Hb Cardarelli [beta86(F2)Ala-->Pro] is a new unstable and high oxygen affinity variant found in several members of a family from Naples, Southern Italy. A detailed structural and functional characterization of the variant was performed on two subjects, at both the protein and DNA level. The first patient exhibited 43% of the variant hemoglobin (Hb) without major hematological problems. The proband showed 82% of the abnormal Hb in association with beta(+)-thalassemia (thal) that caused relevant erythrocytosis requiring frequent phlebotomies. Structural investigation of the Hb variant by mass spectrometric methodologies identified the amino acid replacement as Ala-->Pro at beta86. The corresponding DNA mutation GCC-->CCC at codon 86 of the beta-globin gene was assessed by both DNA sequencing and amplification refractory mutation system (ARMS) techniques. Functional studies carried out on whole blood and diluted hemolysates from both patients demonstrated increased oxygen affinity, decreased Bohr effect, reduced heme-heme interaction and nearly halved 2,3-diphosphoglycerate (2,3-DPG) and chloride effects.

An elongated C-terminal hemoglobin variant, due to the deletion of nucleotide A in codon 144 (nucleotide 63600 GenBank entry UO1317) was found in a 31-year-old woman from Trento (northeastern Italy). This deletion led to the replacement of lysine at beta144 by a serine residue, the disappearance of the stop codon at position 147, and the presence of 12 additional residues, identical to those observed in Hbs Saveme, Tak and Cranston, which result from a similar mechanism. Hb Trento, amounting to 29% of the total hemoglobin, was unstable and had, as the other variants of this group, an increased oxygen affinity. It led to a mild compensated hemolytic anemia with red cell inclusion bodies. Functional studies of the isolated abnormal hemoglobin were difficult to perform because of autoxidation, precipitation, and formation of hybrids with Hb A.

In carriers of abnormal haemoglobins with increased oxygen affinity, polyglobulia is the only possible functional compensation for tissue hypoxia.However, this adaptation is reduced if some Hb-A is used to synthesize hybrid haemoglobins of the type a2bAbX (heterotetramers). In fact, they display increased oxygen affinity. Four patients of this kind were studied: two carriers of Hb-Kempsey: b 99Asp-Asn, one of Hb-Gàmbara: b 82Lys-Glu, and one of Hb- Trento, a new variant with elongated b chains. Moreover, there are some cases, like that of Hb- Tak, also with elongated b chains, and Hb-Casper: b 106Leu-Pro, in which the abnormal Hb does not interact with Hb-A to form hybrids and the patient presents good functional compensation. This is demonstrated by at least three signs: the absence of polyglobulia, the good reactivity of Hb-A towards 2,3-DPG, and the absence of left- shifting of the upper half of the dissociation curve,which is commonly considered the expression of Hb-A oxygenation.

Nine carriers of beta-abnormal haemoglobins with increased oxygen affinity (Hb X) were examined. Their oxygen dissociation curves from whole blood were more or less left-shifted, with six of nine also characterized by biphasism. This refers to an 'inflection point' usually positioned at about 50-60% of Hb O(2) saturation, commonly believed to be a limit between oxygenation of the normal and abnormal components. In effect, the inflection does not always correspond to the Hb X level, which sometimes is much lower than 50% of the total Hb. Moreover, the upper half segment of the dissociation curve could not only be an expression of Hb A oxygenation, since it is always left-shifted. However, a high Hb A level is commonly believed to be the main compensatory factor of these subjects, but many indications suggest that often they have at least three, and not only two, main haemoglobin species: Hb A, Hb X plus hybrids of the type alpha(2)beta(A)beta(X). These would oxygenate after Hb X, but before Hb A. Finally, the interaction of 2,3-diphosphoglycerate with Hb X and/or hybrid tetramers must be altered, and the releasing of oxygen from both is more or less reduced. Unfortunately, it is difficult to demonstrate the presence of hybrids directly, i.e. with amino acid analysis of the abnormal beta-globin.

(1999). Hb Bologna-St. Orsola [β146(HC3)His→Tyr]: a New High Oxygen Affinity Variant with Halved Bohr Effect and Highly Reduced Reactivity Towards 2,3-Diphosphoglycerate. Hemoglobin: Vol. 23, No. 4, pp. 353-359.

Letters| February 26 1999 How to Save Money for Erythropoietin Therapy by Changing from ‘Roller Coaster’ to Continuous Iron Supplementation Subject Area: Nephrology Caterina Canavese; Caterina Canavese Departments of Medical and Surgical Sciences, Section Nephrourology, and Search for other works by this author on: This Site PubMed Google Scholar Anna Grill; Anna Grill Departments of Medical and Surgical Sciences, Section Nephrourology, and Search for other works by this author on: This Site PubMed Google Scholar Ester De Costanzi; Ester De Costanzi Departments of Medical and Surgical Sciences, Section Nephrourology, and Search for other works by this author on: This Site PubMed Google Scholar Guido Martina; Guido Martina Departments of Medical and Surgical Sciences, Section Nephrourology, and Search for other works by this author on: This Site PubMed Google Scholar Enrica Buglione; Enrica Buglione Occupational and Health Diseases, University of Torino, Search for other works by this author on: This Site PubMed Google Scholar Daniela Valente; Daniela Valente Occupational and Health Diseases, University of Torino, Search for other works by this author on: This Site PubMed Google Scholar Onorata David; Onorata David Biochemical Laboratory of Regina Margherita Hospital and Search for other works by this author on: This Site PubMed Google Scholar Maddalena Saitta; Maddalena Saitta Biochemical Laboratory of Regina Margherita Hospital and Search for other works by this author on: This Site PubMed Google Scholar Emanuela Maddalena; Emanuela Maddalena Departments of Medical and Surgical Sciences, Section Nephrourology, and Search for other works by this author on: This Site PubMed Google Scholar Sara Barbieri; Sara Barbieri Departments of Medical and Surgical Sciences, Section Nephrourology, and Search for other works by this author on: This Site PubMed Google Scholar Fabrizio Fop; Fabrizio Fop Departments of Medical and Surgical Sciences, Section Nephrourology, and Search for other works by this author on: This Site PubMed Google Scholar Mario Salomone; Mario Salomone Valletta Hospital, Torino, Italy Search for other works by this author on: This Site PubMed Google Scholar Giuseppe Piccoli Giuseppe Piccoli Departments of Medical and Surgical Sciences, Section Nephrourology, and Search for other works by this author on: This Site PubMed Google Scholar Nephron (1999) 81 (3): 362–363. https://doi.org/10.1159/000045312 Article history Published Online: February 26 1999 Content Tools Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Caterina Canavese, Anna Grill, Ester De Costanzi, Guido Martina, Enrica Buglione, Daniela Valente, Onorata David, Maddalena Saitta, Emanuela Maddalena, Sara Barbieri, Fabrizio Fop, Mario Salomone, Giuseppe Piccoli; How to Save Money for Erythropoietin Therapy by Changing from ‘Roller Coaster’ to Continuous Iron Supplementation. 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An electrophoretically slow-moving hemoglobin, with abnormal beta chains, has been found in a young woman and in three members of her family. This variant amounted to 41% of the total Hb, and did not cause important clinical manifestations, although characterized by decreased oxygen affinity. Structural and aminoacid analyses revealed the mutation of Hb-Agenogi: 90 (F6) Glu-->Lys, a rare variant so far detected in unrelated racial and ethnic groups. This is the first affected family of ascertained Piedmontese ancestry.

The oxygen carrying capacity of dilute solutions of hemoglobin from normal human adults was examined, by using the above indicated Oximeter. The results show that, if the pO2s are compared with those drawn from the Oximeter-539 WTW (a simpler instrument) carrying the same oxygen electrode, there is a good correspondence between the data drawn from both the instruments. The advantage of the former is that pO2s are measure in mBr, whereas the latter measures the oxygen of the aqueous solutions in mg/l; then mg must be converted into Torr pO2. Since both instruments are usually employed in the oxygen measurement of waste waters of earth, another conclusion is that their sensitivity also allows the use in the bio-medical (and zoological) field. In fact, the data obtained agree with those of recent literature on the subject, which are mainly drawn from automated and sophisticated apparatuses specifically built at this purpose.

The whole blood oxygen affinity of a Negro carrier of SC disease was found to be characterized by some right-shifted p50 and clearly increased Bohr effect, whereas the isolated and purified Hb-S and Hb-C exhibited slight deficiencies mainly of the Bohr effect. The right-shifted p50 from whole blood can be easily explained by the mild anemia with a parallel increase of 2,3-diphosphoglycerate (DPG), whereas the functional discrepancies between whole blood function and that of the purified Hb-S and C could be due, at least in part, to the presence in vivo of consistent amounts of hybrid Hb tetramers of the type alpha alpha beta S beta C. Unfortunately, the mechanism promoting the formation (or dissolution) of hybrids are fundamentally unknown; so, either their presence and functional properties are very difficult to be explored.

Dissociation curves for oxygen of dilute samples of human adult Hb-A were evaluated on this occasion, by using the Oximeter-539 WTW with its sensor, and a suitable spectrophotometer. At this purpose, Hb samples were desaturated in oxygen upon given experimental conditions, by bubbling pure nitrogen in them, and their re-oxigenation in air was followed, step by step, by multiple oximetries. In addition, all the spectrophotometric measurements of the saturation of Hb-O2%, corresponding to each individual oximetry, were carried out parallely but separately. Dilution of Hb-A was maintained at 0.1 mM in heme. The p50 at pH 7.3 was 4.435 +/- 0.299 Torr, with the n-value of 2.7 +/- 0.2; Bohr effect was -0.55 +/- 0.08, within a pH range between 6.8, 7.3 and 7.8, whereas chloride and DPG effects at pH 7.3 (the most useful value) were 0.42 +/- 0.44 and 0.453 +/- 0.0187 respectively. In conclusion, these results are similar to those obtained with automated procedures, upon comparable experimental conditions, but do not require expensive and sophisticated instruments. Such a technique could be very useful in the hemoglobinopathies, which are common in Italy, and it could be easily adapted to perform comparative studies on animal hemoglobins not far from human species.

Hb Gàmbara is a new hemoglobin variant with abnormal beta chains that has been found in three out of four members of a family of Lombardy origin (Gàmbara, Brescia, Northern Italy). The affected subjects led a normal life, but they had modest erythrocytosis and mild (compensated) hemolysis with slight splenomegaly. Their abnormal hemoglobin was about 52% of the total hemoglobin, and was shown to be stable by the isopropanol test. Whole blood P50 of the proband was 19.3 Torr, Bohr effect was decreased (-0.25), as well as the molar ratio between the 2,3-diphosphoglycerate level and total hemoglobin of erythrocytes (0.68). The purified abnormal hemoglobin was characterized by an altered oxygen affinity, low n-factor, chloride, and 2,3-diphosphoglycerate effects. The Bohr effect was about 40% of the normal control. The abnormal hemoglobin moved faster than Hb A at alkaline electrophoresis, and split into two fractions, probably due to the formation of hybrid tetramers (alpha 2 beta A beta X). The reversed phase high performance liquid chromatogram from the tryptic digest of the aminoethylated abnormal beta chain subunits indicated the presence of an extra peptide, beta T-9, 10, replacing the individual peptides beta T-9 and beta T-10. Finally, the proband's DNA, drawn from a suitable segment of the beta structural gene (exon 2), revealed a nucleotide sequence carrying the heterozygous mutation AAG-->GAG at codon 82. This led to a Lys-->Glu substitution at position 82(EF6) of the beta chain.

A mixture of NADH, cytochrome-C-reductase and methylene blue was employed to antagonize ferri(met)-Hb formation during oximetries of dilute samples of human Hb-A. Its efficacy is clear at any pH between 6.8, 7.3 and 7.8, in the presence of 100 mM NaCl. In these cases, ferri(met)-Hb decreases of about 2/3, compared with the enzyme-free controls, and p50 increases. On the contrary, when samples are examined at pH 7.3 with 600 mM NaCl and/or 100 mM NaCl plus 1 mM DPG, ferri(met)-Hb of the samples containing the enzymes also decreases, but not more than 1/2 of its initial value, whereas also p50 decreases. Finally, Bohr effect is lower in the samples containing the enzymes. In conclusion, the enzymatic mixture is very useful, but not easy to be handled; so, some observations for its correct use are required.

Functional parameters of diluted Hb-A have been determined before and after addition of catalase and disodium-EDTA to the samples. There are no important differences between the results drawn from catalase added samples and catalase free ones, except for the fact the met-Hb level at pH 7.8 is significantly lower in the samples containing catalase. On the contrary, catalase is almost ineffective against met-Hb at pH 6.8, whereas its activity at pH 7.3 is rather modest. Another limitation is that catalase remains active against met-Hb for not more than 15-20 minutes after addition to the sample, which is just the time necessary for one complete (manual) oximetry.