Britta Landin

Follow-up of plasma semicarbazide-sensitive amine oxidase activity and retinopathy in Type 2 diabetes mellitus

Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO) is high in diabetes. Production of angiotoxic substances (an aldehyde, hydrogen peroxide, and ammonia) in vessel walls is catalysed by SSAO, suggesting a role for SSAO in the development of complications of diabetes. The objective of the present study was to follow up plasma SSAO activity (measured radiometrically), HbA(1c) (using ion exchange chromatography), and retinopathy (by fundus photography) after 2.8 years, in 34 patients with Type 2 diabetes. We also measured urinary levels of an SSAO substrate, methylamine, by fluorometric high-performance liquid chromatography (HPLC). As at baseline, plasma SSAO activity was now higher in subjects with retinopathy (mean 19.5) than in subjects without retinopathy (mean 16.0), 95% confidence interval (CI) for difference 0.6-6.3 nmol benzylamine ml(-1) plasma h(-1). SSAO activity had not changed significantly since baseline, mean difference -1.65 and 95% CI for difference -3.76 to 0.46 nmol benzylamine ml(-1) plasma h(-1). Mean HbA(1c) level remained higher for patients with retinopathy (now 7.9%) compared to those without retinopathy (6.1%), 95% CI for difference 0.6-3.0%. Comparing baseline and the present study, retinopathy was nonproliferative; level had worsened for five and improved for two patients. Urinary methylamine/creatinine ratio was lower in the group of patients with retinopathy (mean 0.99) than in those without retinopathy (mean 1.78), 95% CI for difference 0.1-1.5 microg mg(-1). The results of the present study are compatible with a role for SSAO in the development of diabetic retinopathy.

Electrospray tandem mass spectrometry in the rapid identification of ?-chain haemoglobin variants

The potential use of electrospray tandem mass spectrometry in the rapid characterisation of haemoglobin variants found in the Swedish population has been assessed. Analysis times of the order of 5 -10 min were routinely achieved, and identification of variants using mass spectrometry as the sole analytical technique was possible. However, additional information, readily available from isoelectric focusing experiments, made identification simpler and more secure. In the present communication we report on the identification of the alpha-chain variants, Hb Russ, Hb Le Lamentin and Hb Q-Iran. The identifications were confirmed by the use of nucleotide sequencing techniques.

Bioavailability of cyclosporine in rats after intragastric administration: a comparative study of the L2-phase and two other lipid-based vehicles

The purpose of this study was to evaluate formulations based on surface active dietary lipids only as oral vehicles for cyclosporine. The absolute bioavailability of cyclosporine from two new lipid vehicles was determined in rats after intragastric administration and compared to that of Sandimmun oral solution, which contains non-ionic surface active substances in addition to dietary lipids. In the new vehicles, cyclosporine was dissolved in two different mixtures of glycerides from long-chained fatty acids. One mixture forms an L2-phase, an oil with very low interfacial tension towards water, and was administered both as the oily L2-phase and as a predispersed emulsion formulation. The other mixture forms a liquid crystalline phase and was administered only as an aqueous dispersion. The mean bioavailability of cyclosporine from Sandimmun was 8% while it was 34% from the L2-phase, 38% from the predispersed L2-phase and 27% from the dispersed liquid crystalline phase. The coefficients of variation in area under the blood concentration curve after administration of the two formulations based on the L2-phase were quite low (31% for the L2-phase and 24% for the predispersed L2-phase) and comparable to that after intravenous administration (24%), while the dispersed liquid crystalline phase gave a higher variability (91%), comparable to that of Sandimmun oral solution (101%). The low variabilities found with the two L2-phase vehicles suggest that this formulation is self-emulsifying" in the gastrointestinal tract. Since the L2-phase is based on dietary lipids only, it is expected to be well tolerated and could prove to be a good vehicle for long-term clinical use of oral cyclosporine.

A novel mutation in the β-globin gene causing β-thalassaemia in a Swedish family

To the Editor: P-Thalassaemia is very rarely found in the indigenous population of Scandinavia. Scarce cases due to mutations commonly found in the Mediterranean area are seen, and a few novel mutations have been demonstrated (1,2). Experiences from Japan and Great Britain show that a rather high proportion of the indigenous thalassaemic cases in these temperate countries are classified as thalassaemia intermedia or dominant thalassaemia (3), i.e. the carriers present findings not only of classical pthalassaemia minor but also suffer from varying degrees of haemolysis. The underlying mutations in these cases are usually localized to exon 3 of the P-globin gene (4, 5), thus interfering with the Cterminal structure of the P-globin peptide chain. The more severe phenotype seen with these mutations is explained by the finding that variant P-globin chains are in fact synthesized in several of these cases. These variant P-globin chains retain their haem-binding capacity and the haem-globin complex will be relatively resistant to proteolytic degradation but prone to form intracellular aggregates and thereby lead to dyserythropoietic anaemia. In this report we describe a family where several members turned out to have a novel thalassaemic mutation in exon 2 of the p-globin gene. The index case was a 74-yr-old man with anaemia, who at admission to hospital presented signs of both iron deficiency and haemolysis (Table 1). Because of the pronounced microcytosis the possibility of coinciding thalassaemia was considered. An increased HbA, level was found, although the sample was obtained 6 d after transfusion of 3 units of red blood cells. Isoelectric focusing revealed no haemoglobin variant. Testing of haemoglobin stability in isopropanol-containing buffer gave a normal result. The increased HbA, level thus supported the diagnosis of P-thalassaemia minor. By this time it was also realized that some of the proband’s relatives had been previously investigated because of microcytic anaemia. The alnon-a-globin chain synthesis ratio had been studied in vitro in a sample from his sister and found to be 1.9, i.e. indicating heterozygous p-thalassaemia. The proband’s sister, daughter and grandson all demonstrated typical thalassaemic features with increased HbA, levels but normal HbF. Except for the proband none of the family members had any signs of haemolysis (Table 1). p-Globin cluster haplotyping was performed by ampllfying the regions surrounding 6 different polymorphic restriction enzyme sites (6). The thalassaemic condition was linked to an allele of the haplotype I or V (7). Nucleotide sequencing of double-stranded amplified material obtained from the proband revealed heterozygosity for a deletion (-T) in codon 54 of the second exon of the P-globin gene (Fig. 1). The same mutation was also found in the other thalassaemic family members. This mutation leads to an out of frame reading, introducing an inphase termination at codon 59. If ever translated, the resulting P-globin chain would contain only 58 (instead of normally 146) amino acid residues, and participation in the formation of normal alp-globin dimers would not be possible. However, there is also evidence in the literature (8,9) that mutations in this part of the P-globin gene impedes the stability of the mRNA by a hitherto unknown mechanism. Reduced mRNA survival will explain a typical Po-thalassaemia phenotype but not a dominant thalassaemia. As suggested from the family history the haemolysis found in the proband must then have a cause separate from his thalassaemic mutation. Further investigation of the proband revealed that he suffered from a malignant lymphoma with pronounced splenomegaly as well as a prostatic cancer invading the wall of the urinary bladder. He died 1 month after admission to hospital and at autopsy the spleen, weighing more than 1 kg, was filled with malignant lymphomatous tissue. Phagocytosis of erythroblasts in the abundant histiocytic cells could well explain the haemolysis occurring in this thalassaemic patient.

Characterization of the elusive disulfide bridge forming human Hb variant: Hb Ta-Li β83 (EF7)Gly → Cys by electrospray mass spectrometry

An electrospray mass spectrometric approach to the identification of a human hemoglobin (Hb) variant involving a Cys residue incorporation is presented. In Hb Ta-Li (β83Gly → Cys), Cys83 forms inter-molecular disulfide bridges. Routine analysis of the denatured Hb showed the presence of a minor β chain variant whose mass apparently was 1 Da less than the expected mass difference of 46 Da for a Gly → Cys substitution. Reduction of the globin chains with dithiothreitol gave an intense monomer with the expected mass difference for the Gly → Cys substitution. After reprocessing the original raw data from the denatured Hb and taking into account the possibility of dimer formation, a component was revealed whose mass was consistent with a disulfide linked dimer of Ta-Li β globins. The mutation was localized to peptide βT10 by analysis of a tryptic digest. Tandem mass spectrometry and DNA sequencing confirmed the Gly → Cys substitution occurred at residue 83 of the β chain. Problems encountered in identifying the components in mixtures of monomers and dimers are discussed.

IDENTIFICATION OF Hb KENYA (Aγ81Leu-β86Ala) BY ELECTROSPRAY MASS SPECTROMETRY

Hb Kenya (g through 81; b from 86) was first reported in the early 1970s in association with hereditary persistence of fetal hemoglobin (Hb) (HPFH) (1–3). The variant is characterized by an abnormal globin chain, i.e., a hybrid gb-globin resulting from crossover during meiosis of g and b genes within a gene loci (1–4). The N-terminus (amino acids 1–80) of the hybrid chain corresponds to the g chain, while the C-terminus (amino acids 87–146) corresponds to the b chain. Amino acids 81–86 are homologous for both the g and b chains. Further structural studies have shown that the non-homologous crossover leads to an approximately 22.5 kb gene deletion, including the entire d gene (5,6). In heterozygotes, varying amounts (6.9–23.4%) of Hb Kenya have been found, while Hb F levels are consistently increased (4.7–9.1%) and Hb A2 levels decreased (1–4). The increased level of Hb F is of the g type encoded by the chromosome carrying Hb Kenya. The condition can thus be described as Kenya-g-HPFH (5). Relatively higher proportions of Hb Kenya are found in compound heterozygotes where Hb Kenya occurs in conjunction with Hb S [b6(A3)Glu!Val] (2,7). While Hb Kenya might be suspected when a typical electrophoretic pattern coincides with increased Hb F and decreased Hb A2 levels, a definite diagnosis depends on Southern blotting or DNA sequencing. In this report we demonstrate that electrospray mass spectrometry (ESMS) can also be a very useful tool for rapid identification of Hb Kenya. HEMOGLOBIN, 26(1), 71–75 (2002)

Two different mutations in codon 97 of the β-globin gene cause Hb Malmö in Sweden

An abnormal hemoglobin with increased oxygen affinity, Hb Malmö [α2β297(FG4)His→Gln], was found to cause erythrocytosis in two apparently unrelated Swedish families. Direct nucleotide sequencing of amplified DNA demonstrated a CAC→CAA substitution in one family and a CAC→CAG substitution in the other. Both mutations resulted in a His→Gln substitution in codon 97. This finding prompted us to examine the possible point mutations underlying the different hemoglobin variants reported in the literature.

Compound Heterozygosity for Hb Tacoma [β30(B12)Arg→Ser] and β+-Thalassemia

Hb Tacoma [p3O(B12)Arg+Ser] is a P-globin variant found in several European families (1-4), many of whom are of Finnish extraction, as well as in Afhca (5) and Japan (6). In Sweden, the underlying substitution has been demonstrated to be at codon 30 (AG_G-+ A G D (4). The affected codon is located at the boundary between exon 1 and exon 2, with intron 1 intervening, and hence the substitution is the first nucleotide in exon 2. Hb Tacoma has been subjected to repeated studies at the protein level (7-1 0) since it displays in vitro instability, a reduced Bohr effect and an electrophoretic behavior more similar to native Hb A, than would at first be expected from the substitution. Although some of the affected patients have been slightly anemic, no significant hemolysis has been found in the heterozygous carriers. While Hb Tacoma has been demonstrated with Hb S in an African woman without hematological problems ( 5 ) , an Arabian child with a compound heterozygosity for Hb Tacoma and Po-thalassemia (thal) [codons 36/37 (-T)], was found to