Regulatory mechanisms of hemoglobin oxygen affinity in acidosis and alkalosis.

The recent reports of the effect of 2,3-diphosphoglycerate (2,3-DPG) on hemoglobin affinity for oxygen suggested that this substance may play a role in man's adaptation to acidosis and alkalosis.A study of the effect of induced acidosis and alkalosis on the oxyhemoglobin dissociation curve of normal man was therefore carried out, and the mechanisms involved in the physiological regulation of hemoglobin oxygen affinity examined.In acute changes of plasma pH there was no alteration in red cell 2,3-DPG content. However, there were changes in hemoglobin oxygen affinity and these correlated with changes in mean corpuscular hemoglobin concentration (MCHC). With maintained acidosis and alkalosis, red cell 2,3-DPG content was altered and correlated with the changes in hemoglobin oxygen affinity. Both of these mechanisms shift the hemoglobin oxygen dissociation curve opposite to the direct pH (Bohr) effect, and providing the rate of pH change is neither too rapid nor too large, they counteract the direct pH effect and the in vivo hemoglobin oxygen affinity remains unchanged. It is also shown that approximately 35% of the change in hemoglobin oxygen affinity resulting from an alteration in red cell 2,3-DPG, is explained by effect of 2,3-DPG on the red cell pH.

Hemoglobin olympia ( 20 valine leads to methionine): an electrophoretically silent variant associated with high oxygen affinity and erythrocytosis.

In a family with erythrocytosis, electrophoretic and chromatographic studies failed to demonstrate a hemoglobin variant. However, the oxygen dissociation curves of affected individuals were shifted to the left of normal and this shift persisted when oxygen equilibria were studied in 2.3-diphosphoglycerate-stripped hemolysates. A mutant hemoglobin was evidently present in the red blood cells of the affected persons and was responsible for the increased oxygen affinity and erythrocytosis. Specific staining of tryptic peptide maps of beta-chains from the propositus showed that peptide betaT(3) was positive for a sulfur-containing amino acid. Amino acid analysis yielded a composition identical to that of normal betaT(3), except that there were 2.6 residues of valine and 0.4 residues of methionine (normal composition: Val = 3.0, Met = 0). This suggested that the beta-chains of affected individuals consisted of a mixture of two kinds of chains, 40% of which had a methionyl residue in betaT(3). Structural studies of isolated cyanogen bromide fragments demonstrated unequivocally that, in the abnormal beta-chains, valine in position 20 is replaced by methionine. The new hemoglobin mutant is designated hemoglobin Olympia (beta20 (B2) valine --> methionine).

A logical approach to the investigation of red cell enzymopathies.

This relatively rare group of disorders may cause quite marked morbidity and occasionally be life-threatening. As their inheritance is largely known accurate information in one family member has obvious benefits to other family members as well as the patient. The identification of the defect is dependent on an accurate clinical story which can be used to guide both the use and the interpretation of the various laboratory tests available. From the clinical aspect the enzymopathies can be divided into various broad groups. First, those involving the main glycolysis which produces the red cell's energy requirements in the form of ATP. Defects of this pathway generally cause a non-spherocytic haemolytic anaemia. Second, those involving the pentose phosphate shunt which maintains the redox potential of the cell necessary for its protection against oxidant stress. The commonest enzyme deficiency world wide, G6PD, is in this pathway and is characterized by stress-induced haemolytic crises. Third, defects of the various linked reactions. The most important of these are the methaemoglobin reductases which catalyse the reduction of methaemoglobin to functional haemoglobin and the enzymes in the Rapoport-Luebering shunt which can modulate the 2,3-DPG level. Whilst defects of these metabolic pathways make up the majority of cases associated with haemolysis, defects of other enzymes, on the whole less critical to the red cell's survival, must occasionally be considered. The red cell, because of its relatively easy availability, can be used as a 'biopsy tool' in the diagnosis of some systemic disorders in which the red cell enzymopathy is not the main feature of the disease. Such considerations are particularly important owing to the technological advances that have occurred in the last 10-15 years which have enabled correct assignment of an increased number of difficult cases. Not only is it possible to characterise the variant enzymes more accurately but it is now possible to have a 'metabolic window' on the red cell and examine it for the derangements of metabolism that characterise the various enzyme deficiencies.

Hereditary red cell enzymopathies.

The hereditary red cell enzymopathies are an uncommon but important cause of chronic haemolytic anaemia. Their clinical diversity is mirrored by increasingly evident heterogeneity at the molecular level. The structure, function, and expression of the genes encoding red cell enzymes and the nature of the gene defects in the deficient state are examined.

The osteodystrophy of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare disorder of heme biosynthesis that results in the production of large quantities of photoactive porphyrins. The clinical syndrome is dominated by extreme photosensitivity with mutilation of light exposed extremities and hemolytic anemia. Bone disease has been occasionally noted, but is not well characterised. We describe a man with CEP who developed bone pain and spinal crush fractures at the age of 22. Skeletal radiographs revealed features typical of other severe hemolytic anemias, but in addition there was loss of the terminal phalanges of the hand as a result of photomutilation. Spinal bone density (assessed by DPA) was reduced and at the hip bone density was at the lower limit of normal. The metacarpal cortical bone density was 2.9 standard deviations below normal. Biochemical and histological studies accelerated bone turnover. Although the serum 250H vitamin D concentration was very low (because of light avoidance) there was no evidence that the bone disease was a consequence of this. Treatment for one year with clodronate and a high transfusion regime was associated with small reductions in serum alkaline phosphatase and urine hydroxyproline excretion, but there was no improvement in bone mineral density. We conclude that CEP has a distinctive osteodystrophy comprising osteolysis of light-exposed extremities and a high turnover type of osteoporosis. Privational vitamin D deficiency may also occur. The effect upon bone of the new therapies for CEP should be considered.

Potential use of purine nucleosides and enzyme inhibitors for selective depletion of Thy-lymphoblasts from human bone marrow.

The toxicity of the purine nucleoside, deoxyadenosine in the presence of the adenosine deaminase inhibitor, deoxycoformycin and of deoxyguanosine in the presence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine was measured against two Thy-leukemic cell lines. Toxicity was assessed by the survival of clonogenic cells in a colony assay. The kill of clonogenic Thy-leukemic cells was 99.99% with both nucleoside enzyme inhibitor combinations following 4-h incubations when 50 microM concentration of nucleoside were used. With these nucleoside concentrations some reduction in toxicity was apparent when drug treated cells were cultured in the presence of deoxycytidine (50 microM), however, this reduction in toxicity was not apparent when higher nucleoside concentrations were used (100 microM). Survival of bone marrow myeloid progenitor cells (CFU.GM) was only slightly reduced by these nucleoside concentrations following 4 hour incubations. The presence of a twenty-fold excess of normal bone marrow cells reduced the cytotoxic effect but clonogenic cell incubation still ranged from 99.98 to 99.99% for deoxyguanosine and deoxyadenosine respectively. These combinations of nucleosides and enzyme inhibitors may have a therapeutic role in the elimination of malignant Thy cells from human bone marrow.

The relative sensitivity of peripheral blood T-lymphocyte colony forming cells and bone marrow CFU-GM to deoxyadenosine and 2'deoxycoformycin.

The effects of the nucleoside deoxyadenosine (AdR) towards 2'deoxycoformycin (dCF) treated peripheral blood T-lymphocyte and bone marrow myeloid progenitor cells has been studied. Mononuclear cell preparations were exposed to dCF and AdR in suspension culture prior to washing and culture in a medium free of exogenous nucleosides. The combination of dCF and AdR was found to be highly toxic to peripheral blood T lymphocyte colony forming cells (CFU-TL) following prolonged (18 h) incubation. CFU-GM were markedly less sensitive to the combination of dCF and AdR, concentrations of dCF (10(-5) M) and AdR (10(-5) M) which produced an almost total inhibition of CFU-TL only inhibited CFU-GM by a mean of 10%.

Current problems in haematology. 2: Hereditary spherocytosis.

Hereditary spherocytosis is a relatively common haematological disorder and will be encountered by all haematologists. The abundance of new information, dealing principally with molecular and genetic aspects of pathophysiology, is beginning to have implications for its investigation and management. While these advances have not yet exerted a large influence at therapeutic level, the promise of such advents as prenatal diagnosis make this an exciting field to watch.

Hyaline-vascular variant of angiofollicular lymph node hyperplasia with systemic manifestations and response to corticosteroids.

We report two cases of angiofollicular lymph node hyperplasia of the hyaline-vascular type. The patients were atypical in having systemic complications, which are much more commonly seen in the plasma cell variant of this disorder. In each case, the diagnosis was established some years after the initial presentation. Both patients presented with unusual systemic manifestations. The exact mechanism underlying the associated features is unknown, but these cases provide some evidence that the disease is primarily a vascular proliferative or inflammatory disorder. All manifestations of the disease responded to the administration of prednisolone with rapid reduction of dosage to less than 10 mg daily. Maintenance therapy may be required to prevent relapse.

Parvovirus infection in a family associated with aplastic crisis in an affected sibling pair with hereditary spherocytosis.

There have been many reports of aplastic crisis preceded by a febrile illness in hereditary haemolytic anaemias. Recent research has shown that the human parvovirus may be a causative agent. We report on an adult sibling pair with hereditary spherocytosis who presented in aplastic crisis after a febrile illness. Both had evidence of recent parvovirus infection, shown by the presence of IgM class specific antibody in their sera. The children of one of the sibling pair, one of whom has hereditary spherocytosis but no chronic haemolysis, showed similar evidence of acute parvovirus infection.

Lead poisoning: clinical, biochemical, and haematological aspects of a recent outbreak.

The clinical, biochemical, and haematological aspects of a recent outbreak of lead poisoning, in which exposure was related to the oxyacetylene cutting of red lead painted ironwork, were investigated. Initial suspicion was raised when a blood film showed punctate basophilia which remains a simple and useful method of picking up lead toxicity. Estimations of blood lead concentration and conventional laboratory data confirmed the diagnosis. Although there was prominent punctate basophilia, spectrophotometric analysis showed only negligible accumulation of pyrimidine-5'-nucleotides despite severe suppression of pyrimidine-5'-nucleotidase activity. The pattern of the red cell glycolytic intermediates, investigated for the first time, suggested that lead may also affect glycolysis at the hexokinase step. Once the diagnosis was made intravenous chelation treatment was begun with a rapid improvement in symptoms. Long term follow up is required to assess any sequelae of intoxication. These cases emphasise the classic features of lead poisoning, and despite the currently available diagnostic tests, lead intoxication may still go unrecognised unless a thorough occupational history is taken.

Salmonella bacteraemia in sickle cell disease at King's College Hospital: 1976-1991.

A review of all blood culture isolates for the 16 years from 1976 were collated with prospective laboratory and clinical records of 620 sickle cell patients treated at King's College Hospital. Over half of all salmonella bacteraemias diagnosed in the clinical laboratory occurred in sickle cell disease (SCD) patients. Of 21 bacteraemias in SCD patients, 11 (52.3%) were due to Salmonella spp. compared with 23 (0.4%) of 4884 bacteraemias in patients without SCD (P = < 0.00001). In SCD, Gram-negative bacilli were responsible for 16 (76.2%) bacteraemias, of which 11 (68.8%) were due to Salmonella spp. but there were no cases of S. typhi or S. paratyphi. An increase in the number of salmonella infections over the past 5 years were noted in the SCD and non-SCD patients, nine and 16 cases respectively, compared with two and seven cases in the previous decade. However, the recent increase of S. enteritidis phage type 4 in the UK was not evident in SCD patients. These findings have important preventative and therapeutic implications for the management of SCD patients.

Emergence of ciprofloxacin resistance during treatment of Salmonella osteomyelitis in three patients with sickle cell disease.

The treatment of salmonella osteomyelitis in sickle cell disease (SCD) is difficult and the emergence of antibiotic resistance in Salmonella spp presents further problems for clinicians treating SCD. Three patients presented with salmonella bacteraemia. Treatment with several intravenous antibiotics did not prevent the subsequent development of osteomyelitis. Emergence of resistance to multiple antibiotics, including ciprofloxacin, during the treatment of salmonella osteomyelitis in SCD patients is reported here for the first time. Ceftriaxone 2 g once daily given for 3 months to 3 years was an effective and convenient treatment for osteomyelitis caused by multiply-resistant salmonella. Two of these patients gave a definite history of diarrhoea, and stool cultures confirmed the presence of Salmonella spp in one. Our experience shows that salmonella osteomyelitis may not be prevented by early treatment of bacteraemia in SCD patients. Other measures to reduce the risk of salmonella infection are therefore necessary.

Glycated fibrinogen: a new index of short-term diabetic control.

Fibrinogen is a plasma protein with a short half-life of four days and, therefore, glycated fibrinogen may be valuable as an index of short-term diabetic control. We have developed a simple, rapid method for determining glycated fibrinogen using affinity chromatography. The differences in the percentage of glycated fibrinogen between normal subjects, well-controlled diabetics and poorly-controlled diabetics were highly significant. There was a significant correlation between glycated fibrinogen and glycated haemoglobin for all these subjects. However, in selected subjects with rapidly improving diabetic control the difference between the fall over three days in glycated fibrinogen and glycated haemoglobin was highly significant. In subjects with deteriorating control over an average of four weeks there was a significant difference between the increase in glycated fibrinogen and glycated haemoglobin. We suggest that glycated fibrinogen may be a valuable adjunct to glucose measurements in the assessment of short-term diabetic control due to its rapid change following alterations in control.