Metabolic and Biosynthetic Features of Lymphocytes from Patients with Diabetes Mellitus: Similarities to Lymphocytes in Chronic Lymphocytic Leukaemia

S ummary This study assayed glucose utilization via the hexose monophosphate (HMP) shunt and Embden-Meyerhof pathways in lymphocytes from patients with diabetes mellitus (DM) and chronic lymphocytic leukaemia (CLL) employing the quantitative evolution of ¹⁴CO₂ and production of radioactive glycolytic intermediates from [1-¹⁴C]glucose as respective experimental endpoints. In addition, DNA synthesis, as reflected by [¹⁴C]thymidine incorporation by the same cells in tissue culture, was measured. Most diabetic lymphocytes displayed depressed absolute and proportional glucose metabolism through the direct oxidative pathway and also demonstrated decreased incorporation of radioactive thymidine, particularly when the lymphocytes were stimulated by phytohaemagglutinin (PHA). The lymphocytes in chronic lymphocytic leukaemia showed the same abnormalities. Moreover, in all lymphocytes assayed, a correlative, semiquantitative relationship was present between the amount of glucose passing through the HMP pathway, which generates NADPH, and the synthesis of DNA, a process requiring this enzyme for reduction of oxyribonucleotides to deoxyribonucleotides. It is postulated that normal antigenic recognition and satisfactory initiation of the immune responses, as mediated by the lymphocyte, may have a metabolic basis, and that this function appears to be abnormal in most patients with diabetes mellitus in this particular study group.     

Uptake of Vitamin B12 by Phytohaemagglutinin-Transformed Lymphocytes

Phytohaemagglutinin (PHA)-transformed lymphocytes have been used as a model cell system to study the uptake of radioactive vitamin B₁₂ by haemopoietic cells. Both mature granulocytes and PHA-transformed lymphocytes took up more vitamin B₁₂ than mature, non-transformed lymphocytes. Uptake of vitamin B₁₂ by PHA-stimulated lymphocytes was greatest at 48–72 hr of culture, i.e. at about the time or just before the time of peak DNA synthesis.
Vitamin B₁₂ deficient lymphocytes took up significantly less vitamin B₁₂ than normal lymphocytes. Folate deficient lymphocytes took up an average of about 50% more vitamin B₁₂ than normal but the difference was not statistically significant for the numbers tested. Vitamin B₁₂ uptake by PHA-stimulated lymphocytes was related to their rate of RNA synthesis (measured by ³H-uridine uptake) and was closely related to active cytoplasmic protein synthesis since it was rapidly inhibited by puromycin and cycloheximide.     

Glycogenolysis versus glucose transport in human granulocytes: differential activation in phagocytosis and chemotaxis

Granulocytes depend primarily on anaerobic glycolysis to supply the necessary energy for locomotion and chemotaxis. Either transmembrane transport of extracellular glucose or catabolism of intracellular glucose can supply glycolytic substrate. In this report, using enzymatic analysis of granulocyte glycogen, we describe conditional requirements for glycogenolysis, namely phagocytosis. With abundant extracellular glucose, granulocyte glycogen content (12.2 +/- 1.6 micrograms/10(6) cells) is not depleted whether or not incubations include various soluble chemotaxins (e.g., FMLP, C5ades arg, arachidonic acid). These chemotaxins accelerate transmembrane glucose uptake. With near complete absence (less than 6 mg/dl) of extracellular glucose, both resting and chemotaxin (FMLP, C5ades arg, arachidonic acid) stimulated granulocytes catabolize significant endogenous glycogen. Phagocytosis, however, fails to enhance glucose uptake and promotes glycogen consumption even with abundant extracellular glucose. Simple particle-phagocyte attachment without internalization (produced by cytochalasin-B) also promoted glycogen consumption, suggesting that this membrane deformation alone is a sufficient trigger for glycogenolysis. Resting or chemotactic granulocytes, therefore, can adapt their energy source pending extracellular glucose availability-- often compromised at inflammatory sites-while phagocytic cells depend primarily, if not exclusively, on endogenous glycogen stores. This differential metabolic activation defends the granulocytes energy supply and may be critical in supporting antimicrobial activity in acute inflammation.     

Studies with human erythrocyte pyruvate kinase (PK): effects of modification of sulfhydryl groups

S ummary . Cysteinyl residues of red cell pyruvate kinase (PK; ATP: pyruvate phosphotransferase, EC 2.7.1.40) were modified with methylmethanethiosulfonate (MMTS), p-nitrophenoxycarbonyl methyl disulfide (NPCMD), and sodium tetrathionate (NaTT). At pH>iddot;0, K _0·5s phosphoenol-pyruvate (PEP) was markedly increased. Fructose-1, 6-diphosphate (FDP) increased affinity for PEP, but K _0·5s (PEP) remained elevated and hyperbolic kinetics were not achieved. Inhibition by negative effectors ATP and alanine was not reversed by PEP and FDP concentrations far greater than those abolishing inhibition of unmodified enzyme. At pH < 7·0, PEP affinity was reduced, and FDP markedly increased V _max and diminished K _0·5s (PEP). MMTS greatly impaired the thermostability of PK. Acid pH alone and the simultaneous presence of Mg⁺⁺, K⁺ and PEP prior to MMTS treatment protected against the effects on PEP kinetics, but did not alter the induction of thermolability. No MMTS effect on the FDP binding site, on ADP kinetics or on the relative effectiveness of GDP, UDP or CDP cofactors was demonstrated. The MMTS-induced alterations closely resembled those observed with certain PK mutants associated with haemolytic anaemia.     

The Effect of Collagen on Platelet Glycolysis and Nucleotide Metabolism

S ummary . Glycolytic activity and nucleotide metabolism were studied in normal resting platelets and platelets exposed to collagen. Lactate production was found to be a more sensitive index of glycolytic activity than glucose consumption. In the presence of added glucose, the rate of lactate production was linear for at least 60 minutes. In the absence of glucose, lactate production continued for 60 minutes and appeared to be independent of glucose for the first 10–15 minutes.
Addition of both collagen and pure tropocollagen to the platelet suspension produced a significant and constant increase in glycolytic rate. This was associated with release of ATP and ADP and a small but significant increase in total ATP. Addition of ADP in concentrations similar to those released by collagen had no effect on glycolytic activity. Addition of calcium ions produced release of nucleotides but no increase in glycolytic rate. EDTA inhibited glycolysis both in the resting platelets and in the presence of collagen.
It is suggested that the stimulation of glycolysis by collagen may be due to the action of collagen on one or more rate-limiting glycolytic enzymes.     

A Low Km Phosphoenolpyruvate Mutant in the Amish with Red Cell Pyruvate Kinase Deficiency

S ummary . The biochemical heterogeneity of red cell pyruvate kinase (PK)-deficiency was demonstrated in a study of six patients with this abnormality. Three siblings of an Amish kindred were found to possess a mutant enzyme with a decreased K _m phosphoenolpyruvate. In this kindred the K _m PEP ranged from 0.018 to 0.024 ^mM (normal 0.055–0.067 mm). Two deficient patients were found to have an enzyme with a normal K _m PEP, while in the sixth patient the K _m PEP was increased to 0.120 mM.
In all patients the red cells possessed increased quantities of the glycolytic intermediates proximal to the PK step, reduced levels of ATP and reduced glycolysis for cell age.
When reconstituted haemolysates from these patients were supplemented with PK, a correction of the glycolytic rate was observed. No acceleration of glycolysis occurred in PK-supplemented normal or reticulocyte-rich haemolysates.
These studies re-emphasize the heterogeneity of PK deficiency and demonstrate that this deficiency alone can result in an impairment in red cell glycolysis.     

Do HbSS erythrocytes lose KCl in physiological conditions?

KCl cotransporter activity in sickle (HbSS) red blood cells (RBCs) was measured in cells suspended in 'simple' physiological saline, saline augmented with inorganic salts, and autologous plasma. Our results showed that the transporter was only functioning at 20% of the level of cells in saline when cells were resuspended in autologous plasma. Kinetic analysis of the data showed that plasma decreased both V _max and K _m for K⁺ of the transporter. The plasma factor(s) responsible was heat-stable and dialysable (i.e. size &60; 10 kD).
Adding magnesium, calcium, inorganic phosphate or bicarbonate to 'simple' saline to mimic the effect of plasma revealed that Mg²⁺ and Ca²⁺ had no significant effect at physiological concentrations. P_i was not effective at 1.1 m M  , but did inhibit significantly (42±2%) at 5.6 m M  . HCO⁻₃   had a major inhibitory effect on K⁺ influx when added to saline, and was identified as the principal candidate for the plasma effect.
We suggest bicarbonate may play a significant role in modifying KCl cotransport, and hence HbSS cell volume in vivo . It acts by altering the set point of the transporter via the signalling systems involved in its regulation.     

Abnormal erythrocyte metabolism in hepatic disease.

Erythrocyte (RBC) metabolic studies were done on 114 patients with severe hepatic disease. Heinz body formation after incubation of RBCs with acetyl phenylhydrazine was found to be significantly higher in patients than in controls. RBC-reduced glutathione levels were lower than those of controls both before and after incubation with acetyl phenylhydrazine, and patients with the highest Heinz body counts had the lowest reduced glutathione levels. RBC methylene blue-stimulated hexose monophosphate (HMP) shunt metabolism and glucose recycling through the shunt were significantly lower in patients with active hepatic disease than in controls. There was no difference in resting HMP shunt activity or in resting recycling of glucose. Despite impairment of shunt metabolism, total glucose consumption was greater in patients than in controls. The patients with the lowest stimulated HMP shunt metabolism and glucose recycling had the highest Heinz body counts, lowest reduced glutathione, and highest total glucose consumption. A continuum of abnormal shunt metabolism was seen, from a mild reduction of stimulated HMP shunt activity to a severe combined decrease in both the HMP shunt and glucose recycling. When measured, glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, and transketolase were normal or increased. Sequential studies were done on 11 patients who had abnormal metabolic studies. Coincident with improvement of HMP shunt metabolism, the Heinz body counts became lower, reduced glutathione higher, hematocrit higher, and liver function improved. Impaired HMP shunt metabolism appears to be a common, acquired RBC abnormality in patients with severe, active liver disease.     

Proliferation of Haemopoietic Stem Cells in Vitro

S ummary . A liquid culture system for murine haemopoietic cells is described where proliferation of granulocytes (G-cultures) or mononuclear cells (M-cultures) can be maintained for many weeks. In M-cultures there is a rapid loss of spleen colony forming cells (CFUs). Agar colony forming cells (CFUc) are present for 6 weeks, then decline rapidly. Colonies formed from these CFUc contain only large mononuclear cells. In G-cultures there is proliferation of CFUs, grafts of which (after 7-9 weeks in culture) protect lethally irradiated mice from haemopoietic death. Similarly, there is proliferation of CFUc, which form colonies in agar consisting of granulocytes, in all stages of differentiation.     

Redox regulation of apoptosis in interleukin-3-dependent haemopoietic cells: absence of alteration in both mitochondrial membrane potential (Δψm) and free radical production during apoptosis induced by IL3 withdrawal

Apoptosis in haemopoietic cells has been linked to the production of free radicals and reduction in mitochondrial transmembrane potential, Δψ_m. We have found that apoptosis in murine IL3-dependent haemopoietic cells induced by IL3 withdrawal lowers a principal source of anti-oxidant (reduced glutathione) but does not increase free radicals nor affect Δψ_m. Increased free radicals are, however, produced during IL3 signalling. Artificially increasing intracellular free radicals by depleting internal glutathione mimics IL3 signalling in that cells maintain shape and are transiently stimulated into S-phase in the absence of IL3. Conversely, depletion of free radicals by anti-oxidants accelerates apoptosis both in the presence and absence of IL3. Our studies suggest that redox conditions rather than free radicals or mitochondrial membrane potential per se may play a significant role in maintaining cell and replicative integrity, and in determining apoptosis in haemopoietic cells.     

Studies of the Oxidation of Ethanol to Acetaldehyde by Oxyhemoglobin Using Fluorigenic High-Performance Liquid Chromatography

We noted a rise in acetaldehyde levels in clinical samples of venous whole blood containing ethanol that did not occur in samples from teetotalers. Experiments were performed to define the mechanism involved in acetaldehyde production. The addition of 0.10% ethanol to whole blood produced an immediate increase in acetaldehyde due to acetaldehyde in the stock solution followed by a subsequent increase that became statistically significant by 48 hr. Separation of blood into components documented that the increase in acetaldehyde was associated with the red cell but not plasma fraction. Incubation of isolated hemoglobin with ethanol produced a rise in acetaldehyde levels. Incubation of oxygenated whole blood with ethanol produced a linear increase in acetaldehyde, whereas nitrogen-exposed blood produced no increase. The rise of acetaldehyde in the presence of ethanol was dependent on the concentration of oxygenated hemoglobin A₀. Addition of inhibitors of catalase, alcohol dehydrogenase, and glycolytic enzymes (aminotriazole, azide, pyrazole, sodium fluoride, sodium citrate, and iodoecetate) did not inhibit the rise of acetaldehyde, but addition of the hemoglobin ligand cyanide abolished the rise in acetaldehyde. Kinetic analysis with oxygenated whole blood plus inhibitors revealed a K _m of 2.5 mM and V _max of 1.42 μM/min. We conclude that oxyhemoglobin contributes to the metabolism of ethanol to acetaldehyde. These findings may explain in part the high levels of acetaldehyde found in red cells compared with plasma. The results also have implications for the optimum storage of blood samples for acetaldehyde analysis.     

Selective in Vitro Cytotoxicity of a Glycolytic Enzyme Inhibitor for Immature Lymphoid Cells

S ummary . A specific lactate dehydrogenase inhibitor, sodium oxamate, which prevents glycolytic resynthesis of ATP, has been shown to possess a significant, in vitro cytotoxic effect for EB₂ cells and for the acute leukaemia lymphoblast. This result was obtained at an oxamate concentration of 0.08 M in TC 199 medium adjusted to physiological osmolality, in comparison with control cultures containing an equimolar concentration of sodium chloride. No significant oxamate cytotoxicity was found for normal marrow myeloid precursors or for blood lymphocytes and neutrophils, although a significant reduction in glycolysis-dependent neutrophil phagocytosis was obtained.     

Permanent Mixed-Field Polyagglutinability (PMFP)

Abstract. With the use of endless belt fluid electrophoresis, these studies confirm previous reports of two cell populations in PMFP having different mobilities. Our method shows two clearly separated bands, while with the microelectrophoresis method a distinctly bimodal distribution is reported. The abnormal (A) cells show a 33- to 42-percent reduction in mobility and a 47- to 48-percent reduction in sialic acid as compared with standard cells. The serologically normal (N) cell population, on mixture with standard cells and subjected to electrophoresis, exhibits no increase in streak width indicative of increased electrophoretic heterogeneity. This is consistent with the findings in the preceding report of no serological abnormalities in the (N) cells. Results of hematological studies, red cell isozyme determinations and assays of glycolytic enzymes activity were not significantly different between the two cell populations.
These observations lead us to suggest that the basic abnormality in PMFP (A) cells (Tn transformation) represents a point mutation involving a single genetic step in erythrocyte membrane glycoprotein biosynthesis. A block in the transfer of terminal D-galactose to N-acetylgalactosamine in a substantial proportion of (A) cell heterosaccharides is postulated to be common to all of the serological and biophysical aberrations. This would be consistent with the observed reduction of T, N_VG, N_Hmn (also M_Hmn), electrostatic charge, sialic acid, and increased 'saline agglutinability' in incomplete antisera. The defect in galactose linkage could also leave exposed underlying N-acetylgalactosamines with the consequent manifestation of A_Hmn, A_DB, A_HP, N_BV and Tn.     

Problems in Pharmacokinetic Analysis of Alcohol Disposition: A Trial of the Bayesian Least-Squares Method

The technical problems of the pharmacokinetic analysis of alcohol disposition were studied using the Michaelis-Menten elimination kinetic model. This model was defined by two forms of equations: differential and integrated, with the latter being derived by integration of the differential equation. We compared the parameter values, estimated by one-line curve-fitting, using these two equation forms. We concluded that, for the kinetic analysis of alcohol disposition, curve-fitting with the differential equation was superior to that with the integrated equation. We also studied the methodological problems involved in one-line fitting. The ordinary least-squares (OLS) method was compared with the Bayesian least-squares (BLS) method. Correlation between the V _max and β (ethanol elimination rate) values, and between the V _max and K _m values, was seen when the parameter values were estimated by the OLS method. These results suggested that one-line fitting by the OLS method was not adequate for Michaelis-Menten-type elimination kinetic analysis. BLS analysis resulted in no correlation between the estimated parameter values that did not change with the level of the dose. The BLS method seemed to be more useful than the OLS method for the estimation of individual pharmacokinetic parameter values.     

A novel surface marker (B203.13) of human haemopoietic progenitors, preferentially expressed along the B and myeloid lineages

We used a monoclonal antibody (mAb) (B203.13, IgM) generated from a mouse immunized with the human B/myeloid bi-phenotypic B1b cell line, to analyse haemopoietic cells. The antigen recognized by this mAb is expressed on most adult and umbilical cord blood CD21⁺ B cells, at minimal density on mature monocytes, and is undetectable on granulocytes, T, natural killer (NK) cells, and erythrocytes. Within umbilical cord blood and adult bone marrow haemopoietic progenitor cells, the B203.13 mAb recognized a surface marker, present on progenitor cells of several haemopoietic lineages, that was transiently expressed on early erythroid and T/NK progenitors, and was preferentially maintained on cells of the B and myeloid lineages. Within the CD34⁺ cells, B203.13 was expressed on early committed myeloid (CD33⁺) and erythroid (CD71^dim) progenitor cells, as confirmed in colony formation assays. The mAb also reacted with cells of B and myeloid chronic leukaemias and cell lines. These data define B203.13 mAb as a novel reagent useful for the characterization of haemopoietic progenitors and leukaemias.     

Effects of 3-5 log10 pre-storage leucocyte depletion on red cell storage and metabolism

SUMMARY. A new, in-line high-efficiency 3-5 log₁₀ leucodepletion filter system (Leukotrap° RC system) was used to investigate the effect of pre-storage white cell removal on the quality of AS-3 red cell concentrates stored for 42 d at 4°. Median residual white cell content was 4 × 10⁵ when filtration was performed at 22° within 8 h of phlebotomy ( n = 20) and 3.2 × 10⁴ when filtration was performed at 4° 12-24 h after phlebotomy ( n = 24). None exceeded 1 × 10⁶ WBC per red cell product. Filtration was rapid (median 28 min), and red cell loss averaged (mean ± 1 SD) 6.4 ± 0.7%. In a paired study design, post-transfusion recoveries of 42 d stored red cells in the filtered units averaged 84 ± 6% v 82 ± 8% for unfiltered units ( P < 0.05) and post-storage haemolysis, ATP, osmotic fragility, K+ and pH were significantly ( P < 0.05) better in the filtered units. Reduced glycolytic activity was also observed in the filtered units, and there was a correlation between osmotic fragility, glucose consumption, and lactate produced in standard units that was not present in leucodepleted units. In conclusion, this study suggests that leucodepletion of AS-3 red cell concentrates prior to storage results in better maintenance of the integrity of the red cell membrane with reduced glycolytic activity. There was a modest improvement in post-infusion viability sufficient to offset the filtration-induced loss and to result in an equivalent red cell product.     

Blood Volume Changes in Chronic Bronchitis and Emphysema

S ummary The profound anaemia previously seen in splenectomized irradiated LAF₁ mice injected with isogenic spleen nodule cells, has been confirmed in two other mouse strains. The anaemia does not invariably cause death, but a slow recovery to normal haematocrit levels occurs in about 80 days.
By splenectomy at various times after irradiation, some modification of the severity of the anaemia was achieved, but recovery was not accelerated.
Implantation of normal splenic tissues in Millipore filter chambers intraperitoneally failed to modify the severity of the anaemia, but an improvement in the rate of recovery was observed. ⁵⁹Fe 5-hour uptake data indicate that spleen nodule cells do not help in the re-population of erythroid elements of bone marrow, though there is some acceleration of re-population of spleen as a result of injection of these cells. Both bone marrow cells and normal spleen cells are seen to be more effective than spleen nodule cells in the re-populating bone marrow.
Spontaneous recovery of the bone marrow erythroid population occurs in mice irradiated and injected with spleen nodule cells but an anaemia persists unless the spleen is present.
It is concluded that an erythrocyte defect occurs in splenectomized, irradiated, spleen nodule cell-injected mice — a defect which only slowly recovers.     

Glucose transport and hypoxia–reoxygenation injury in the perfused rat liver

Abstract During liver transplantation, oxidative stress occurs during hypoxia and reoxygenation of the donor organ. Chemical oxidative stress impairs cell membrane transport. Therefore, in this study the influence of hypoxia and reoxygenation on hepatocellular membrane transport was investigated. Specifically, glucose transport was studied in the perfused rat liver using the multiple indicator-dilution technique. First, it was observed that in normal rat livers, glucose transport was rapid but saturable ( K _m48 ± 10 mmol/L and V _max9.4 ± 0.9 μmol/s per g of liver). To simulate hypoxia and reoxygenation, livers were perfused for 30 min with nitrogen-saturated buffer and then with oxygen-saturated buffer for 20 min. The livers from fed rats were protected from hypoxia-reoxygenation injury whereas those from fasted rats were highly susceptible to injury as determined by lactate dehydrogenase release. After reoxygenation, the rate of glucose influx decreased significantly by ∼50% in the fasted livers ( P < 0.001) but was unaffected in the fed livers. This impairment of the hepatocellular transport of glucose, which could be secondary to oxidative injury to the hepatocyte membrane, has implications for the function of donor livers that have sustained hypoxia-reoxygenation ('preservation') injury during transplantation.     

Abnormal erythrocyte metabolism in hepatic disease: effect of NADP repletion.

Erythrocytes from ten patients with severe liver disease displayed low methylene blue-stimulated hexose monophosphate (HMP) shunt activity and glucose recycling despite elevated total glucose consumption when compared to controls. Heinz body formation was increased and reduced glutathione concentration significantly decreased. After hemolysis, no differences in methylene-blue estimulated HMP shunt activity or glucose recycling could be demonstrated between patients and controls. The addition of 2- and 4-mM NADP to the hemolysates produced significantly greater HMP shunt activity and glucose recycling in the patients' hemolysates. The addition of NADPH to the incubation mixture produced no significant stimulation of either HMP shunt activity or glucose recycling, unless methylene blue was also added. Omission of NAD or phosphate from the incubation mixture produced no change in shunt metabolism. The absence of supplemental ATP resulted in extremely low shunt metabolism and refractoriness to NADP stimulation in both patients and controls. In the absence of additional magnesium, a reduction of shunt metabolism was noted. These data suggest that the defect in stimulated shunt metabolism in the intact erythrocytes of patients with hepatic disease does not result from an absolute enzyme deficiency, but rather from an unavailability of NADP or other cofactor.