Robert C. Hartmann

The Sugar-Water Test for Paroxysmal Nocturnal Hemoglobinuria

THE diagnosis of paroxysmal nocturnal heinoglobinuria is relatively easy in patients who give the classic story of hemoglobinuria on arising, with subsequent clearing during the day. In many patien...

Divergent effects of epinephrine and prostaglandin E1 on the level of cyclic AMP in human blood platelets

Clinical studies were conducted into the effects of PGs (prostaglandins) E1 and E2 and 2 adrenergic blocking agents phentolamine and propranolol on the level of cyclic AMP in blood platelets. The methodology of the studies is explained and the results are graphed. Epinephrine stimulates platelet aggregation by decreasing the intracellular level of cyclic AMP. This cyclic AMP-lowering effect of epinephrine is inhibited by the alpha-adrenergic blocking agent phentolamine but not by the beta-adrenergic blocking agent propranolol. PGE1 was shown to be a powerful stimulant of levels of cyclic AMP in human blood platelets; PGE2 was much less powerful in its stimulatory effect. While PGE1 increased the level of cyclic AMP at concentrations of 10 ng/ml PGE2 was only effective at concentrations of 1000 ng/ml. The study results add to the evidence that the level of cyclic AMP in blood platelets is an important determinant of the aggregation tendency of these cells. Alpha-adrenergic responses in general are seen to be mediated by decreased levels of cyclic AMP.

Paroxysmal nocturnal hemoglobinuria: II. Erythrocyte acetylcholinesterase defect☆

Abstract Markedly reduced erythrocyte acetylcholinesterase activity was demonstrated in all eight cases of paroxysmal nocturnal hemoglobinuria studied. Consistent reduction of this degree has not been noted in other blood dyscrasias, either in studies cited in the literature or in our own investigations. The defect was localized to the stroma of the PNH red cell. The enzyme activity of reticulocytes was markedly impaired, indicating that the immature cells were formed with defective acetylcholinesterase. The activity of normal cells was not reduced following transfusion into patients with PNH. No evidence was found for a circulating inhibitor of acetylcholinesterase or for one which could be readily eluted from the PNH erythrocyte membrane. However, our studies do not exclude the possibility of an inhibitor, particularly one bound firmly to the enzyme. In the one case in which a family study was carried out, normal erythrocyte acetylcholinesterase activity was present in all members tested. The previous work of many investigators has suggested that erythrocyte acetylcholinesterase plays an important role in the preservation of the integrity of the red cell. The significance of the defective enzyme activity in the PNH cell is discussed in this connection.

Paroxysmal nocturnal hemoglobinuria: I. Clinical studies

Abstract The clinical and laboratory findings in six patients with paroxysmal nocturnal hemoglobinuria are presented. Some unusual or little recognized features are discussed. These include occurrence of paroxysmal nocturnal hemoglobinuria in the Negro, presence of leg ulcers and skin lesions, hemorrhagic complications, and the development of iron deficiency. In accord with previous suggestions of others, a carefully planned program of rehabilitation is advocated, based on periodic outpatient transfusions of washed red cells.

EFFECTS OF PROSTAGLANDINS ON FUNCTION AND CYCLIC AMP LEVELS OF HUMAN BLOOD PLATELETS

platelet adenyl cyclase. This was noted several years ago by R.E.Scott and his colleagues(Butcher et a1,1967;Scott, 1970),and has been confirmed since by at least three other groups of investigators(Wo1fe and ShulmanIl969;Marquis et al,l969;Moskowitz et a1,1970). The order of potency of different prostaglandins,to the extent that this has been studied,is the same as their order of potency as inhibitors of platelet aggregation. With the exception of the fluoride ion,to be mentioned laterlagents which do not inhibit aggregation are not known to be capable of stimulating platelet adenyl cyclase. The second line of evidence derives from studies with intact platelets. As expected from their effect on adenyl cyclase,the prostaglandins increase the steady state level of cyclic AMP in these cells(Fig.l)(Robison et a1,1969). The greater potency of PGEl over PGE2 is in line with their known potencies as inhibitors of aggregation(Weeks et all 1969;Robison et a1,1969). Other investigators(Moskowitz et all 1970;Vigdahl et all 1969)have used 3H-adenine or 14Cadenosine to label endogenous ATP,and have obtained essentially similar results: the prostaglandins were found to stimulate the incorporation of radioactivity into cyclic AMP,reflecting an increased rate of synthesis of the cyclic nucleotide in the intact cells. Conversely,several agents which stimulate platelet aggregation have been shown to produce a fall in the level of cyclic AMP in these cells. These are epinephrine (Moskowitz et alIl970;Robison et alIl969;Salzman & Neri, 1969;Marquis et a1,1970)and adenosine diphosphate(ADP), (Salzman & Neri,l969;Cole et alIl970a),both of which are thought to play an important role in regulating platelet function in vivo(Mustard ti Packham,l970). The effect of epinephrine is mediated by adrenergic a-receptors, First,the prostaglandins are potent stimulants of

POTASSIUM TRANSPORT IN THE ACETYLCHOLINESTERASE-DEFICIENT ERYTHROCYTES OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)*

The mechanism by which certain mammalian red cells accumulate potassium has been the subject of extensive investigation. Maintenance of a high intra-erythrocytic potassium concentration against a relatively low plasma concentration has been attributed to an active transport system. The precise relationship of such a transport system to cellular metabolism in general is obscure. Glycolysis, an important energy-yielding process in erythrocytes, has been held responsible for potassium reaccumulation and sodium extrusion by cold-stored human red blood cells when rewarmed to 37.50 (1, 2). Certain metabolic poisons which inhibit glycolysis also affect cation transport in red blood cells (3). These observations support the concept that the active transport mechanism may be geared to and utilize energy from glycolysis. Yet the hypothesis of an active transport system in human erythrocytes has never been absolutely established. Some investigators have attempted to incriminate a single enzyme, acetylcholinesterase, as responsible for maintaining normal permeability in human and canine erythrocytes (4-6). These workers noted a retarding effect of acetylcholine on cation movement and on hemolysis and a reversal of this effect by physostigmine. These reports stimulated much interest in the possible role of acetylcholinesterase in governing sodium and potassium transport in erythrocytes. Other investigators, however, were unable to find evidence of a significant relationship between erythrocyte acetycholinesterase activity and potassium movement (7-10).

Solubilization and Purification of Human Erythrocyte Membrane Acetylcholinesterase

Summary A variety of commonly employed membrane solubilizing agents were studied for a possible effect on erythrocyte AChE activity. Only sodium deoxycholate had any appreciable inhibitory effect. Stepwise solubilization of red cell membrane with a variety of agents showed no selective removal of membrane AChE. Rather the release of enzymic activity closely paralleled the solubilization of membrane proteins, suggesting that AChE is an integral part of the membrane. A relatively simple method was described for the isolation and purification of human red cell AChE which resulted in a 4200-fold purification. The enzyme appears to be associated with lipids and gave a single precipitin band on immunodiffusion.

Erythrocyte diphosphopyridine nucleotidase (NADase) in paroxysmal nocturnal hemoglobinuria.

Summary Erythrocyte NADase activity was found to be normal in nine PNH patients, including with one with concomitant G-6-PD deficiency. Thus the erythrocyte AChE deficiency in PNH does not likely reflect any “general poverty” of stromal-bound enzymes in this disorder.