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The antioxidant activity of haptoglobin towards haemoglobin-stimulated lipid peroxidation.

Haemoglobin stimulates the peroxidation of lipids in two discernable phases. The first phase is inhibited by binding haemoglobin to the protein haptoglobin. The second phase is stimulated by complexable iron released from the haemoglobin molecule during the process of lipid peroxidation. This latter peroxidation is inhibitable by transferrin and the iron chelator desferrioxamine. Heat-denatured haemoglobin and haemin both stimulated lipid peroxidation but this is not inhibitable by haptoglobin. It is suggested that the haptoglobins play an important antioxidant role in vivo by preventing iron-stimulated formation of oxygen radicals.

Antioxidants↗

The effects of limited proteolysis by trypsin on human haptoglobin.

Trypsin digestion of haptoglobin resulted i four glycopeptides. The glycopeptides were characterized by amino acid composition and molecular weight, as determined by thin-layer chromatography, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. Hemoglobin-binding capacity and immunological properties were investigated. glycopeptides I and II did not form an active complex with hemoglobin and they inhibited the reaction of haptoglobin with specific antiserum by over 70%. Glycopeptides III and IV showed 11 and 4% of the hemoglobin-binding capacity and 82 and 67% of antigenic reactivity of native haptoglobin, respectively. Glycopeptide IV contained three antigenic determinants, whereas glycopeptides III contained four, one of them being exposed by trypsin digestion. In crossed two-dimensional immunoelectrophoresis, glycopeptide III showed at least four components reacting with antihaptoglobin serum, and glycopeptide IV, two components.

Amino Acids↗

Biosynthesis of rabbit haptoglobin: chemical evidence for a single chain precursor.

The primary translation product of the mRNA for rabbit haptoglobin was obtained from a rabbit reticulocyte lysate cell-free system by immunoprecipitation with an antiserum that was directed to the beta chain of haptoglobin. Analysis of the translation product by gel electrophoresis and by protein sequencing analysis identified a single polypeptide of Mr 41 000. Sequence analysis established a signal region of 18 residues that was immediately followed by the alpha chain sequence. These results give strong evidence that haptoglobin is initially synthesized as a single chain composed of a signal peptide followed by alpha and beta chain regions, respectively.

Amino Acid Sequence↗

Protein-chemical analysis of pertussis toxin reveals homology between the subunits S2 and S3, between S1 and the A chains of enterotoxins of Vibrio cholerae and Escherichia coli and identifies S2 as the haptoglobin-binding subunit.

The purified toxin of Bordetella pertussis was dissociated in 5 M urea in the presence of immobilized haptoglobin. The toxin was dissociated in free S1, free S5 and the free complexes S2-S4 and S3-S4, with S2-S4 as the only haptoglobin-binding moiety, identifying S2 as the haptoglobin-binding protein. Partial NH2-terminal amino acid sequences were obtained from the dissimilar toxin subunits, after separation by SDS-polyacrylamide gel electrophoresis followed by electroblotting onto polybrene-coated glass-fiber sheets. The sequences reveal extensive homology of the N-terminal portions of the constitutive subunits S2 and S3 and between S1 and the enterotoxin A chains of Vibrio cholerae and Escherichia coli.

Amino Acid Sequence↗

Augmentation of haptoglobin production in Hep3B cell line by a nuclear factor NF-IL6.

The nuclear factor NF-IL6 had been suggested to be responsible for the IL-6-mediated induction of several acute-phase proteins. To obtain evidence for the involvement of NF-IL6 in the induction of acute-phase proteins, we introduced the NF-IL6 gene and its truncated mutant (delNFIL6) gene into a hepatoma cell line Hep3B. Then, we examined the effect of the overproduced NF-IL6 and delNFIL6 on the expression of haptoglobin, fibrinogen and albumin. As a result, basal production as well as induction of haptoglobin by IL-6 were augmented by the expression of NF-IL6, whereas delNFIL6 blocked the production of haptoglobin, fibrinogen and albumin.

Acute-Phase Proteins↗

Uptake of iron from hemoglobin and the haptoglobin-hemoglobin complex by hemolytic bacteria.

The abilities of Staphylococcus aureus and Streptococcus pyogenes to remove iron from mouse 59Fe hemoglobin that was either in free form or complexed with human haptoglobin, were evaluated. 59Fe hemoglobin from the amphibian Taricha granulosa was also used in free form or complexed with the amphibian's hemoglobin-binding proteins. Contrary to what was reported from a study using pathogenic Escherichia coli, haptoglobin failed to exhibit a bacteriostatic influence when complexed with hemoglobin. In our study, more 59Fe was removed by the bacteria from the haptoglobin-hemoglobin complex than from free mouse hemoglobin. The hemoglobin and hemoglobin-plasma protein complexes of Taricha were stripped of 59Fe at similar rates and extents by both bacterial species.

Amphibians↗

Haptoglobin response of cattle infected with foot-and-mouth disease virus.

Haptoglobin, a major bovine acute phase protein, was evaluated as a marker of the primary replication of foot-and-mouth disease virus in 12 naturally infected cattle from which blood was collected daily. An acute phase response, as measured by an increase in serum haptoglobin concentration and the presence of fever, was not detected during the previraemic stage of disease, but there was a significant increase in serum haptoglobin after the onset of viraemia. It occurred on the same day as the first appearance of clinical signs in three of the cattle, one day later in four and two days later in the other five cattle.

Animals↗

Evaluation of C-reactive protein and haptoglobin as malaria episode markers in an area of high transmission in Africa.

Field studies of malaria in endemic areas frequently use the presence or levels of parasitaemia, together with the measurement of fever, as the primary criteria with which to identify cases. However, since malaria cases do not always present with measurable fever, and since asymptomatic parasitaemia occurs, additional episode markers might be useful epidemiological tools. We have measured the C-reactive protein and haptoglobin levels in paediatric patients presenting to a village health post in the Kilombero District in Tanzania and in convalescent sera from the same patients, in order to evaluate these acute-phase reactants as alternative markers of Plasmodium falciparum episodes. Among afebrile patients, C-reactive protein levels were highly correlated with parasite density. High C-reactive protein levels are therefore probably indicative of recent clinical malaria episodes in currently afebrile individuals with high parasite densities. An appropriate case definition for malaria in epidemiological studies in endemic areas might therefore be hyperparasitaemia accompanied by either, or both, measurable fever and raised C-reactive protein levels. This would give less biased estimates of the overall burden of malaria morbidity than does a definition which requires measurable fever. Levels of haptoglobin were highly negatively correlated with parasitaemia, but did not appear to be useful episode markers because this correlation was probably not related to acute morbidity. However, haptoglobin can be useful to assess at community level the impact of interventions on parasitaemia.

Biomarkers↗

Haptoglobin: an immunoregulatory role in tuberculosis?

A significant correlation was found between levels of haptoglobin in sera from Indonesian patients with tuberculosis and the extent to which these sera suppressed mitogen-driven activation of normal lymphocytes in vitro. As in a previous study we showed that there was an inverse relationship between haptoglobin levels and the circulating lymphocyte count in the same group of patients, it is now suggested that this protein has an immunoregulatory role in tuberculosis. Similar observations have been made by other workers on patients with cancer. There was no difference in the distribution of the 3 allotypes of haptoglobin in patients and healthy subjects nor was there any association between such allotypes and the lymphocyte count, the dermal reactivity to tuberculin or the degree of suppression.

Agglutination Tests↗

Prostaglandin synthesis inhibition by maternal and fetal sheep plasma and its relation to haptoglobin and albumin levels.

Mammalian plasmas and sera have been reported to contain endogenous inhibitors of prostaglandin synthesis (EIPS), but the identity and role of these suggested inhibitors is as yet undetermined. Albumin and haptoglobin have been proposed as possible inhibitors, and it has been suggested that EPIS may have a part to play in the control of PG production during pregnancy and in the neonatal period. As part of a series of studies aimed at elucidating the identity and role of EIPS, maternal and fetal blood samples were collected from chronically catheterized pregnant ewes, and plasma levels of albumin, haptoglobin and EIPS activity determined. Pregnant ewe plasma possessed high EIPS activity and fetal lamb plasma little or no EIPS activity. Levels of albumin and/or haptoglobin did not consistently parallel that of EIPS activity. A post-operative rise (4 sheep studied) and a pre-parturition nadir (1 sheep studied) in maternal plasma EIPS activity were also noted. The possible physiological significance of these results is discussed.

Animals↗

Production and characterization of a monoclonal antibody against bovine haptoglobin and its use in an ELISA.

Haptoglobin (Hp) is the major acute phase reactant found in cattle. As such, it is an excellent indicator of early disease processes and could be used as a marker for pre-clinical illness in cattle. The production of monoclonal antibodies (mAbs) directed against bovine Hp and bovine hemoglobin is described. The anti-haptoglobin mAbs (Hap1, Hap2, Hap3) and the anti-bovine hemoglobin (Hb) mAb (BoHem1) were characterized and tested for cross-reactivity by means of enzyme-linked immunosorbent assay (ELISA) and immunoblotting analyses. Additionally, the development of an ELISA based on an anti-haptoglobin mAb is discussed.

Animals↗

Identification of the products of the haptoglobin-related gene.

A novel haptoglobin, which is undetectable using conventional peroxidase staining techniques, has been detected using immunoblotting. The behaviour of this novel haptoglobin after electrophoresis under native, denaturing and reducing conditions indicates that it is the product of the haptoglobin-related gene. This protein is detectable in normal human plasma.

Alleles↗

Haptoglobin polymorphisms and iron homeostasis in health and in disease.

Haptoglobin (Hpt) is a plasma protein with hemoglobin-binding capacity. It is a well-known marker of hemolysis. Hpt is also an acute-phase protein that functions as a bacteriostatic agent, an inhibitor of prostaglandin synthesis and angiogenesis. However, the best-known biological function of Hpt is capture of hemoglobin (Hb). The identification of functional differences in haptoglobin molecules resulting from relatively common polymorphisms has further elucidated the importance of haptoglobin in iron homeostasis and in disease processes influenced by iron metabolism. In this review the effect of Hpt polymorphism on these different disease entities will be discussed.

Disease↗

Changes in C-reactive protein and haptoglobin in dogs with lymphatic neoplasia.

Acute phase proteins (APP) are regarded as a useful diagnostic tool in humans with lymphomas, leukaemias and multiple myeloma. C-reactive protein (CRP) and haptoglobin concentrations were measured in dogs with malignant multicentric (high grade) lymphoma (n=16), acute lymphoblastic leukaemia (ALL) (n=11), chronic lymphocytic leukaemia (CLL) (n=7) and multiple myeloma (n=8). Twenty-five healthy dogs served as controls. Measurements of the CRP plasma concentration were performed using a commercial ELISA and haptoglobin was measured with an assay based on its haemoglobin binding capacity. Global group comparisons using Kruskal-Wallis-test revealed significant group differences for both APPs (P<0.0001). Median CRP concentrations were increased in all groups with neoplastic lymphatic disorders (lymphoma: 37.2mg/L, ALL: 47.8mg/L, CLL: 35.5mg/L, myeloma: 17.6mg/L) compared to controls (1.67mg/L; P<0.001). Compared to the healthy controls (median=0.59g/L), haptoglobin was especially increased in dogs with ALL (6.8g/L, P<0.0001) followed by dogs with malignant lymphoma (3.8g/L, P<0.0001), CLL (3.2g/L, P=0.0008), and multiple myeloma (3.0g/L, P=0.0163). For both APPs, a wide range of values was found in all patient groups. The results indicate that particularly severe and acute lymphatic neoplasia, such as high grade lymphoma and ALL, cause significant acute phase reactions in dogs and must be included in the differential diagnoses of increased blood levels of these APPs.

Animals↗

Marked induction of IL-6, haptoglobin and IFNgamma following experimental BRSV infection in young calves.

Bovine respiratory syncytial virus (BRSV) has been identified worldwide as an important pathogen associated with acute respiratory disease in calves. An infection model has been developed reflecting accurately the clinical course and the development of pathological signs during a natural BRSV-infection. In the experiments described in the present study, calves were infected at 13-21 weeks of age and reinfected 14 weeks later. Blood samples from the entire infection period were analysed for acute phase protein (haptoglobin) by ELISA and for expression (mRNA level in peripheral blood mononuclear cells) of the cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-gamma (IFNgamma) by quantitative real-time reverse transcribed polymerase chain reaction (RT-PCR). IFNgamma, interleukin-6 and haptoglobin were markedly induced together with development of clinical signs in response to the first infection with BRSV. The IFNgamma response was biphasic, with an early peak at day 1-3 post infection (p.i.) and a later increase between day 5 and 8 p.i. Reinfection also resulted in an induction of IFNgamma, but without induction of clinical signs, IL-6 and haptoglobin. These results indicate that early mediators connected with the innate responses are induced on a first encounter with the pathogen, but not on a second encounter (reinfection) where the adaptive immune system may act as the first line defence.

Animals↗

Haptoglobin phenotype as a predictor of restenosis after percutaneous transluminal coronary angioplasty.

We have demonstrated that a genetic polymorphism in the antioxidant protein haptoglobin is important in determining which patients develop restenosis after percutaneous transluminal coronary angioplasty. Knowledge of the haptoglobin phenotype may be useful in the assessment and utilization of new therapies to reduce restenosis, particularly in patients who are homozygous for the haptoglobin 2 allele.

Adult↗

Activation of haptoglobin gene expression by cAMP involves CCAAT/enhancer-binding protein isoforms in intestinal epithelial cells.

CCAAT/enhancer-binding protein (C/EBP) isoforms are expressed in rodent intestine and in the rat intestinal epithelial cell line IEC-6 but their role remains to be determined. Treatment of IEC-6 cells with the adenylate cyclase activator forskolin led to coordinate induction of C/EBP isoforms alpha, beta and delta at the mRNA and protein levels. Transient transfection assays showed that their expression is controlled at the transcriptional level. Forskolin treatment induced haptoglobin mRNA levels. Electrophoretic mobility shift and supershift assays demonstrated an increase in DNA-binding activities of the three C/EBP isoforms to the haptoA and haptoC C/EBP DNA-binding sites of the proximal haptoglobin promoter. Site-specific mutations of both sites led to a decrease in transcriptional induction by forskolin, suggesting that C/EBP isoforms are involved in the cAMP-dependent regulation of the acute-phase protein gene haptoglobin in intestinal epithelial cells.

Acute-Phase Reaction↗

Haptoglobin phenotype and coronary artery collaterals in diabetic patients.

Cross-cultural epidemiological studies of incident cardiovascular disease in the diabetic patient have demonstrated marked differences in susceptibility that may be due to a genetic factor. The coronary artery collateral circulation is the chief determinant of the size of a myocardial infarction and is highly variable between patients. We recently demonstrated that a functional allelic polymorphism in the haptoglobin gene is correlated with a number of diabetic vascular complications. We thus set out to test the hypothesis that haptoglobin phenotype is associated with collateral formation in the setting of diabetes. We correlated the Hp phenotype (1-1, 2-1 or 2-2) as determined by polyacrylamide electrophoresis with the presence or absence of coronary collaterals by angiography in 82 consecutive diabetic patients and 138 consecutive non-diabetic patients undergoing catheterization. We found that diabetic patients with the Hp phenotype 2-1 were more likely to have collaterals than diabetic patients with the Hp phenotype 2-2 (P=0.007). There was no correlation between Hp phenotypes and the presence of collaterals in non-diabetic patients. Hp phenotype thus appears to be associated with the development of the coronary collateral circulation in diabetic patients with coronary artery disease. Haptoglobin 2-2 may predispose to less compensation for coronary artery stenosis in diabetic patients, and thereby portend a worse prognosis.

Aged↗