Marion S. Murphy

Detection of Mutant Hemoglobins with Altered Affinity for Oxygen: A Simplified Technique

The detection of high- or low-affinity hemoglobins in subjects with polycythemia or anemia is difficult for most physicians because of the requirement for special equipment to do oxygen-hemoglobin dissociation curves. Measurement of the pH, oxygen tension, and oxygen saturation of antecubital venous blood with instruments present in most clinical chemistry laboratories permits an estimate of the strength of oxygen binding to hemoglobin. An equation can be used to convert the venous oxygen tension (standardized to pH 7.4) and the oxygen saturation to the P50 of the oxygen-hemoglobin dissociation curve on which the observed point falls. The data indicate that this method is a reliable initial step in the identification of a hemoglobin with abnormal affinity for oxygen and may be applied to population studies, since reliable results are obtained with venous blood stored at 4 degrees C for up to 24 hours.

Reduced Red Cell 2,3-Diphosphoglycerate and Adenosine Triphosphate, Hypophosphatemia, and Increased Hemoglobin-Oxygen Affinity after Cardiac Surgery

Serum inorganic phosphorous was decreased significantly on the third postoperative day following cardiac surgery in 18 patients initially studied. Reduced plasma inorganic phosphate has been shown to cause a reduced concentration of red cell organic phosphates, an important determinant of hemoglobin-oxygen affinity. Therefore, 10 consecutive patients were studied to determine if reduced 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) concentration and increased hemoglobin-oxygen affinity accompanied the fall in serum inorganic phosphate concentration.A significant fall in 2,3-DPG and an increase in hemoglobin-oxygen affinity was present in red cells of patients studied on the first postoperative day. A reduction in red cell ATP was also present and persisted for 5 days during which time red cell 2,3-DPG returned to levels which were in excess of preoperative values. The reduction in serum inorganic phosphorous followed the reduction in red cell 2,3-DPG and correlated with the reduction in ATP. The latter changes may indicate the diversion of glucose and more specifically 1,3-DPG into the Rapoport-Leubering pathway away from ATP generation at the phosphoglycerate kinase step and the utilization of plasma inorganic phosphate for 2,3-DPG resynthesis. Neither the transfusion of stored blood nor the effect of cardiopulmonary bypass fully explained the reduction in red cell 2,3-DPG and the inefficiency of hemoglobin function postoperatively. Further studies in postsurgical patients are needed to clarify the cause of the changes observed since they are potentially deleterious, especially in the subject with compromised cardiovascular and pulmonary function.

Effect of Propranolol on Oxygen Binding to Hemoglobin In Vitro and In Vivo

We have shown that propranolol reduces oxygen binding by hemoglobin in intact red cells by increasing the selective permeability of the red cell membrane resulting in an exodus of potassium, chloride, and water. The latter effects result in a new distribution of hydrogen ion between cell and plasma, and thereby a reduction in red cell pH. The reduction in pH can fully explain the change in hemoglobins affinity for oxygen based on the Bohr effect. Either D- or DL-propranolol can produce the change in red cell pH and oxygen binding by hemoglobin. The drug action on permeability is not prevented by epinephrine, although it is by chlorbutanol. Hence, the membrane action of propranolol does not appear to be related to its activity as a beta-adrenergic receptor blocking agent.Propranolol produced a marked alteration in red cell shape as well as in hydration (hypovolumic stomatocytes). The two effects were separable since dehydration of the cell by the addition of sucrose to plasma did not result in stomatocytes and chlorbutanol blocked the enhancement of permeability of the red cell membrane by propranolol without preventing the shape change (isovolumic stomatocytes). This suggests that propranolol may have two separate sites of membrane interaction.Propranolol (10 to 360 mg) administered to human subjects did not affect hemoglobin-oxygen affinity. This is explained by the fact that the concentration in blood after such doses is nearly 4,000 to 100-fold lower than that required to achieve changes in blood in vitro.

The relationships between arterial oxygen flow rate, oxygen binding by hemoglobin, and oxygen utilization after myocardial infarction.

The interrelationships of arterial oxygen flow rate index, oxygen binding by hemoglobin, and oxygen consumption have been examined in patients with acute myocardial infarction. Proportional extraction of oxygen increased in close association with decreasing oxygen flow rate, and hence, whole body oxygen consumption was constant over nearly a three-fold variation in arterial oxygen flow rate. A reduction in hemoglobin-oxygen affinity at in vivo conditions of pH. Pco(2) and temperature also occurred in proportion to the reduction in arterial oxygen flow rate. Therefore, the increased proportional removal of oxygen from arterial blood at low oxygen flow rates, required to maintain oxygen consumption, may have been facilitated by the reduced affinity of hemoglobin for oxygen at in vivo conditions. However, the decrease in affinity did not appear to explain more than 30-40% of the increased extraction. Respiratory alkalosis was a frequent occurrence in these patients and 2,3-diphosphoglycerate was positively associated with blood pH as well as with the time-averaged proportion of deoxyhemoglobin in arterial and venous blood.Hemoglobin-oxygen affinity measured at standard conditions and the mixed venous oxygen saturation were equally good indicators of reduced arterial oxygen flow rate in patients without shock. However, Svo(2) is more easily measured and is a more useful indicator of reduced oxygen flow rate, since its relationship to oxygen flow appears to be independent of affinity changes and time.

Oxygen Binding to Haemoglobin in Subjects with Hypoproliferative Anaemia, with and without Chronic Renal Disease: Role of pH

Summary. The relationships of haenioglobin concentration and blood pH to red cell 2,3‐dipliosphoglycerate and oxygen binding by haenioglobin have been studicd in healthy subjects and subjects with hypoproliferative anaemia with or without severe chronic renal disease. Red cell 2,3‐DPG was inversely correlated with haemoglobin deficit and directly and equally strongly associated with blood pH in anaemic subjects without chronic renal disease. In subjects with chronic renal disease receiving regular haemodialysis, predialysis pH was not increased despite severe anaemia, and red cell 2,3‐DPG was not significantly elevated, except in subjects who had a sustained alkalosis due to the use of sodium bicarbonate.

The Use of a Single Venous Blood Sample to Assess Oxygen Binding to Haemoglobin

The measurement of pH, Po2, Pco2 and So2 in a single venous blood sample can be used to determine the P50 at standard or at in vivo conditions. This technique makes it feasible for a physician, firstly, to make an assessment of the net adaptation of the red cell to reductions in blood oxygen content or flow and, secondly, to make an initial assessment of whether a haemoglobin with altered affinity for oxygen is present in subjects with polycythaemia or anaemia.

Acidification of plasma by the red cell due to radiographic contrast materials.

The effect of water-soluble radiographic contrast material on pH when added to blood in clinical dosages in vitro or when used in vivo for diagnostic purposes was examined. Contrast material caused a reduction of blood pH. The mechanism of this occurence was found to be the balancing of the negative charge of intracellular organic anions by the extracellular anionic contrast material molecules. The normal negative potential of about 10 mV across the red cell membrane was reduced, nullified, or reversed depending on the concentration of contrast material added to blood. As the inside of the cell became more positive with respect to the outside, protons were, in effect, repelled into plasma, although the apparent exodus of protons occurs by the generation and outward diffusion of carbon dioxide. Since the acidemia is dependent on rehydration of carbon dioxide in plasma, a reaction measured in seconds, the site of injection and transit time of dye will contribute to the pH of the plasma during passage through a regional capillary bed. We speculate that an alteration in membrane potential and/or the acute acidemia may contribute to the adverse effects of contrast material, particularly on tissues dependent on membrane electrical rhythmicity such as the myocardium.

Factitious changes in binding of oxygen to hemoglobin when based on extracellular pH in the presence of certain blood additives like radiographic contrast media.

Construction of oxygen-hemoglobin DISSOCIATION CURVES BASED ON EXTRACELLULAR PH and using blood tonometered with 5 per cent CO2, is misleading under certain experimental conditions. These include the presence in blood of poorly penetrating non-ionic molecules like sucrose of poorly penetrating anionic aompounds like radiographic contrast materials. False conclusions regarding the position of the oxygen-hemoglobin dissociation curve can result because of the disturbance of the normal pH gradient between plasma and red cell induced by such chemicals.