Hemoglobin-oxygen-affinity and acid-base properties of blood from the fossorial mole-rat, Cryptomys hottentotus pretoriae.

Oxygen affinity and other hematological parameters in strictly subterranean mole-rats, Cryptomys hottentotus (subspecies pretoriae) were measured immediately upon capture and after 14-21 days in captivity. The pH, hematocrit, hemoglobin (Hb) concentration, blood oxygen content, 2,3 bisphosphoglycerate (2,3 BPG) concentration and oxygen dissociation curves (ODC), as well as tonometric measurements, were determined using whole blood. Additionally ODCs were also determined for stripped hemolysates of individual animals. Compared to other mammals, blood of freshly caught animals had low pH (7.32+/-0.22), elevated hematocrits (48.4+/-3.8 %) and significantly lower P50 values for whole blood (21.1+/-1.6 mm Hg at pH 7.4) than those reported for other similar-sized fossorial and terrestrial mammals. Blood carbon dioxide content (22.4+/-3.9 mMol L(-1)), hemoglobin concentration (1.9+/-0.15 mMol L(-1)), oxygen content (164.8+/-26 mL L(-1)), bicarbonate concentrations (22.5+/-3.5 mMol L(-1)) were within the range of values reported for similar-sized mammals. We conclude that high blood-oxygen affinity, low body temperature and possibly also high hematocrit enable C. h. pretoriae to maintain an adequate oxygen supply to the tissues in a potentially hypoxic burrow atmospheres, but that the blood of this species shows no exceptional CO2 sensitivity or buffering capacity.

Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin.

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.

The oxyhemoglobin dissociation curve in liver cirrhosis.

STUDY OBJECTIVES: To trace the entire oxyhemoglobin dissociation curve (ODC) in a cohort of cirrhotic patients in stable condition who were candidates for orthotopic liver transplantation (OLT). DESIGN: Prospective cohort study. SETTING: A large academic hospital. PATIENTS AND METHODS: We traced the entire ODC in whole blood in standard conditions (pH 7.4; PCO2, 40 mm Hg; temperature, 37 degrees C) for 50 cirrhotic candidates for OLT (27 men and 23 women) and 50 age- and height-matched healthy subjects (27 men and 23 women). All subjects were nonsmokers or ex-smokers for at least 5 years. We also measured 2,3 diphosphoglycerate (2,3 DPG) in RBCs, plasma ions, and arterial blood gases in all subjects according to standard methods. Mixed venous blood was also obtained from the 50 cirrhotic patients. RESULTS: Mean ODC was the same in the two groups. However, for the cirrhotic patients, the dispersion of the PO2 values of oxygen saturation percentage (SO2%) from 20 to 80% was significantly larger (p < 0.01 to p < 0.0001). In the cirrhotic patients, the mean PO2 for half-saturation of hemoglobin (P50) was 7.11 + 0.14 mEq/L chloride (p < 0.001) plus 0.36 mEq/L inorganic phosphate (p < 0.05) plus 0.25 micromol/gram of hemoglobin (gHb) 2,3 DPG (p < 0.00002) in absolute numerical values. Sodium, potassium, and calcium, three plasma ions disturbed in cirrhotic patients, did not contribute to determine the mean P50. DISCUSSION: In patients with cirrhosis, increased dispersion of PO2 values for a given level of SO2% may be related to four factors: (1) an observed alteration of the enzymes controlling the phosphoglycerate shunt; (2) hypothyroidism, which may affect 7 to 20% of patients with primary biliary cirrhosis; (3) the type of ongoing treatment, eg, diuretics and/or propranolol; and (4) plasma ion disturbances. CONCLUSIONS: We describe the ODC by three indexes: shape, position, and an index of dispersion of the PO2 values for a given level of SO2%. In addition, when the latter is increased, we suggest that other factors than pH, temperature, carbon dioxide, and inorganic phosphates are acting on the position of the ODC.

AANA journal course: update for nurse anesthetists--the ruddy globule: the erythrocyte--its biology, chemistry, and functional variations.

Red blood cells were first observed in the mid-1600s and termed ruddy globules for their conspicuous shape and color. Since that time, the functional significance of this extraordinary membranous pouch of proteins and electrolytes has been carefully detailed. The architecture of the red blood cell, the biochemistry of hemoglobin, the mechanics of the oxygen-hemoglobin affinity-altering diphosphoglycerate compounds, and the variants of hemoglobin are reviewed. Far from being a simple transport vehicle of oxygen to and from tissues, the red blood cell is a multifunctional entity whose absence would make life as we know it impossible.

Automated blood component collection with the MCS 3p cell separator: evaluation of three protocols for buffy coat-poor and white cell-reduced packed red cells and plasma.

BACKGROUND: Automated collection of blood components with a cell separator (MCS 3p, Haemonetics), was performed according to three protocols. STUDY DESIGN AND METHODS: The first protocol provided 2 units of fresh-frozen plasma (FFP); and one buffy coat-poor red cell (RBC) concentrate in additive solution. The second protocol included an additional in-line filtration of the RBC in a closed system after storage at 4 degrees C for 24 hours. In the third protocol, an additional platelet concentrate (PC) was recovered from the buffy coat. Cell counts and biochemical characterization of the RBCs (n = 20 each) were determined on Days 0, 1, 14, 28, and 49. RESULTS: The RBC volume was 336 +/- 9 mL (first protocol), 337 +/- 7 mL (second protocol) and 293 +/- 12 mL (third protocol) with a hematocrit of 59 +/- 2, 53 +/- 3, and 61 +/- 5, percent respectively. On Day 49, hemolysis was 0.24 +/- 0.1 percent (first protocol), 0.33 +/- 0.32 percent (second protocol), and 0.38 +/- 0.1 percent (third protocol). The filtered RBC concentrate met the international standards for white cell-reduced RBCs. Filtration resulted in a clinically irrelevant increase of hemolysis. The in vitro RBC values (lactate dehydrogenase, 2-hydroxybutyrate dehydrogenase, hemolysis, potassium, 2,3 DPG, ATP) were at least equal to those in RBCs collected by conventional whole-blood donation. There is a trend toward extended preservation of 2,3 DPG in RBCs collected by apheresis. Two units of FFP could be collected with each donation (first protocol: 420 +/- 55 mL, 5.4 +/- 7 WBCs/microL, 6.5 +/- 5 x 10(3) platelets/microL; second protocol: 440 +/- 33 mL, 3 +/- 5.2 WBCs/microL, 32 +/- 12 x 10(3) platelets/microL; third protocol: 398 +/- 32 mL, 5 +/- 12 WBCs/microL; 3.4 +/- 3.5 x 10(3) platelets/microL). PCs prepared from the buffy coat collected by the third protocol contained 90 +/- 30 x 10(9) platelets in 88 +/- 14 mL of plasma. In vitro test results in these PCs were superior to those in PCs collected by conventional whole-blood donation. The procedure was well tolerated by all donors. No adverse reactions appeared. CONCLUSION: Erythroplasmapheresis with the MCS 3p cell separator is a useful alternative to conventional whole-blood donation and separation.

The presurgical management with erythrocytapheresis of a patient with a high-oxygen-affinity, unstable Hb variant (Hb Bryn Mawr).

BACKGROUND: Hemoglobin (Hb) Bryn Mawr is an unstable Hb variant resulting in congenital hemolytic anemia. This variant Hb also has an increased affinity for oxygen. The perioperative transfusion management of this disorder is described, and the first genomic analysis of this Hb variant is given. CASE REPORT: An 11-year-old boy, heterozygous for Hb Bryn Mawr, was referred for cholecystectomy. Sequence analysis of genomic DNA confirmed that the patients was heterozygous for a T-->C transition in the codon for amino acid 85, causing a substitution of serine for phenylalanine in the beta-globin chain. On the basis of whole-blood O2 dissociation studies, projected tissue O2 delivery would have been suboptimal during general anesthesia; therefore, a partial red cell exchange transfusion was performed to lower variant Hb and prevent tissue hypoxia during surgery. The red cell mass to be exchanged (50%) was determined from the calculated increase in O2 delivery capacity required to maintain an O2 extraction of 4 to 5 mL of O2 per dL of whole blood. The p50 of whole blood from the patients immediately after the exchange transfusion was 16.0 torr. At the time of surgery, the p50 was normal (25.9 torr). The patient's whole blood 2,3 DPG levels were 4.70 mmol per mL of red cells (before transfusion) (normal range = 4.8 +/- 0.3 mmol/mL red cells), 4.07 mmol per mL of red cells (immediately after transfusion), and 4.55 mmol per mL of red cells (48 hours after transfusion). CONCLUSION: This patient with Hb Bryn Mawr was prepared for surgery with a partial exchange transfusion to prevent tissue hypoxia during anesthesia. Decreased 2,3 DPG levels immediately after transfusion resulted in increased O2 affinity of whole blood; however, 48 hours after exchange transfusion, a normal p50 (due to both removal of variant Hb and regeneration of 2,3, DPG) was observed. Partial exchange transfusion is useful in the preoperative management of patients with Hb variants characterized by increased O2 affinity.

Cell populations in the bone marrow of guinea pig fetuses with intrauterine growth retardation.

Bone marrow smears and blood samples were examined in guinea pig fetuses in which intrauterine growth retardation (IUGR) had been induced by uterine artery ligation and compared with those of control (well-grown) fetuses from uterine horns with intact circulation. Differential bone marrow cell counts were obtained from a count of 300 cells per smear and blood samples were assayed for hemoglobin concentration and 2,3-diphosphoglycerate (DPG). Results of blood assays showed no difference in hemoglobin concentration. DPG levels were reduced in the IUGR guinea pigs (P < .05), which could be a consequence of decreased glucose availability or represent an adaptation to reduced oxygen availability. Comparisons of bone marrow counts revealed an increase in total erythrocyte precursors (P < .05) and a decrease in total granulocytic precursors (P < .05) in IUGR fetuses. Within the erythroid lineage there was a significant increase in late (orthochromatic) erythroblasts (P < .005) in the IUGR animals compared with control animals. The granulocytic lineage of the IUGR fetuses showed a significant decrease in mature neutrophils (P < .05) and eosinophilic precursors (P < .05) compared with controls. These data suggest that the hypoxic stress of uterine artery ligation leads to an increase in medullary erythropoiesis. In concert with a previous study that showed a reduction in hepatic erythropoiesis, these data suggest a precocious shift of the anatomic site of erythropoiesis from the liver to the bone marrow under conditions of hypoxia.

Functional characterisation of Eskimo dog hemoglobin: I. Interaction of Cl- and 2,3-DPG and its importance to oxygen unloading at low temperature.

The oxygen binding properties of hemoglobin and some hematological parameters in Eskimo dogs (belonging to Canis lupus familiaris) in Ilulissat/Jacobshavn, Greenland were analysed. The average [2,3-DPG] and [Hb] (n = 16) were 3.14 +/- 0.34 mmol l-1 blood and 9.53 +/- 0.65 g dl-1 (1.49 mmol l-1), respectively, giving a stoichiometric ratio of 2.11 mol 2,3-DPG/mol Hb. Oxygen binding analysis carried out on hemolysate in HEPES buffer at 20 and 37 degrees C revealed a high oxygen affinity (1.2 mmHg at pH 7.4, 20 degrees C) in the desalted condition, which decreased markedly in the presence of chloride and 2,3-DPG. A low apparent equilibrium constant for the binding of 2,3-DPG (1.0 x 10(-5) mol l-1) was found at pH 7.2 and 20 degrees C in the absence of chloride. Moreover, we show that chloride ions have an additive effect on oxygen affinity in the concentration range 10-300 mmol l-1 in the presence of 3 mmol l-1 2,3-DPG at low pH and temperature (pH < 7.4 and 20 degrees C). This feature may be of physiological importance to oxygen unloading under acidotic conditions when tissue temperature is low. Thermodynamic analysis reveal that in the presence of 3 mmol l-1 2,3-DPG and 100 mmol l-1 chloride, the Eskimo dog hemoglobin exhibits a low heat of oxygenation, which places this animal close to arctic ruminants with respect to the influence of temperature on oxygen binding in vivo.

Effect of an aldose reductase inhibitor on abnormalities of electroretinogram and vascular factors in diabetic rats.

The effect of an aldose reductase inhibitor, [5-(3-thienyl) tetrazol-1-yl] acetic acid (TAT), on the electroretinogram was determined in rats with streptozotocin-induced diabetes. Laboratory chow containing 0.05% TAT was given to rats for 2 months, while other diabetic rats were untreated. Groups of TAT-treated and untreated normal rats were also studied. Treatment with TAT produced significant improvement of the electroretinogram. TAT shortened the peak latencies of the b-wave oscillatory potentials, which were significantly prolonged in untreated diabetic rats (P < 0.0001 vs. untreated normal rats). This was accompanied by a significant decrease in the retinal sorbitol and fructose concentrations (by 46.5% and 25.7%, respectively). TAT treatment of diabetic rats also markedly reduced ADP-induced platelet aggregation and significantly increased the red blood cell 2,3-diphosphoglycerate level, accompanied by a marked reduction in sorbitol and fructose concentrations of platelet and red blood cells. There were significant correlations between the summed b-wave peak latencies and platelet aggregation or the 2,3-diphosphoglycerate level in diabetic rats. These findings suggest that an aldose reductase inhibitor, TAT, has therapeutic value for diabetic retinopathy.

Right shift of the oxyhemoglobin dissociation curve in acute respiratory distress syndrome.

Deep hypoxia is known to increase the intraerythrocytic 2,3 diphosphoglycerate (DPG) level and therefore to induce a right shift of the oxyhemoglobin dissociation curve (ODC), which is considered to be a protective mechanism against tissular hypoxia. Our purpose was to assess whether the ODC is shifted to the right in patients with acute respiratory distress syndrome (ARDS) and whether this shift had a beneficial effect on tissue oxygenation. We have determined the whole ODC and related indices in 29 control subjects and 29 patients suffering from ARDS for at least 5 days. The ODC of the patients were shifted to the right from 20 to 95% saturation. Their DPG level was increased (19.9 +/- 3.9 mumol/gHb, mean +/- SD) as compared to control subjects (12.5 +/- 2.1 mumol/gHb). There was a highly significant relation in patients between their P50 (pO2 necessary to achieve 50% saturation) and their DPG according to the equation: P50 (kPa) = 0.03 DPG (mumol/gHb) + 3.24 (r = 0,54) (p < 0,001). It is likely that in such patients hypoxia is severe enough to stimulate the DPG synthesis and induce a right shift of the ODC. It is not at all certain that this shift has a favourable effect on tissue oxygenation. Indeed, the extreme values for PaO2 were from 3.43 to 7.96 kPa that correspond to SO2 of 48 to 91% where the ODC has not yet his upper part. A right shift decreases therefore the captation of oxygen from the lung. On the other hand theoretical studies have shown that at low arterial PO2 values a right shift of the ODC has a detrimental effect on release of oxygen from hemoglobin if the mixed venous PO2 is decreased. In order to quantify the amount of oxygen actually transmitted to the tissues, others factors are to be investigate as the PCO2 or the temperature.

The effect of hypotonicity, glutamine, and glycine on red cell preservation.

BACKGROUND: Red cells (RBCs) stored in hypo-osmolar additive solutions with the same concentrations of adenine, dextrose, mannitol, and sodium chloride and varied amounts of ammonium, phosphate, glycerol, and glutamine were better preserved than RBCs in the standard additive solution (Adsol). Cell swelling occurred in all the experimental additives. This observation prompted the evaluation of glutamine and glycine alone, as well as a combination of glutamine and glycine, all of which have been described as producing swelling of rat liver cells. STUDY DESIGN AND METHODS: Aliquots of RBCs were stored at 4 degrees C in Adsol or experimental additive solutions (EASs) all containing adenine, 2 mM; dextrose, 110 mM; mannitol, 55 mM; and sodium chloride, 50 mM. EAS 42 had, in addition, glutamine, 10 mM; glycine 5 mM, and phosphate, 20 mM. EAS 43 had glutamine, 10 mM; glycine, 10 mM; and phosphate 20 mM. EAS 44 had glutamine, 10 mM; EAS 45 had glutamine, 10 mM, and phosphate, 20 mM, and EAS 46 had only glycine, 10 mM. At intervals, measurements were made of mean corpuscular volume, mean corpuscular hemoglobin concentration, morphology, ATP, hemolysis, supernatant potassium, ammonia, pH, and microvesicles shed. RESULTS: The initial mean corpuscular volumes were larger in all EASs than in Adsol, but the greatest difference was between EASs 44 and 46 (108 fL) and Adsol (86 fL) (p < 0.001). The morphology scores were significantly better in all the EASs (p < 0.04). The ATPs were significantly greater in all the EASs (p < 0.001), and highest in those with phosphate. potassium leakage and hemolysis were less in the EASs (p < 0.001). The ammonia levels higher in all the EASs than in Adsol, with the exception of EAS 46. During storage, the extracorpuscular and intracorpuscular pH levels were essentially identical. The shedding of microvesicles was greatly reduced in all the EASs. CONCLUSION: Cell swelling induced in RBCs after collection appears to improve preservation. Ammonia and phosphate enhance RBC ATP maintenance. Glycine decrease the formation of ammonia by RBCs stored in a hypotonic medium.

Membrane potential and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.

Rejuvenation of irradiated AS-1 red cells.

BACKGROUND: Gamma irradiation of blood components is used to prevent transfusion-associated graft-versus-host disease. The demand for irradiated blood components is increasing because of the increase in directed donation by family members. Irradiated units currently have a recommended maximum storage life of 28 days. Since in vivo recovery is related to red cell ATP levels, rejuvenation of stored irradiated units using a pyruvate-inosine phosphate-adenine additive was explored. STUDY DESIGN AND METHODS: Units of AS-1 red cells from 16 volunteer donors were divided into two equal volumes and one split unit from each was irradiated with 25 Gy. Ten units were irradiated on Day 5, 6, or 7 of 4 degrees C storage and 6 units were irradiated on Day 1 of 4 degrees C storage. All units were rejuvenated for 1 hour at 37 degrees C using a pyruvate-inosine-phosphate-adenine additive on Day 42 of 4 degrees C storage. Units were assayed for ATP, 2, 3 DPG and supernatant sodium, potassium, and glucose. RESULTS: ATP and 2, 3 DPG levels were restored equally well in irradiated and non-irradiated units. The previously reported irradiation-induced red cell potassium-sodium shift was demonstrated. Supernatant potassium and sodium levels did not reverse 1 hour after rejuvenation was completed. There was no significant difference in results between units irradiated on Day 1 or Day 5, 6, or 7. CONCLUSION: Red cell ATP and 2, 3 DPG levels were restored in irradiated AS-1 units stored at 4 degrees C for 42 days using a pyruvate-inosine-phosphate-adenine rejuvenation additive.

Reduced tissue oxygenation and its reversibility by glycemic control in diabetic patients.

Relationship of transcutaneous oxygen pressure (TcP(O2)) to glycemic control and diabetic complications was investigated in patients with non-insulin dependent diabetes mellitus. TcP(O2) was measured in 103 patients with non-insulin dependent diabetes mellitus. Correlation of TcP(O2) to HbA1c, fasting blood sugar (FBS), age, duration of diabetes, serum lipids, hypertension, and diabetic complications were examined. We divided the patients into three groups according to their glycemic control: good control group (HbA1c < 7.0%), fair control group (HbA1c, 7.0-8.9%) and poor control group (HbA1c > or = 9.0). We compared TcP(O2) of these three groups with 19 non-diabetic controls. In 103 patients, TcP(O2) at baseline correlated with HbA1c, FBS and age (P < 0.001, P < 0.01 and P < 0.05, respectively), but did not correlate with duration of diabetes mellitus, neuropathy, nephropathy or retinopathy. TcP(O2) of good and fair control group was not reduced comparing to the non-diabetic control (63 +/- 11, 59 +/- 10 and 64 +/- 12 mmHg, respectively). The poor control group had significantly reduced TcP(O2) (55 +/- 10 mmHg) comparing to non-diabetic control (P < 0.005) and good control group (P < 0.005). Furthermore, in an independent study, TcP(O2), arterial oxygen pressure (Pa(O2)), oxygen pressure of dorsal pedal vein (PV(O2)) and erythrocyte 2,3-diphosphoglycerate (2,3-DPG) in eight patients with poor glycemic control were followed prospectively. Six patients with improvement of glycemic control showed a significant increase of TcP(O2) and Pa(O2) (P < 0.001 and P < 0.005, respectively). However, two patients without improvement of hyperglycemia had no change in TcP(O2) and Pa(O2). PV(O2) and 2,3-DPG levels of erythrocytes were not changed in six patients. These findings suggest that tissue oxygenation in diabetic patients was deteriorated in relation to hyperglycemia and was reversed with glycemic control. Improvement of Pa(O2) might contribute partly to the increase of TcP(O2).

[Effect of x-irradiation on the structural and functional characteristics of rat erythrocytes and activity of individual enzymes of glycolysis]. / Doslidzhennia vplyvu renthenivs'koho vyprominiuvannia na strukturno-funktsional'ni kharakterystyky erytrotsytiv shchuriv i aktivnist' okremnykh fermentiv hlikolizu.

The activity of glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, hexokinase), content of metabolites (lactate, pyruvate, ATP, 2,3-DPG) and haemolytic stability of rat erythrocytes at the action of chronic X-irradiation at a daily dose 0.258 mC/kg, 2.58 mC/kg, 5.16 mC/kg during 90, 60 and 30 days, accordingly, have been investigated. It was shown, that the glycolytic enzymes activity of female rats in different seasons may vary in wide limits. A general tendency to the decrease of lactate dehydrogenase activity is revealed, but the beginning effects under the irradiation at a daily dose of 2.58 mC/kg and 5.16 mC/kg may be differently directed. The analogous tendencies are found in the change of hexokinase activity. The changes in pyruvate kinase activity correlated with erythrocytes haemolytic stability. The data obtained prove that changes in the direction of glycolytic enzymes activity under the irradiation depend on the initial level, which is caused by seasonal peculiarities of physiological state.

Adenosine causes cAMP-dependent activation of chick embryo red cell carbonic anhydrase and 2,3-DPG synthesis.

In late chick embryos, coordinate activation of red cell carbonic anhydrase II (CAII) and 2,3-diphosphoglycerate (2,3-DPG) synthesis is initiated by hypoxia. The effects are mediated by unidentified hormonal effectors resident in chick plasma. In the present investigation, we have analyzed the effect of adenosine receptor stimulation on embryonic red cell CAII and 2,3-DPG synthesis. We find that primitive and definitive embryonic red blood cells from chick have an A2a adenosine receptor. Stimulation of the receptor with metabolically stable adenosine analogues causes a large increase of red cell adenosine 3',5'-cyclic monophosphate (cAMP) and subsequent activation of red cell CAII and 2,3-DPG production in definitive red blood cells and of CAII synthesis in primitive red blood cells. Direct stimulation of adenylyl cyclase with forskolin has the same effect. Analysis of red cell protein pattern after labeling with [35S]methionine shows that stimulation of red cell cAMP levels activates synthesis of several other proteins aside from CAII. Presence of actinomycin D inhibits cAMP-dependent changes of protein synthesis, indicating that cAMP-dependent transcriptional activation is required. In contrast to the stable adenosine receptor analogues, adenosine itself was a very weak agonist, unless its metabolism was significantly inhibited. Thus, besides adenosine, other effectors of the adenylyl cyclase system are likely to be involved in the O2 pressure-dependent regulation of red cell metabolism in late development of avian embryos.

Norepinephrine-mediated hypoxic stimulation of embryonic red cell carbonic anhydrase and 2,3-DPG synthesis.

Hypoxia is the stimulus for activation of red cell carbonic anhydrase II (CAII) and 2,3-diphosphoglycerate (2,3-DPG) synthesis of chick red blood cells during late embryonic development. We have tested whether plasma catecholamines are involved as hormonal mediators, because hypoxia is a well-known stimulus for catecholamine release in mammalian fetuses. Plasma catecholamines were measured in 8- to 16-day-old chick embryos. Plasma levels of norepinephrine (NE) were initially low, but its concentration increased rapidly from 2.7 nM (day 12) to 13.4 nM at day 13 and 25.5 nM at day 16. Epinephrine (E) was not detectable before day 13. Short-term hypoxic exposure of day 11 embryos (1-h incubation at 13.5% O2) increased plasma NE concentration fivefold compared with the controls but had no effect on E. During 15-h in vitro incubation of red blood cells from day 11, addition of 1 microM NE to the incubation medium increased the red cell 2,3-DPG concentration nearly threefold and CAII activity sixfold compared with the control. The CAII activity and 2,3-DPG concentration were also increased when cells were incubated with plasma from late chick embryos. The activation was induced by beta-adrenergic stimulation of adenylyl cyclase. Atenolol and propranolol blocked the effects of NE and embryonic chick plasma. Analysis of de novo protein synthesis ([35S]methionine incorporation) demonstrated that catecholamines stimulate the synthesis of several proteins besides CAII. The results indicate that developmental changes of plasma NE concentration are instrumental in the adenosine 3',5'-cyclic monophosphate-dependent activation of CAII and 2,3-DPG synthesis of red blood cells from late chick embryos.

Nerve function and blood flow in Otsuka Long-Evans Tokushima Fatty rats with sucrose feeding: effect of an anticoagulant.

To investigate the pathogenesis of diabetic neuropathy in non-insulin-dependent diabetes mellitus, Otsuka Long-Evans Tokushima Fatty rats, an animal model of non-insulin-dependent diabetes mellitus, and non-diabetic Long-Evans Tokushima Otsuka rats were fed with or without sucrose and/or cilostazol, an anticoagulant, for 8 weeks. Sucrose-fed diabetic rats showed a delayed motor nerve conduction velocity, decreased R-R interval variability of electrocardiogram, reduced sciatic nerve blood flow, increased platelet aggregability and a decreased erythrocyte 2,3-diphosphoglycerate concentration compared with non-sucrose-fed diabetic rats and non-diabetic rats. These abnormalities were significantly prevented by treatment with cilostazol without changes in the nerve tissue levels of polyols. These findings indicate that sucrose-fed Otsuka Long-Evans Tokushima Fatty rats may be a useful animal model of neuropathy in non-insulin-dependent diabetes mellitus, and that cilostazol may prevent the development of diabetic neuropathy by modifying vascular factors.

Effect of inosine supplementation on aerobic and anaerobic cycling performance.

Ten competitive male cyclists completed a Wingate Bike Test (WIN), a 30-min self-paced cycling performance bout (END), and a constant load, supramaximal cycling spring (SPN) to fatigue following 5 d of oral supplementation (5,000 mg.day-1) with inosine and placebo. Blood samples were obtained prior to and following both supplementation periods, and following each cycling test. Uric acid concentration was higher (P < 0.05) following supplementation with inosine versus placebo, but 2,3-DPG concentration was not changed. The data from WIN demonstrate that there were no significant differences in peak power (8.5 +/- 0.3 vs 8.4 +/- 0.3 W.kg body mass-1), end power (7.0 +/- 0.3 vs 6.9 +/- 0.2 W.kg body mass-1), fatigue index (18 +/- 2 vs 18 +/- 2%), total work completed (0.45 +/- 0.02 vs 0.45 +/- 0.02 kJ.kg body mass-1.30-s-1), and post-test lactate (12.2 +/- 0.5 vs 12.9 +/- 0.6 mmol.l-1) between the inosine and placebo trials, respectively. No difference was present in the total amount of work completed (6.1 +/- 0.3 vs 6.0 +/- 0.3 kJ.kg body mass-1) or post-test lactate (8.4 +/- 1.0 vs 9.9 +/- 1.3 mmol.l-1) during END between the inosine and placebo trials, respectively. Time to fatigue was longer (P < 0.05) during SPN for the placebo (109.7 +/- 5.6 s) versus the inosine (99.7 +/- 6.9 s) trial, but post-test lactate (14.8 +/- 0.7 vs 14.6 +/- 0.8 mmol.l-1) was not different between the treatments, respectively. These findings demonstrate that prolonged inosine supplementation does not appear to improve aerobic performance and short-term power production during cycling and may actually have an ergolytic effect under some test conditions.