition by haptoglobin compared with
the absence of haptoglobin. Relative inhibition was calculated by integrating the area under the curve of the TBARS assay using the
MATLAB program. All values are expressed as mean ± SEM.
P values were determined using the paired Student
t test, with P < .05 considered statistically significant.
Determination of the free hemoglobin concentration in the
haptoglobin hemoglobin incubation conditions used to assess the
antioxidant activity of haptoglobin.
Haptoglobin and met-hemoglobin were used at the concentrations
described above for oxidation of LDL or linolenic acid. After a
10-minute incubation at room temperature, the haptoglobin-hemoglobin mixture was placed in a Centricon ultrafiltration apparatus with 100 kd
cutoff (YM-100; Millipore, Bedford, MA). The apparatus was then
subjected to centrifugation for 30 minutes at 1000g
according to the manufacturer's instructions, resulting in the
retention of complexes larger than 100 kd in the upper chamber and
smaller than 100 kd in the lower chamber (filtrate). Because only free hemoglobin (molecular weight of the hemoglobin monomer is 64 kd) could pass into the filtrate, the concentration of hemoglobin in
the filtrate, determined spectrophotometrically, was used to determine
the concentration of free hemoglobin. For these studies, we used an
extinction coefficient for met-hemoglobin of EmM 179 at 405 nm.12
Preparation of recombinant truncated haptoglobin
Enzyme-linked immunosorbent assay for qualitative determination of
binding of truncated haptoglobin to hemoglobin
Antioxidant activity of truncated haptoglobin Recombinant GST-fusion proteins were analyzed in the linolenic acid oxidation assay for their ability to inhibit the oxidation of linolenic acid by hemoglobin as described above. GST alone had no effect on the oxidation of linolenic acid by hemoglobin even when used at concentrations 10 times greater than that used for the recombinant GST-haptoglobin fusion proteins.
Inhibition of oxidation of linolenic acid by purified haptoglobin As previously demonstrated, we found that hemoglobin can oxidize linolenic acid in a time-dependent fashion as assessed using conjugated diene (A232) formation (Figure 2A). This oxidation of linolenic acid by hemoglobin was shown to be inhibited by stoichiometric amounts of a mixture of the different haptoglobins prepared from pooled human sera.16,17 We sought to determine whether the ability to inhibit the oxidation of linolenic acid by hemoglobin as assessed by diene formation was different between the haptoglobin 1-1 and 2-2 proteins. Figure 2A provides a representative example of the differences in diene formation produced by the oxidation of linolenic acid in the presence of no haptoglobin and in the presence of haptoglobin 1-1 or haptoglobin 2-2, each at a concentration of 0.6 µM haptoglobin. At this concentration, haptoglobin 1-1 provided statistically significant greater protection against linolenic oxidation than haptoglobin 2-2 (Figure 2B). The percentage inhibition of the hemoglobin-induced oxidation of linolenic acid by both haptoglobin type 1-1 and haptoglobin type 2-2 was linearly related to the concentration of haptoglobin in our assay over a range of haptoglobin concentrations, from approximately 0.1 to 0.7 µM. Over this concentration range, haptoglobin 1-1 consistently demonstrated more inhibition of oxidation of linolenic acid than haptoglobin 2-2 (Figure 2B). At haptoglobin concentrations greater than 1.0 µM, there was complete (100%) inhibition of linolenic acid oxidation by both types of haptoglobin, whereas at haptoglobin concentrations less than 0.1 µM, there was no significant inhibition of linolenic acid oxidation by either type of haptoglobin.
Inhibition of low-density lipoprotein oxidation by haptoglobin As previously demonstrated,13 we found that hemoglobin can oxidize LDL in a time-dependent fashion as assessed by measuring TBARS (Figure 3A). This oxidation of LDL by hemoglobin was previously shown to be inhibited by stoichiometric amounts of a mixture of haptoglobin proteins prepared from pooled human sera.13 We sought to determine whether the ability to inhibit the oxidation of LDL by hemoglobin was different between haptoglobin 1-1 and 2-2 proteins. Figure 3A provides a representative example of the differences in TBARS formation produced by the oxidation of LDL in the presence of no haptoglobin and in the presence of haptoglobin 1-1 or haptoglobin 2-2, each at a concentration of 5 µM haptoglobin. At this haptoglobin concentration, haptoglobin 1-1 provided statistically significant greater protection against LDL oxidation than haptoglobin 2-2 (Figure 3B). The percentage inhibition of the hemoglobin-induced oxidation of LDL by haptoglobin 1-1 or 2-2 was linearly related to the concentration of haptoglobin in the assay over a range of haptoglobin concentrations, from 1 to 20 µM. Over this concentration range, haptoglobin 1-1 consistently demonstrated greater inhibition of oxidation of LDL than haptoglobin 2-2 (Figure 3B). Outside this concentration range for haptoglobin, where either haptoglobin was present in extreme molar excess or hemoglobin was present in extreme molar excess, the 2 types of haptoglobin were not different in protecting against hemoglobin-induced oxidation of LDL, analogous to what was described for linolenic acid above.
Effective hemoglobin-binding capacity of haptoglobin 1-1 and haptoglobin 2-2 Differences in the antioxidant protection provided by our haptoglobin preparations could be a result of a systematic error made in estimating haptoglobin hemoglobin-binding capacity. We used haptoglobin monomer molar concentrations in these studies because it has been established that every haptoglobin monomer can bind 1 hemoglobin molecule (alpha-beta dimer) (stoichiometry of 1-1).1 This stoichiometry of the binding reaction is thought to be identical for all forms of haptoglobin.1,18,19 However, it has been proposed that not every haptoglobin monomer in the larger cyclic polymers found in haptoglobin type 2-2 is capable of binding hemoglobin because of steric considerations.20 If every haptoglobin monomer in a cyclic 2-2 polymer cannot bind hemoglobin, then over the concentration range of haptoglobin used to inhibit the oxidation of LDL or linolenic acid we predicted there would be a greater amount of free hemoglobin in reactions using haptoglobin 2-2 than in those using haptoglobin 1-1. The excess free hemoglobin in the reaction using haptoglobin 2-2 would be expected to result in more oxidation of LDL or linolenic acid than what was observed with haptoglobin 1-1.To investigate the effective hemoglobin-binding capacity of our
haptoglobin 1-1 and 2-2 preparations, we developed a filtration assay
designed to monitor the amount of unbound hemoglobin, as described in
"Materials and methods." The filtration assay used the Centricon
microfiltration system by way of a membrane with a 100-kd cutoff.
Haptoglobin and hemoglobin were mixed together at the same molar ratios
used in the oxidation studies described above and then were subjected
to Centricon ultrafiltration. The amount of free or unbound hemoglobin
was determined spectrophotometrically in the ultrafiltrate. If the
greater amount of oxidation in the reactions involving haptoglobin 2-2 was attributed to less hemoglobin-binding capacity than those involving
haptoglobin 1-1, we would have expected to see more free hemoglobin in
the ultrafiltrate when using haptoglobin 2-2. This was not the case.
There was no significant difference in the free hemoglobin
concentrations in the ultrafiltrate of haptoglobin 1-1-hemoglobin
solutions than in haptoglobin 2-2-hemoglobin solutions. At a
concentration of 10 µM hemoglobin and 5 µM haptoglobin (concentrations at which we found haptoglobin 1-1 was significantly superior to haptoglobin 2-2 in protecting against hemoglobin-induced LDL oxidation), we found that the molar ratio of free hemoglobin in the
ultrafiltrate for haptoglobin 1-1-hemoglobin solutions compared to
haptoglobin 2-2-hemoglobin solutions was 1.09 ± Identification of putative hemoglobin-binding sites on haptoglobin by ELISA using truncated haptoglobin The hemoglobin-haptoglobin complex has not as yet been crystallized; thus, the residues involved in binding are not definitively known. Findings from gel permeation studies with purified haptoglobin have suggested that the -chain of haptoglobin is
responsible for binding to hemoglobin.1 The importance of
several residues in the -chain has been suggested by the use of
proteolytic peptides of haptoglobin and the ascertainment of their
ability to bind to hemoglobin in native polyacrylamide
gels.21 Further assessment of the putative residues on
haptoglobin capable of binding to hemoglobin using recombinant
haptoglobin-truncated mutants or haptoglobin peptides has not been
performed. Therefore, we developed a simple ELISA capable of
differentiating qualitative differences in the binding of haptoglobin
to hemoglobin. A battery of  - and -chain recombinant fusion
proteins was made and is shown schematically in Figure 1. We were able
to identify binding not only in the -chain but, surprisingly, also
in the -chain (qualitatively denoted by 0-2+ binding in Table
3). This assay was only used to detect
the presence or absence of binding of specific haptoglobin mutants before ascertaining whether they had activity as antioxidants and could not be used to demonstrate quantitative differences in
binding affinity between the different mutants.
Truncated haptoglobin can prevent the oxidation of linolenic acid by
hemoglobin. We tested the ability of the truncated haptoglobin fusion
proteins that were shown to bind to hemoglobin in the ELISA for their
ability to inhibit the oxidation of linolenic acid by hemoglobin as
described in "Materials and methods." Using progressive deletion
analysis, we were able to identify in the
We have demonstrated that there are functional differences in the antioxidant capacity of the different haptoglobin proteins toward hemoglobin, suggesting that those with haptoglobin 1-1 protein may have superior antioxidant protection than those with haptoglobin 2-2 protein. These data are consistent with earlier findings showing that the consumption of vitamin C in the plasma in vitro of persons with haptoglobin 2-2 was more rapid than in the plasma of those with haptoglobin 1-1 and that vitamin C levels are significantly lower in those with haptoglobin 2-2.22 The stoichiometries of haptoglobin 1-1 and 2-2 binding to hemoglobin are identical.1,18,19 Our results showing a difference between the amount of hemoglobin-inducible oxidation between our 2 different preparations was not caused by a systematic error in the determination of the hemoglobin-binding capacity of our preparations of the 2 haptoglobin types. Using concentrations of haptoglobin and hemoglobin identical to what was used in the oxidation reactions described in this study, we found that there was no significant difference between the amount of free hemoglobin using our haptoglobin 1-1 preparations and our haptoglobin 2-2 preparations. This was important to demonstrate because if our haptoglobin 2-2 had lower hemoglobin-binding capacity than our haptoglobin 1-1, the amount of free hemoglobin available to oxidize linolenic acid or LDL in the oxidation reactions using haptoglobin 2-2 would have been greater. Therefore, the inferior antioxidant capacity of our haptoglobin 2-2 could not be attributed to lower hemoglobin-binding capacity. The antioxidant activity of haptoglobin results, at least in part, from the binding of haptoglobin to hemoglobin, thereby preventing the dissociation of ferric heme from globin.23 Demonstration that iron is one of the species directly responsible for the oxidation of linolenic acid has been demonstrated by 2 groups using the iron chelator, desferrioxamine.16,17 However, Gutteridge16 has also shown that hemoglobin is capable of stimulating lipid peroxidation for a short period of time by a reaction independent of free heme (ie, inhibited by haptoglobin but not inhibited by desferrioxamine) and that heme release only occurs after the products of lipid peroxidation damage the hemoglobin molecule, causing it to release iron. The difference between haptoglobin 1-1 and haptoglobin 2-2 in inhibiting the oxidation of linolenic acid, according to these mechanistic schemes, may be the result of differences in the ability of the different types of haptoglobin to prevent the release of heme. We have not observed that the differences between the 2 types of haptoglobin are more exaggerated at earlier time points in the oxidation reaction, which might have suggested that the alternative mechanism proposed by Gutteridge16 is a point of difference between the 2 types of haptoglobin. The haptoglobin-hemoglobin complex has been demonstrated to have a potent peroxidase activity24 that could also serve as a point of difference between the 2 haptoglobin types and could provide an explanation for their differences in apparent antioxidant activity. The statistically significant but relatively modest differences we have
described here in antioxidant capacity between the different
haptoglobin types may be dramatically amplified in vivo because of
differences in the ability of the different haptoglobin types to gain
access to the vessel wall. A schematic drawing of the different
haptoglobin polymers1,25 in persons with haptoglobin 1-1 or 2-2, as shown in Figure 5,
demonstrates the large differences in size between the different
haptoglobin types likely to account for these differences in sieving
capacity. At sites of blood vessel injury (ie, after coronary
angioplasty), there is a sudden release of free hemoglobin into the
blood vessel wall. Haptoglobin is not normally found in appreciable
concentrations in the normal vessel wall. Therefore, the ability of
haptoglobin to sieve into the vessel wall to neutralize hemoglobin is
likely to be of great importance. In the patient with diabetes,
already burdened with increased oxidative stress from
hyperglycemia,9,10 differences in genetically determined
endogenous antioxidant protection may have exaggerated importance.
We have identified 2 peptides derived from haptoglobin that can independently bind to hemoglobin and prevent it from oxidizing substrate, analogous to the full-length haptoglobin molecule. We have not determined the affinity of these constructs for hemoglobin, nor have we demonstrated that the stoichiometry of binding of these constructs is identical to native haptoglobin. Our studies suggest that these truncated haptoglobins are similar in their potency to native haptoglobin in terms of their ability to inhibit the oxidation of linolenic acid by hemoglobin. Such a mini-haptoglobin may be expected to have improved access to the extravascular space and thus may be proposed as a candidate drug in animal models of diabetic vascular complications. We are performing progressive truncation of these mini-haptoglobins to define the absolute minimal haptoglobin that can inhibit oxidation by hemoglobin. We have learned from epidemiologic studies that haptoglobin type is fundamentally important in the development of diabetic vascular disease. Elucidation of the biochemical basis for differences between the haptoglobin types is the first step necessary for the development of new drugs and strategies to limit diabetic vascular complications.
Submitted June 18, 2001; accepted August 1, 2001.
Supported by National Heart Lung and Blood Institute grants RO1 HL-58510 and RO1 HL66195 (A.P.L.), the Israel Cancer Research Fund (A.P.L.), the Israel Cancer Association (A.P.L.), the Israel Science Foundation (A.P.L.), and the Bruce Rappaport Fund for Biochemical Research (A.P.L.).
M.M.-F., O.L., and B.I.E. contributed equally to the preparation of this manuscript.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Andrew P. Levy, Technion Faculty of Medicine, POB 9649, Haifa 31096, Israel; e-mail:alevy{at}tx.technion.ac.il.
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© 2001 by The American Society of Hematology.
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G. Silva, V. Jeney, A. Chora, R. Larsen, J. Balla, and M. P. Soares Oxidized Hemoglobin Is an Endogenous Proinflammatory Agonist That Targets Vascular Endothelial Cells J. Biol. Chem., October 23, 2009; 284(43): 29582 - 29595. [Abstract] [Full Text] [PDF]
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 F. M. Nakhoul, R. Miller-Lotan, H. Awad, R. Asleh, K. Jad, N. Nakhoul, R. Asaf, N. Abu-Saleh, and A. P. Levy Pharmacogenomic effect of vitamin E on kidney structure and function in transgenic mice with the haptoglobin 2-2 genotype and diabetes mellitus Am J Physiol Renal Physiol, April 1, 2009; 296(4): F830 - F838. [Abstract] [Full Text] [PDF]
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 M. T. Gladwin and E. Vichinsky Pulmonary Complications of Sickle Cell Disease N. Engl. J. Med., November 20, 2008; 359(21): 2254 - 2265. [Full Text] [PDF]
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 R. Asleh, S. Blum, S. Kalet-Litman, J. Alshiek, R. Miller-Lotan, R. Asaf, W. Rock, M. Aviram, U. Milman, C. Shapira, et al. Correction of HDL Dysfunction in Individuals With Diabetes and the Haptoglobin 2-2 Genotype Diabetes, October 1, 2008; 57(10): 2794 - 2800. [Abstract] [Full Text] [PDF]
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 P. R. Moreno, K. R. Purushothaman, M. Purushothaman, P. Muntner, N. S. Levy, V. Fuster, J. T. Fallon, P. A. Lento, A. Winterstern, and A. P. Levy Haptoglobin Genotype Is a Major Determinant of the Amount of Iron in the Human Atherosclerotic Plaque J. Am. Coll. Cardiol., September 23, 2008; 52(13): 1049 - 1051. [Abstract] [Full Text] [PDF]
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 S. Blum, U. Milman, C. Shapira, R. Miller-Lotan, L. Bennett, M. Kostenko, M. Landau, S. Keidar, Y. Levy, A. Khemlin, et al. Dual Therapy With Statins and Antioxidants Is Superior to Statins Alone in Decreasing the Risk of Cardiovascular Disease in a Subgroup of Middle-Aged Individuals With Both Diabetes Mellitus and the Haptoglobin 2-2 Genotype Arterioscler Thromb Vasc Biol, March 1, 2008; 28(3): e18 - e20. [Full Text] [PDF]
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U. Milman, S. Blum, C. Shapira, D. Aronson, R. Miller-Lotan, Y. Anbinder, J. Alshiek, L. Bennett, M. Kostenko, M. Landau, et al. Vitamin E Supplementation Reduces Cardiovascular Events in a Subgroup of Middle-Aged Individuals With Both Type 2 Diabetes Mellitus and the Haptoglobin 2-2 Genotype: A Prospective Double-Blinded Clinical Trial Arterioscler Thromb Vasc Biol, February 1, 2008; 28(2): 341 - 347. [Abstract] [Full Text] [PDF]
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 K. L. Chaichana, A. P. Levy, R. Miller-Lotan, S. Shakur, and R. J. Tamargo Haptoglobin 2-2 Genotype Determines Chronic Vasospasm After Experimental Subarachnoid Hemorrhage Stroke, December 1, 2007; 38(12): 3266 - 3271. [Abstract] [Full Text] [PDF]
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 A. P. Levy, K. R. Purushothaman, N. S. Levy, M. Purushothaman, M. Strauss, R. Asleh, S. Marsh, O. Cohen, S. K. Moestrup, H. J. Moller, et al. Downregulation of the Hemoglobin Scavenger Receptor in Individuals With Diabetes and the Hp 2-2 Genotype: Implications for the Response to Intraplaque Hemorrhage and Plaque Vulnerability Circ. Res., July 6, 2007; 101(1): 106 - 110. [Abstract] [Full Text] [PDF]
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M. J. Nielsen, S. V. Petersen, C. Jacobsen, S. Thirup, J. J. Enghild, J. H. Graversen, and S. K. Moestrup A Unique Loop Extension in the Serine Protease Domain of Haptoglobin Is Essential for CD163 Recognition of the Haptoglobin-Hemoglobin Complex J. Biol. Chem., January 12, 2007; 282(2): 1072 - 1079. [Abstract] [Full Text] [PDF]
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S. Blum, R. Asaf, J. Guetta, R. Miller-Lotan, R. Asleh, R. Kremer, N. S. Levy, F. G. Berger, D. Aronson, X. Fu, et al. Haptoglobin Genotype Determines Myocardial Infarct Size in Diabetic Mice J. Am. Coll. Cardiol., January 2, 2007; 49(1): 82 - 87. [Abstract] [Full Text] [PDF]
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A. P. Levy, J. E. Levy, S. Kalet-Litman, R. Miller-Lotan, N. S. Levy, R. Asaf, J. Guetta, C. Yang, K. R. Purushothaman, V. Fuster, et al. Haptoglobin Genotype Is a Determinant of Iron, Lipid Peroxidation, and Macrophage Accumulation in the Atherosclerotic Plaque Arterioscler Thromb Vasc Biol, January 1, 2007; 27(1): 134 - 140. [Abstract] [Full Text] [PDF]
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R. Asleh, R. Miller-Lotan, M. Aviram, T. Hayek, M. Yulish, J. E. Levy, B. Miller, S. Blum, U. Milman, C. Shapira, et al. Haptoglobin Genotype Is a Regulator of Reverse Cholesterol Transport in Diabetes In Vitro and In Vivo Circ. Res., December 8, 2006; 99(12): 1419 - 1425. [Abstract] [Full Text] [PDF]
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 P. R. Moreno, K-R. Purushothaman, M. Sirol, A. P. Levy, and V. Fuster Neovascularization in Human Atherosclerosis Circulation, May 9, 2006; 113(18): 2245 - 2252. [Full Text] [PDF]
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 T R Zaccariotto, E T Rosim, D Melo, P M D Garcia, R R Munhoz, F H Aoki, and M de Fatima Sonati Haptoglobin polymorphism in a HIV-1 seropositive Brazilian population. J. Clin. Pathol., May 1, 2006; 59(5): 550 - 553. [Abstract] [Full Text] [PDF]
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M. J. Weiss Handling heme Blood, October 1, 2005; 106(7): 2225 - 2226. [Full Text] [PDF]
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M. Suleiman, D. Aronson, R. Asleh, M. R. Kapeliovich, A. Roguin, S. R. Meisel, M. Shochat, A. Sulieman, S. A. Reisner, W. Markiewicz, et al. Haptoglobin Polymorphism Predicts 30-Day Mortality and Heart Failure in Patients With Diabetes and Acute Myocardial Infarction Diabetes, September 1, 2005; 54(9): 2802 - 2806. [Abstract] [Full Text] [PDF]
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 N. L. Lohr, D. C. Warltier, W. M. Chilian, and D. Weihrauch Haptoglobin expression and activity during coronary collateralization Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1389 - H1395. [Abstract] [Full Text] [PDF]
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N. S. Levy and A. P. Levy ELISA for Determination of the Haptoglobin Phenotype Clin. Chem., November 1, 2004; 50(11): 2148 - 2150. [Full Text] [PDF]
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 S. Mustafa, T. Vukovich, T. Prikoszovich, C. Winzer, B. Schneider, H. Esterbauer, O. Wagner, and A. Kautzky-Willer Haptoglobin Phenotype and Gestational Diabetes Diabetes Care, September 1, 2004; 27(9): 2103 - 2107. [Abstract] [Full Text] [PDF]
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A. P. Levy, P. Friedenberg, R. Lotan, P. Ouyang, M. Tripputi, L. Higginson, F. R. Cobb, J.-C. Tardif, V. Bittner, and B. V. Howard The Effect of Vitamin Therapy on the Progression of Coronary Artery Atherosclerosis Varies by Haptoglobin Type in Postmenopausal Women Diabetes Care, April 1, 2004; 27(4): 925 - 930. [Abstract] [Full Text] [PDF]
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 J. N. Fain, S. W. Bahouth, and A. K. Madan Haptoglobin release by human adipose tissue in primary culture J. Lipid Res., March 1, 2004; 45(3): 536 - 542. [Abstract] [Full Text] [PDF]
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P. Philippidis, J.C. Mason, B.J. Evans, I. Nadra, K.M. Taylor, D.O. Haskard, and R.C. Landis Hemoglobin Scavenger Receptor CD163 Mediates Interleukin-10 Release and Heme Oxygenase-1 Synthesis: Antiinflammatory Monocyte-Macrophage Responses In Vitro, in Resolving Skin Blisters In Vivo, and After Cardiopulmonary Bypass Surgery Circ. Res., January 9, 2004; 94(1): 119 - 126. [Abstract] [Full Text] [PDF]
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A. Roguin, W. Koch, A. Kastrati, D. Aronson, A. Schomig, and A. P. Levy Haptoglobin Genotype Is Predictive of Major Adverse Cardiac Events in the 1-Year Period After Percutaneous Transluminal Coronary Angioplasty in Individuals With Diabetes Diabetes Care, September 1, 2003; 26(9): 2628 - 2631. [Abstract] [Full Text] [PDF]
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R. Asleh, S. Marsh, M. Shilkrut, O. Binah, J. Guetta, F. Lejbkowicz, B. Enav, N. Shehadeh, Y. Kanter, O. Lache, et al. Genetically Determined Heterogeneity in Hemoglobin Scavenging and Susceptibility to Diabetic Cardiovascular Disease Circ. Res., June 13, 2003; 92(11): 1193 - 1200. [Abstract] [Full Text] [PDF]
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A. P. Levy, I. Hochberg, K. Jablonski, H. E. Resnick, E. T. Lee, L. Best, and B. V. Howard Haptoglobin phenotype is an independent risk factor for cardiovascular disease in individuals with diabetes: the strong heart study J. Am. Coll. Cardiol., December 4, 2002; 40(11): 1984 - 1990. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
0.15 (P = .835; n = 6). Therefore, the inferior antioxidant
capacity of our preparation of haptoglobin 2-2 could not be explained
by a lower effective hemoglobin-binding capacity than our preparation of haptoglobin 1-1.
