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Letters to the Editor |
1
Dept. of Forensic Sci., School of Allied Health Sci., Kitasato Univ., 1-15-1 Kitasato, Sagamihara City, 228 Japan ,
2
Dept. of Biochem., Mitsubishi Kagaku Bio-Clin. Labs., Inc., Shimura 3-30-1, Itabashi-ku, Tokyo, 174 Japan
To the Editor:
Langlois and Delanghe (1) described several functional differences between haptoglobin phenotypes in this journal. They reviewed the functional properties of haptoglobin phenotypes such as ability for hemoglobin (Hb) binding, antioxidative capacity, and inhibition of prostaglandin synthesis, which were strong in Hp-1, intermediate in Hp2–1, and weak in Hp2–2. However, we do not agree with their results about the ability for Hb binding.
Hp2–1 and Hp2–2 phenotypes form many polymers, and analysis of the functional properties of each of the isolated polymer proteins had been very difficult (2). We designed new analytical methods such as crossed Hb electrophoresis (3) and two-dimensional affinity electrophoresis, using Hb (4) to identify the Hb-binding ability of each of the haptoglobin polymer proteins of Hp2–1 and Hp2–2 phenotypes. From the results of crossed Hb electrophoresis, we found that a haptoglobin molecule combined with equimolar amounts of Hb to form at least a hexamer. We confirmed by two-dimensional affinity electrophoresis that the larger polymer of the Hp2–2 phenotype had a lower affinity to Hb, but a Hp2–1 polymer of very high molecular mass had high affinity to Hb. The electrophoretic pattern of Hp2–1 polymers changed in the serum of patients with hemolytic disease because of the different affinities to Hb and the different turnover rates of each polymer. From these phenomena, we concluded that the total affinity to Hb changes with the alteration of the Hp2–1 polymer pattern and that the Hb binding ability in Hp2–1 is not always intermediate.
It is well known that a Hb molecule adheres to the beta subunit of haptoglobin (5), but the kind of alpha subunits among haptoglobin phenotypes may affect the affinity of haptoglobin molecules to Hb (4)(6). The heteroconjugate polymer of Hp2–1 phenotype may have a characteristic formation with high affinity to Hb. One must therefore consider the functional differences of each of the polymers of high molecular mass in a discussion on the functional properties of each Hp phenotype.
Footnotes
References
-chain of human hemoglobin. J Protein Chem 1990;9:735-742.
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Dept. of Clin. Chem., University Hosp. Gent, De Pintelaan 185, B 9000 Gent, Belgium
a Author for correspondence.
To the Editor:
The data provided by Okazaki and Nagai regarding the affinity between hemoglobin (Hb) and haptoglobin (Hp) polymers could not have been incorporated in our review article (1) since their paper [2] had not yet appeared at the time our review was accepted for publication in this journal (June 1996).
In our review, we discussed the available literature about quantitative Hb binding as a function of Hp type and Hp concentration. In their letter above, Okazaki and Nagai confuse the term "affinity" with hemoglobin-binding capacity (HBC), the latter being an expression of the protective capacity of human serum Hp against hemolysis. Existing literature data about the stoichiometry of Hp–Hb binding are mainly based on older immunodiffusion methods (3)(4), which underestimate serum Hp 2–2 concentration because the diffusion of high-Mr immune complexes is impaired in gels (typically in the analysis of polymeric Hp 2–2) (5). However, the more recent immunonephelometric methods for determining Hp concentration are not phenotype-dependent. Making use of fixed-time nephelometry (from Behringwerke AG), we found a good correlation between serum Hp concentration and HBC. After addition of Hb to human serum, the HBC was determined by gel permeation chromatography on a Protein PAK Glass 300 SW column (Waters Nihon Millipore), followed by photometric detection of the eluting Hp–Hb complexes at 418 nm. For Hp 1–1 (n = 16), Hp 2–1 (n = 48), and Hp 2–2 (n = 36), we determined the following ranges for HBC (defined as grams of Hb per liter of serum) by Hp phenotype: 1.70–6.76 for Hp 1–1, 1.30–5.41 for Hp 2–1, and 1.22–4.81 for Hp 2–2 (P <0.05, ANOVA) (unpublished results). In these findings, Hp 2–2 is associated with the lowest HBC, whereas Hp 2–1 shows an intermediate HBC between Hp 1–1 and Hp 2–2.
These data are further confirmed by the biological effects of Hp phenotypes on the metabolism of vitamin C (6). The Hb-catalyzed vitamin C oxidation is related to the serum Hp concentration. In Hp 2–2 plasma, vitamin C concentrations are lower than in the other types because of less-effective protection against the Hb/iron-driven peroxidation (6). Consequently, the conclusions summarized in our review regarding quantitative Hb binding of Hp phenotypes remain valid.
We have also studied the in vitro effect of Hb addition to human Hp 2–1 serum on the distribution of Hp 2–1 polymers on starch electrophoresis. Addition of increasing concentrations of Hb (from 0.05 to 3 g/L) did not induce any change in the typical electrophoretic pattern of Hp 2–1 polymers (unpublished results). The observation of Okazaki and Nagai that the electrophoretic pattern of Hp 2–1 changes after transfusion has been used as an argument for the existence of different affinities for Hb between Hp polymers (2). However, observations made after blood transfusions should always interpreted with caution in light of the complexation of albumin with the heme from the transfused erythrocytes [7], which gives rise to additional bands of methemalbumin on the gels. Banding patterns may indeed change in the hours after transfusion. In contrast to the fast removal of the Hp–Hb complexes by the liver [8], these methemalbumin complexes can be observed in plasma by 5 h after hemolysis and persist in the plasma for a few days.
References
The following articles in journals at HighWire Press have cited this article:
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  H. IMRIE, F. J.I. FOWKES, P. MICHON, L. TAVUL, J. C.C. HUME, K. P. PIPER, J. C. REEDER, and K. P. DAY HAPTOGLOBIN LEVELS ARE ASSOCIATED WITH HAPTOGLOBIN GENOTYPE AND {alpha}+-THALASSEMIA IN A MALARIA-ENDEMIC AREA Am J Trop Med Hyg, June 1, 2006; 74(6): 965 - 971. [Abstract] [Full Text] [PDF]
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F. J. I. FOWKES, H. IMRIE, F. MIGOT-NABIAS, P. MICHON, A. JUSTICE, P. DELORON, A. J. F. LUTY, and K. P. DAY ASSOCIATION OF HAPTOGLOBIN LEVELS WITH AGE, PARASITE DENSITY, AND HAPTOGLOBIN GENOTYPE IN A MALARIA-ENDEMIC AREA OF GABON Am J Trop Med Hyg, January 1, 2006; 74(1): 26 - 30. [Abstract] [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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