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A new approach to the antenatal diagnosis of beta-thalassaemia major (homozygous beta-thalassaemia) has been developed. Using rhodamine-labelled antibodies directed against gamma-globin, fetal erythrocytes can be identified in fetal blood samples contaminated with maternal cells, and using fluorescein-labelled antibodies directed against beta-globin, the presence of beta-chains (and thus Hb-A) may be determined in the same cells by double labelling immunofluorescence microscopy. The results obtained using this technique on blood samples from fetuses at risk for beta-thalassaemia major show that if fetal beta-chains are detected immunochemically the fetus is normal or heterozygous for beta-thalassaemia and over 85% of the fetuses unaffected by beta-thalassaemia major may be identified in this way. The technique is rapid, applicable to small numbers of fetal erythrocytes heavily contaminated with maternal cells and eliminates the necessity of determining chain synthesis ratios in those cases where beta-chains are detected immunochemically.

Restriction endonuclease analysis has been performed on the alpha and beta globin gene clusters of 57 Cypriots homozygous for beta thalassaemia, 30 with the transfusion dependent form of the condition (thalassaemia major) and 27 who are less severely affected (thalassaemia intermedia). There was a significant difference in the incidence of alpha thalassaemia between the two groups: 14/27 of the patients with thalassaemia intermedia also had deletion forms of alpha thalassaemia, while only 4/30 of the patients with thalassaemia major were similarly affected. Thus in Cypriot patients who are homozygous for beta thalassaemia the co-inheritance of alpha thalassaemia is an important factor in determining the clinical course.

Monoclonal antibodies for the beta-globin chain of human HbA (UCH beta) and the gamma-globin chain of human HbF (UCH gamma) have been made. UCH beta indirectly labelled with rhodamine-labelled goat anti-mouse Ig and directly flouresceinated UCH gamma have been used, via double labelling immunofluorescence microscopy, to assay for the presence of beta-chains in fetal erythrocytes obtained at fetoscopy from fetuses at risk for beta-thalassaemia major. The results from 111 cases demonstrate that beta-chain synthesis of as low as 1.8% can be detected in fetal erythrocytes. The immunochemical labelling can be rendered more sensitive by the use of biotinylated UCH beta and avidin-FITC conjugates. This method is rapid and can be used for a sample that is highly contaminated with maternal erythrocytes.

We report an Asian Indian family in which two daughters have Hb Sun Prairie, a known unstable alpha 2-globin variant [codon 130, GCT--CCT; alpha 2 130(H13)Ala--Pro beta 2]. While the homozygous probands have chronic hemolysis-the same phenotype as previously reported, the heterozygous parents are asymptomatic with a thalassemia carrier phenotype, distinct from the chronic hemolytic state previously described in a heterozygote. Unlike the earlier cases in which family studies were not available, this family clearly exhibits autosomal recessive inheritance, unusual amongst variants within the same region of helix H. Globin chain biosynthesis ratios initially suggested a beta-thalassemic hemoglobinopathy-this was excluded by normal sequence analysis of both beta-globin genes. This case report further illustrates the complexity of phenotypes in the thalassemic hemoglobinopathies. It also demonstrates inversion of the alpha/beta-globin chain biosynthesis ratio, a phenomenon which had been noted in other alpha-globin variants and can be a confounding factor in the investigation of thalassemic hemoglobinopathies.

A fixation and permeabilization procedure using formaldehyde and acetone has been developed which allows immunostaining of intracellular haemoglobin for fluorescence activated cell sorter (FACS) analysis of erythrocytes. The treatment preserves antigenicity and light-scattering properties. Validation of the method was given by the correlation of F cell number in adults determined by FACS analysis with that assessed by microscopic examination of cell smears, and by the direct relationship between beta chain synthesis and intensity of beta chain/Hb A immunofluorescence within fetal erythrocyte samples known to vary in their beta chain/Hb A content. The procedure is rapid, non-subjective and sensitive, and makes analysis of haemoglobin content, type and distribution amongst red cell populations possible.

Summary. In order to define a predictive animal model for the effects of hydroxypyridinone (HPO) iron chelators in humans, we have compared the 28 d oral efficacy and toxicology of the HPO, 1,2-diethyI-3–hydroxypyridin-4–one (CP94) in rats and guinea-pigs and related the results to the contrasting metabolism of this compound in the two species. CP94 was highly effective at mobilizing liver iron in rats but showed toxicity at higher doses, whereas in the guinea-pig the compound lacked toxicity but was ineffective at mobilizing liver iron. These differences can be explained by the contrasting metabolism of the drug between the two species. In rats, at the top dose of 300 mg/kg intragastrically, all animals died before the end of the study, with no deaths or weight loss at lower doses. At 100 mg/kg, rat liver non-haem iron concentrations were reduced by 53% and 44% in females and males respectively (P

Liposomes containing the photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) were coupled to polyclonal sheep anti-mouse-Ig antibody and bound to cells coated with specific mouse monoclonal antibody. When illuminated with red light, the AlSPc in the liposomes was activated to produce singlet oxygen and the antibody and liposome targeted cells were destroyed. DW-BCL cells (an Epstein Barr virus immortalised B-cell line) were targeted with an anti-B-cell antibody (8A) and killed specifically, both alone and in the presence of bone marrow mononuclear cells (BM-cells), without phototoxic effects on the untargeted bone marrow CFU-GM progenitor cells. The presence of an excess of non-target cells did not interfere with antibody and liposome binding, or light access to target cells. Similar results were obtained with T-lymphocytes as target cells using anti-CD3 antibody. Specific targeting to the B-cells was demonstrated in the cell mixtures by use of fluorescent microscopy combined with a sensitive technique to detect low levels of AlSPc fluorescence, a cooled charge couple device (CCD) camera. This was also able to show low levels of non-specific background binding of AlSPc to BM-cells and a small population of cells that took up AlSPc in the absence of antibody. The latter were shown to be monocytes by flow cytometry. Images Figure 5

Five orally effective iron chelators of the 3-hydroxypyridin-4-one series have been administered intraperitoneally to iron-overloaded and nonoverloaded male mice at a dose of 200 mg/kg/24 h for a total of 60 days to investigate the effect on iron loading and toxicity. There was a significant reduction in hepatic iron at the end of the study in the iron-overloaded mice with all compounds studied using chemical iron quantitation (P less than .001) and with Perls' stain (P less than .01). Liver iron removal with the hydroxypyridinones ranged from 37% with CP20 to 63% with CP51, compared with 46% removal for desferrioxamine (DFO). There was no significant reduction in splenic or cardiac iron with any chelator. There were no deaths in iron-overloaded animals receiving any of the hydroxypyridin-4-ones, but significantly more deaths in the nonoverloaded groups as a whole (P less than .03). No weight loss was observed with any chelator. Significant reductions in hemoglobin and white cell count were observed with CP20(L1). No histologic abnormalities of kidney, spleen, bone marrow, or stifle joints were observed. Intracytoplasmic inclusion bodies were observed in the centrilobular hepatocytes of animals administered each of the hydroxypyridin-4-ones, while the DFO-treated and control groups showed no such changes.

We report two siblings with congenital spherocytosis, multiple phenotypic abnormalities and an inherited interstitial deletion of the short arm of chromosome 8 (8p). The propositus came to our attention with acute bone marrow hypoplasia secondary to B19 parvovirus infection. The bone marrow trephine biopsy appearances of intranuclear eosinophilic degeneration in the erythroblasts may be pathognomonic of B19 parvovirus induced acute bone marrow aplasia. The presence of B19 parvovirus DNA was demonstrated in erythroblasts by in situ hybridization. Chromosome analysis of peripheral blood lymphocytes from both siblings showed an interstitial deletion of the short arm of chromosome 8, del (8) (p11p21). This abnormal chromosome was inherited from their mother, who showed this deletion as well as a small fragment representing the deleted 8p chromosome portion, del (8) (p11p21), +f. Centromeric material from chromosome 8 was detected in this chromosome fragment by in situ hybridization using an alpha satellite probe (pJM 128), but not by C banding. Chromosome analysis of skin fibroblasts from the mother and a third sibling with a similar karyotype showed the deleted fragment in over 80% of cells. Cells in which the fragment was absent exhibited the deleted 8p, suggesting there was no mosaicism. The mother and the third sibling were phenotypically normal without spherocytosis. A fourth sibling and the father were normal. The chromosome abnormality was not observed in five of the mother's siblings, suggesting that it arose de novo in the mother. Our findings strongly support a locus for congenital spherocytosis on the short arm of chromosome 8. The frequency of defects at this locus is unknown.

The relationship between the oral efficacy and the acute toxicity of hydroxypyridin-4-one iron chelators has been investigated to clarify structure-function relationships of these compounds in vivo and to identify compounds with the maximum therapeutic safety margin. By comparing 59Fe excretion following oral or intraperitoneal administration of increasing doses of each chelator to iron-overloaded mice, the most effective compounds have been identified. These have partition coefficients (Kpart) above 0.3 in the iron-free form with a trend of increasing oral efficacy with increasing Kpart values (r = .6). However, this is achieved at a cost of increasing acute toxicity, as shown by a linear correlation between 59Fe excretion increase per unit dose and 1/LD50 (r = .83). A sharp increase in the LD50 values is observed for compounds with Kpart values above 1.0, suggesting that such compounds are unlikely to possess a sufficient therapeutic safety margin. Below a Kpart of 1.0, acute toxicity is relatively independent of lipid solubility. All the compounds are less toxic by the oral route than by the intraperitoneal route, although iron excretion is not significantly different by these two routes. At least five compounds (CP51, CP94, CP93, CP96, and CP21) are more effective orally than the same dose of intraperitoneal desferrioxamine (DFO) (P less than or equal to .02) or orally administered L1(CP20) (P less than or equal to .02).

The ability of a number of heterocyclic metal chelators to deliver zinc into red cells, to release the liganded zinc to haemoglobin and thereby cause a left shift in the oxygen dissociation curve of intact red cells has been investigated. Incubation of neutrally charged zinc-pyrone and zinc-pyridin-2-one complexes with red cells led to the rapid accumulation of zinc within cells, whereas unliganded zinc in the form of zinc acetate, zinc chloride or zinc sulphate accumulated only slowly. The rate at which zinc was delivered to red cells by pyrone and pyridin-2-one ligands increased with increasing lipid solubility of the ligands. The uptake of zinc into both normal adult and sickle red cells was associated with a dose-dependent increase in the oxygen affinity of haemoglobin. The degree of left shift in the oxygen dissociation curve following the incubation of red cells with zinc-pyrone and -pyridin-2-one complexes suggests that these complexes may find application as agents to increase the oxygen affinity of haemoglobin in sickle cell disease and thereby decrease the probability of intravascular sickling at low tissue oxygen tensions. Ethylmaltol appears to be a particularly useful agent due to its known low toxicity.

In order to define a predictive animal model for the effects of hydroxypyridinone (HPO) iron chelators in humans, we have compared the 28 d oral efficacy and toxicology of the HPO, 1,2-diethyl-3-hydroxypyridin-4-one (CP94) in rats and guinea-pigs and related the results to the contrasting metabolism of this compound in the two species. CP94 was highly effective at mobilizing liver iron in rats but showed toxicity at higher doses, whereas in the guinea-pig the compound lacked toxicity but was ineffective at mobilizing liver iron. These differences can be explained by the contrasting metabolism of the drug between the two species. In rats, at the top dose of 300 mg/kg intragastrically, all animals died before the end of the study, with no deaths or weight loss at lower doses. At 100 mg/kg, rat liver non-haem iron concentrations were reduced by 53% and 44% in females and males respectively (P0.001). At this dose, adrenal medullary cell vacuolation, increased mammary secretory activity, vacuolation of corpora luteal cells and single cell hepatocyte necrosis were seen. There were no reductions in the white cell count. At 50 mg/kg rat liver non-haem iron concentrations were decreased by 50% and 34% in females and males respectively (P0.02). In female rats this was associated with increased mammary secretory activity. In iron-overloaded rats given 100 mg/kg by gavage for 28 d, liver non-haem iron concentration was reduced by 39% (P0.01) and serum ferritin by 71% (P0.001). Ovarian and mammary changes were not influenced by iron loading. In guinea-pigs, CP94 was evaluated at 50 mg/kg, 100 mg/kg or 200 mg/kg by oral insufflation for 28 d. No reduction in liver iron was seen and no systematic dose related histological, biochemical or haematological effects were observed. Whereas in guinea-pigs 99% of urinary recovery following an oral dose of CP94 (100 mg/kg) was as the inactive glucuronide metabolite, in the rat only 23% of the dose was excreted in the urine as the glucuronide with remainder as the free drug or an iron binding metabolite. The lack of both efficacy and toxicity in the guinea-pig may therefore be explained by the rapid inactivation of CP94 by glucuronidation. This metabolism of CP94 in the guinea-pig is closer to humans than the rat, suggesting that both the efficacy and toxicity of this compound in humans may also be limited by glucuronidation.

The relationship between the oral efficacy and the acute toxicity of hydroxypyridin-4-one iron chelators has been investigated to clarify structure-function relationships of these compounds in vivo and to identify compounds with the maximum therapeutic safety margin. By comparing 59Fe excretion following oral or intraperitoneal administration of increasing doses of each chelator to iron-overloaded mice, the most effective compounds have been identified. These have partition coefficients (Kpart) above 0.3 in the iron-free form with a trend of increasing oral efficacy with increasing Kpart values (r = .6). However, this is achieved at a cost of increasing acute toxicity, as shown by a linear correlation between 59Fe excretion increase per unit dose and 1/LD50 (r = .83). A sharp increase in the LD50 values is observed for compounds with Kpart values above 1.0, suggesting that such compounds are unlikely to possess a sufficient therapeutic safety margin. Below a Kpart of 1.0, acute toxicity is relatively independent of lipid solubility. All the compounds are less toxic by the oral route than by the intraperitoneal route, although iron excretion is not significantly different by these two routes. At least five compounds (CP51, CP94, CP93, CP96, and CP21) are more effective orally than the same dose of intraperitoneal desferrioxamine (DFO) (P less than or equal to .02) or orally administered L1(CP20) (P less than or equal to .02).

To determine whether beta-thalassemia can be detected in the fetus, blood was obtained from abortuses of normal mothers and of mothers with beta-thalassemia trait. The red cells were incubated with radioactive leucine and the globin chains were analyzed by radiochromatography. Two independent methods were utilized to correct the results for contamination by maternal radioactive beta-chain, and the corrected beta/gamma ratios were compared to a previously established range of normal fetal beta/gamma synthetic ratios obtained by similar measurements in pure fetal cells. In the erythroid cells of three fetuses from mothers with beta-thalassemia trait, the beta/gamma synthetic ratio was normal in two. The third had a beta/gamma ratio of 0.04 at 10 1/2 weeks, a 50% reduction, consistent with fetal beta-thalassemia trait. Two other fetuses, derived from parents both of whom had beta-thalassemia trait, were also studied. One had a beta/gamma ratio of 0.029 at 8 weeks, a 65% reduction, also consistent with beta-thalassemia trait. The cells of the other had a ratio of essentially zero at 11 weeks, highly suggestive of homozygous beta-thalassemia. Although further experience will be needed to distinguish the homozygous and heterozygous states reliably, it now appears that the beta-thalassemia gene is expressed in the first trimester. Therefore these data suggest that the antenatal diagnosis of beta-thalassemia is becoming an attainable goal.

In a group of young patients with thalassaemia and iron overload treated by subcutaneous infusions of desferrioxamine we have found a number of minor alterations in retinal function. The incidence of such changes is not related to drug dosage or to ferritin level but to abnormality of the extended glucose tolerance test.

A dipeptide L-lysine-L-phenylalanine is shown to inhibit both cell sickling and the gelation of solutions of sickle-cell haemoglobin. The effect on deoxyhaemoglobin solutions and gels was followed by centrifugation; a progressive inhibition of gelation was observed up to 30 mM Lys-Phe. The haemoglobin concentration at the plateau (26 g/dl) suggests that an effect might be seen in vivo if suitable concentrations of Lys-Phe (about 20 mM) can be maintained in the blood stream. Additional studies of lag time before onset of gelation support this. Oxygen dissociation curves of sickle cells showed an effect of Lys-Phe only after incubation for 3 h before measurement, the P50 decreasing from 51 mmHg (6.8 MPa) to 41 mmHg (5.5 MPa) for cells depleted of 2,3-bisphosphoglycerate. The effect of Lys-Phe on intact sickle cells was more rapid. A marked increase in the number of unsickled cells in the presence of Lys-Phe was observed after only 15 min incubation. This result, together with measurements of uptake both into the cell and onto the cell membrane shows that the compound produces a membrane-mediated antisickling effect in addition to the effect on haemoglobin in solution within the cell. The membrane effect is not due to a change in cell volume. The properties of this dipeptide may be of value in the treatment of impending and early sickle crisis.

The polymerisation of deoxyhaemoglobin S at 37 °C has been characterised by the supernatant concentration remaining after ultracentrifugation of the gel. Other haemoglobins, that are often found to occur with haemoglobin S, influence the polymerisation process. The effects of such haemoglobins have been quantitated in terms of a parameter, which estimates the difference in tendency to polymerise between a particular haemoglobin and haemoglobin S. The interpretation also assesses the participation of hybrid molecules present in mixtures of haemoglobins. The results suggest that, while each of the haemoglobins can form mixed polymers with haemoglobin S, haemoglobin F shows a greater inhibition of polymer formation than haemoglobin A, and haemoglobin A 2 has the maximum inhibitory effect. Hybrids show the same gelling potential as an equimolar mixture of each of these haemoglobins (A, F or A 2 ) with haemoglobin S. Haemoglobin C shows a large inhibition of polymerisation, intermediate to haemoglobins F and A 2 . However, hybrids of haemoglobins C and S behave like haemoglobin S and contribute preferentially to polymer formation. This behaviour could explain the severity of SC-disease relative to haemoglobin AS trait.

Desferrioxamine was given intravenously, at higher doses than previously reported, to counter the effects of transfusion-induced iron overload in four patients with beta thalassaemia major. In two of them retinal abnormalities developed, presenting with night blindness and field defects, which improved on withdrawal of the drug.

Three members of an Oxfordshire family have polycythaemia. In each case their whole-blood oxygen affinity is increased. This is due to a previously undescribed haemoglobin variant which has been named haemoglobin Radcliffe (alpha2beta299(Gl)Ala). In addition to having a high oxygen affinity haemoglobin Radcliffe shows virtually no haem-haem interaction and a diminished Bohr effect. It is synthesized at the same rate and is as stable as haemoglobin A. X-ray analysis indicates that crystals of deoxyhaemoglobin Radcliffe are isomorphous with those of deoxyhaemoglobin A. Solutions of haemoglobin Radcliffe were also studied by high-resolution proton nuclear magnetic resonance spectroscopy. The structure/function relationships of haemoglobin Radcliffe are discussed in the light of these studies.

A chromatographic procedure for the separation of human globin chains is described. This method uses CM-Sepharose CL-6B as ion-exchanger and NaCl gradients formed in sodium phosphate buffers containing urea and 0.05M mercaptoethanol to elute the chains. One advantage of this system is that a column, once packed, may be used repeatedly for several chain separations, thus reducing cost and practical work. The resolution in our hands was also better than that on CM-Cellulose using phosphate buffer gradients.

The effect of urea on the oxygen affinity of sickle cells and normal cells was studied up to a concentration of about 1.0 M. Besides the increase in oxygen affinity found in both normal and sickle cells there was a further increase found only in the sickle cells. This specific increase was caused by the direct inhibition of the polymerization of deoxygenated Hb-S by the urea and was used to measure the extent of this inhibition. Even at concentrations of 1.0 M the urea did not fully inhibit the polymerization. At the urea concentrations recommended for treatment of sickle cell crises and for the oral treatment of the disease there was only a very slight inhibition of polymerization. The small increase in oxygen affinity brought about by these concentrations of urea (equivalent to a log P50 change of 0.016) will cause some additional minimal inhibition of sickling at physiological partial pressures of oxygen. Oxygen dissociation measurements of Hb-A in dilute solution showed that 0.95 M urea had no effect on the interaction of 2,3-DPG and chloride with haemoglobin nor on the haem-haem interactions. This implies that the quaternary 'deoxy' form of the haemoglobin may still form and that the action of urea in inhibiting the polymerization is through interference with the intermolecular bonds rather than by inducing a conformational change which disrupts the bonding. Unless urea at the concentrations recommended for treatment inhibits the sickling of the cells by some mechanism other than its effect on the haemoglobin (such as by an effect on the membrane), these results suggest that it is of little use for the treatment of sickle cell disease.

Five patients with heterozygous beta-thalassaemia with an unusually severe clinical picture, low haemoglobin levels occasionally requiring blood transfusion, splenomegaly and unusually prominent basophilic stippling were found to have co-inherited a triple alpha-globin gene arrangement on one chromosome (alpha alpha alpha/alpha alpha). It seems probable that the expression of a single extra alpha-globin gene is sufficient in some patients with heterozygous beta-thalassaemia to give rise to a clinically significant degree of dyserythropoietic anaemia.

In all of six cases of congenital dyserythropoietic anaemia, type II (HEMPAS), gel electrophoresis in the presence of SDS revealed abnormally rapid migration of the preponderant integral membrane protein, band 3. After proteolysis of intact cells, the remaining part of the band 3, comprising the intramembrane segment and the cytoplasmic domain, migrated electrophoretically as a single band, identical in mobility to that from normal cells treated in the same manner. The anomaly thus resides in the extracellular domain of the protein, which is the glycosylated part of the chain. Peptide digests of the band 3 showed no evidence of a missing protein segment in the abnormal cells and the amino acid composition of the peptides derived from proteolysis of the extracellular protein of intact cells was also normal. We infer that the anomaly is one of glycosylation. The major glycoproteins, detected by carbohydrate-specific (PAS) stain appear normal in SDS gels. However, when the more sensitive procedure of reacting after electrophoresis with radioiodinated lentil lectin is employed, some additional minor protein components are revealed. In particular one species of apparent subunit molecular weight about 150 000 appeared in all cases of HEMPAS examined and in no normals. This component is not accessible to proteolysis by chymotrypsin or Streptomyces griseus protease, and may be associated with the inner membrane patches, characteristic of the HEMPAS condition. Overall cell shape and microviscosity of the membrane bilayer, as measured by fluorescence polarization of a lipid-soluble fluorophore, were substantially normal in HEMPAS cells.

A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4- ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2- ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.

The hydroxypyridin-4-one group of iron chelators show promise as potential compounds for the treatment of iron overload by the oral route. In the search for the compounds best suited for long term clinical use, a balance has to be struck between the desire to mobilise the maximum amount of iron and the wish to minimise the potential toxicity of such compounds. In this article we review the approach we have used to evaluate which of the hydroxypyridinones have the properties best suited for further development prior to clinical trials in man. The diversity of a number of closely related compounds substituted on the ring nitrogen have allowed us to study the properties of chelators responsible for cellular mobilisation of iron(III), as well as those which may contribute to their toxicity. The primary hepatocyte culture model has facilitated the investigation of the contribution of their iron binding constant, as well as the critical importance of their relative lipid solubility to both cellular iron mobilisation and toxicity. Similarly studies in mice have confirmed that the factors affecting cellular iron release also control iron excretion in whole animals. Further we have demonstrated that the acute toxicity of this group of compounds is closely linked to the size of the available iron pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary. Oxygen dissociation studies were carried out on red cells and lysates from a patient heterozygous for Hb Hammersmith. The oxygen affinity of the cells at pH 7.1 was decreased, partly by an increased cellular concentration of 2, 3-DPG and more importantly by an intrinsic low affinity of Hb Hammersmith. Haemhaem interactions were reduced and an abnormal Bohr effect (pH 7.1–7.4) was found in the cells which would bring about an additional decrease in oxygen affinity at physiological pH. Oxygen dissociation studies on the lysates showed a low oxygen affinity, a normal Bohr effect at 50% saturation, slightly decreased haem-haem interactions and a normal interaction with 2, 3-DPG. Estimation of the percentage of Hb Hammersmith in the lysates of the patient's cells by selective precipitation of the abnormal β chain with p-chloromercuribenzoate (PCMB) showed the presence of 30–33% abnormal haemoglobin. Assuming no interaction between the Hb A and the Hb Hammersmith, oxygen dissociation curves were calculated for Hb Hammersmith alone and these indicated that it has greatly decreased oxygen affinity, a normal Bohr effect, normal 2, 3-DPG interaction and (somewhat) decreased haem-haem interactions.

High levels of committed erythroid and granulocytic/monocytic progenitor cells have been demonstrated in fresh blood obtained at fetoscopy. The fetal progenitor cells were more sensitive to appropriate stimuli (erythropoietin and colony-stimulating factor) than adult progenitor cells grown under the same conditions, and this was shown to be due to intrinsic differences in the progenitor cells at the different developmental stages.

A partially purified fraction of the water soluble photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) was encapsulated in liposomes which were then linked to a targeting monoclonal antibody 791T/36 using a heterobifunctional linking agent. The photocytotoxic effects of the liposomes were determined on two cell lines bearing an antigen with which the targeting antibody binds: 791T, an osteosarcoma and C170, a colorectal carcinoma; and a control cell line not bearing the antigen; DW-BCL, an Epstein-Barr virus immortalised B-cell line. Antibody dependent cytotoxicity was observed in 791T and C170 cells and was proportional to the number of antigens on the cells, the AlSPc concentration and the time of exposure to activating red light. No significant toxicity was seen using untargeted liposomes, control cells or free AlSPc fraction under similar conditions. Targeted cells and controls kept in the dark also showed no significant toxicity. A possible mechanism of action is postulated and simple adaptations which demonstrate the versatility of the model are discussed. Some suggestions as to the clinical situations to which this system might be applied in the form of photodynamic therapy (PDT) are made.

The amino acid compositions of the tryptic peptides of both chains (α Ami and β Ami ) of the normal major haemoglobin of Macaca irus (Hb-A mi ) have been analysed (excluding αT 12B and αT13). The most probable sequences of these chains have been constructed by comparison with the α- and β-chain sequences of human Hb-A and Macaca mulatta Hb-A. A minor component (α 2 Xmi β 2 Ami ) is found as a polymorphism in M. irus . Amino acid analysis of the soluble tryptic peptides of the α Xmi -chain has elucidated the positions of four amino acid differences between this chain and the α Ami -chain.

Neutral 2:1 ligand:zinc(II) complexes in which at least one ligand is provided by a compound being 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms are of value for use in effecting an enhancement of the oxygen binding ability of a patient's haemoglobin, this being of particular application in the treatment of sickle cell disease.

Summary. It is shown that the haemolytic anaemia in the original patient with ‘idiopathic Heinz body anaemia’ is due to the presence of 36% unstable haemoglobin: Hb-Bristol (β67 (E11) ValAsp). This haemoglobin has a low oxygen affinity which accounts for the normal development of this patient in spite of the presence of permanent severe anaemia. The relative rates of synthesis of βBristol- to βA-chains were identical, indicating that the reduced amount of Hb-Bristol found in the haemolysate is due to precipitation of the abnormal protein. The inability to separate this haemoglobin by electrophoresis is due to interaction of the abnormal aspartic acid with the adjacent histidine at β63 (E7).

Mild to moderate chronic hemolytic anemia in a mother and her two sons was found to be associated with the presence of about 40 per cent abnormal hemoglobin of rapid electrophoretic mobility: Hb-Seattle. Hb-Seattle was found to be unstable in vitro. It was shown to be due to a replacement of alanine by glutamic acid at position 76 of the β hemoglobin chain. The substitution occurs at the surface of the β hemoglobin chain. The hemoglobin instability was attributed to an abnormal interaction between the substituted glutamic acid and histidine at position β77.

Recent work on the structure and function of transferrin indicates that it is not only an inert carrier of iron but that it has a complex function in the absorption and distribution of iron in the body.

The reaction between p-chloromercuribenzoate (PCMB) and haemoglobin has been investigated to define the optimum conditions for dissociation to the subunits. The production of various molecular species has been followed by electrophoresis, gel filtration and ultracentrifugation. The reagent was applied to human haemoglobins A, S, C, Ja (Glasgow), A2, H and F, and also to the haemoglobins of dog, rabbit, horse and pig. Evidence is presented for the occurrence of haemoglobins without α-chains containing dissimilar β-chains, i.e. β2Yβ2X The behaviour of haemoglobins in the presence of PCMB was found to correlate qualitatively and quantitatively with the nature and number of their sulphydryl groups. It is shown that attachment of reagent at position β-93 causes initial dissociation of the tetramer to the dimer: α2β2 ag 2αβ. From the location of these reactive sulphydryls it is concluded that the initial dissociation is along the bc plane of the haemoglobin molecule with the formation of two α1β1 dimers. Further dissociation by PCMB at pH 6.0 only occurred with haemoglobins containing a second sulphydryl group on the β-chain (β-112). From the data it is concluded that substitution at both unreactive thiols β-112 and α-104 causes final dissociation to the monomeric species. The contribution of the various sulphydryl groups to dissociation was also indicated by the behaviour of alkylated haemoglobins when treated with PCMB. Evidence is presented that dissociation of haemoglobin to half-molecules in several media occurs across the same plane as that initially affected by PCMB. The connection between the sigmoid oxygen tension curve and dissociation is discussed.

Summary. An unstable haemoglobin, Hb-Perth, β32 (B14) LeuPro, is described. The patient had a haemolytic anaemia with a T½51Cr RBC survival of 8 days, Hb 10–12 g/100 ml and had post splenectomy Heinz body formation.

Haemolysates from 202M.irus, imported mainly from Thailand and Vietnam, were examined by starch-gel electrophoresis. In addition to the normal haemoglobin, Hb-Ami, two major haemoglobin variants designated as Hb-Pmiand Hb-Qmiand two minor components were found. Hb-Pmi, which occurred in 12% of the sample, forms molecular aggregates, especially when released from the red cell. Peptide analysis showed that it differs from Hb-Amiin the absence of peptidesα.TP III andα-Tp IV. Sera from animals with this haemoglobin in their red cells show two haeme-positive bands in addition to the usual single haptoglobin band; this pattern can be produced in the sera of some animals which do not possess it, by addition of Hb-Pmi. Hb-Qmi, which occurred in 24% of the animals, migrates anodally to Hb-Amiat alkaline pH and does not form aggregates. It is found in two ranges of concentration when present with Hb-Ami. It was shown by recombination experiments to have normalβAmi-chains. The sample was polymorphic for a minor component which was shown to have normalβAmichains. Some animals have two major haemoglobins and also this minor component and therefore possesses three different non-β-chains. It is suggested that the minor component is the product of a mutated duplicate of theα-locus. The population genetics of these variant haemoglobins and the possible selective role of simian malaria are discussed.

Diseases of protein synthesis can be divided into two main groups: in the first group the rate of synthesis of the protein or one of its constituent poly­ peptide chains is reduced, while in the second an abnormal protein is pro­ duced. For hemoglobin, the first group is the thalassaemias, and these have already been reviewed in this series (1). This article will deal with the other group, those caused by the structurally abnormal hemoglobins. These dis­ eases are important for two reasons. First, some of them, such as sickle cell disease, are very common in certain parts of the world and present an im­ portant clinical problem. The second reason is the relative ease, compared to other human proteins, with which the abnormal hemoglobins can be isolated and their structural abnormality determined. In this way, the abnormal hemoglobins have provided a model indicating the type of protein abnor­ malities which can cause disease and also a natural "testbed" for the rules thought to govern the assembly of protein molecules. Recent work on the structure and function of hemoglobin has not only increased our understanding of the mechanism of sickle cell disease but has led to the discovery of two further groups of diseases (Table 1). The first consists of hemoglobins which cause disease because of abnormal oxygen dissociation properties. The second group is the "unstable hemoglobins" which lead to hemolytic anemia. Some of these hemoglobins also have al­ tered oxygen dissociation. In this short review only the more recent advances in the diseases caused by abnormalities of structure of hemoglobin will be discussed. Previous work has been reviewed elsewhere (2-6), and a compre­ hensive list of the variations in structure of the abnormal hemoglobins is given by Lehmann & Carrell (7).