Leon Ellenbogen

Intravenous arachidonate in the mouse: A model for the evaluation of antithrombotic drugs

Abstract The intravenous administration of arachidonate to mice offers a rapid, convenient, and effective in vivo model for the study of platelet aggregation and thrombosis, as well as the evaluation of potential antithrombotic drugs. Following intravenous administration in the mouse, there is dose-dependent cyanosis and respiratory distress with death at doses about 100 mg/kg. The rabbit is much more sensitive to the effects of intravenous arachidonate with death occurring at doses of 1 mg/kg; the rat shows an intermediate sensitivity. There is a relative specificity for the arachidonate effect: administration of certain other unsaturated fatty acids produces only minimal respiratory distress at equivalent doses. Respiratory distress in the mouse can be correlated with histologic evidence of platelet aggregation in the microvasculature of the lungs, but not in the heart or brain. Several nonsteroidal anti-inflammatory drugs (NSAID) which are known to be inhibitors of collagen-induced platelet aggregation and release, as well as inhibitors of prostaglandin synthetase, are able to block the effect of arachidonate in vivo . ADP inhibitors (dipyridamole, adenosine, and VK 744) are also effective in blocking the effects of arachidonate in vivo . Both the NSAID and the ADP inhibitors also block arachidonate-induced platelet aggregation in vitro .

Tyrosine hydroxylase inhibition in vitro and in vivo by chelating agents

Abstract A variety of metal chelating agents inhibited bovine adrenal tyrosine hydroxylase in vitro. Bipyridyl, o-phenanthroline, TPTZ (2,4,6-tripyridyl-s-triazine) and bathophenanthroline (4,7-diphenyl-1,10-phenanthroline), which have high affinities for divalent iron, were the most effective inhibitors. Inhibition by o-phenanthroline was noncompetitive with tyrosine or pteridine cofactor, but dependent on iron concentration. Bipyridyl, administered to rats, inhibited adrenal tyrosine hydroxylase activity and markedly lowered adrenal, heart and brain catecholamines.

Preparation of highly purified intrinsic factor.

Summary A highly potent intrinsic factor preparation has been obtained from desiccated hog stomach by ammonium sulfate fractionation, digestion with proteolytic enzymes, alcohol fractionation and finally ultrafiltration. The ultrafiltration residue fraction proved effective in pernicious anemia patients in relapse at a daily dose of 1 or 2 mg together with vit. B12. Electrophoresis and ultracentrifuge studies of this fraction showed that the major portion of the material was a component with a molecular weight of about 5000. Preliminary separation in ultracentrifuge and clinical evaluation of this homogeneous component suggested strongly that it was intrinsic factor. The ultrafiltration residue fraction contained 15.2% glucosamine and 11.8% nitrogen. In confirmation of our analysis for hexosamine, UFR fraction No. 21-2 was found by Dr. Saul Roseman, University of Michigan, Ann Arbor, to contain 14% hexosamine HCl. By a new differential colorimetric method developed by Dr. Roseman about half of the hexosamine was glucosamine HCl while the remainder was presumably chondrosamine HCl.

Studies on the mechanism of vitamin B12 absorption: in vivo transfer of vitamin B12 to intrinsic factor.

Abstract Evidence is presented that the binding of vitamin B 12 by intrinsic factor prior to oral administration is unnecessary for the intestinal absorption of vitamin B 12 . In a mixture of intrinsic factor and physiologically inactive binding protein, vitamin B 12 is not preferentially bound to the former. The vitamin is randomly distributed among its binding sites and subsequently transferred in the gut from nonintrinsic factor protein to intrinsic factor. In vitro experiments show that vitamin B 12 , bound to intrinsic factor or nonintrinsic factor protein from hog gastric mucosa, does not become dialyzable after exposure to an acid medium or treatment with pepsin, trypsin-chymotrypsin, or pancreatin.

Effect of D-sorbitol on absorption of vitamin B12 by pernicious anemia patients.

Summary 1) In the urinary excretion test 5 pernicious anemia patients excreted radioactivity corresponding to an average of 0.02 μg of Vit. B12 when fed 2 μg of Vit. B12 orally and excreted an average of 0.02 μg when fed 2 μg of Vit. B12 together with 0.1 to 10 g of sorbitol. These patients excreted an average of 0.23 μg when fed 2 μg of Vit. B12 with intrinsic factor and an average of 0.22 μg when 2 μg of Vit. B12 were fed together with intrinsic factor and 10 g of sorbitol. 2) These 5 anemia patients excreted an average of 0.29 μg when fed 30 μg of Vit. B12 and 0.12 μg when fed 30 μg of Vit. B12 together with 10 to 21 g of sorbitol. The same 5 patients excreted an average of 0.35 μg when fed 30 μg of Vit. B12 with intrinsic factor, and excreted 0.33 μg when 10 to 21 g of sorbitol was fed together with 30 μg of Vit. B12 and intrinsic factor. 3) Sorbitol had no effect in promoting absorption of Vit. B12 in pernicious anemia patients.

The inhibition of monoamine oxidase by styrylquinoliniums

Abstract A large group of styrylquinoliniums was found to inhibit the oxidative deamination of dopamine and other biogenic amines by guinea pig liver monoamine oxidase. 4-(p-Chlorostyryl)-1-methyl-quinolinium iodide was nearly five times more potent than harmaline and 1200 times more potent than iproniazid. The position of attachment of the styryl moiety to the quinolinium affected potency, the 4-styrylquinoliniums being more potent than the 2-styrylquinoliniums. Removal of the styryl moiety decreased potency, suggesting its participation in the inhibition. The effects of other structural modifications of the quinolinium and styryl moieties on the potency of inhibition are discussed.

Studies on the inhibition of histidine decarboxylase, aromatic-l-amino acid decarboxylase and acid secretion by brocresine and its metabolites

Abstract Brocresine (NSD 1055) is rapidly metabolized in vivo. The probable metabolites are 4-bromo-3-hydroxy-benzyl alcohol, 4-bromo-3-hydroxy-benzoic acid and 4-bromo-3-hydroxy-hippuric acid. Brocresine and these metabolites inhibit both rat fetal and rat gastric histidine decarboxylase ( l -histidine carboxylase, EC 4.1.1.22) in vitro with a molar I50 of about 10−8, 10−4, lO−3 and 10−5, respectively, for both enzymes. Brocresine and the metabolites also inhibit aromatic- l -amino acid decarboxylase (3,4-dihydroxy- l -phenylalanine carboxylase EC 4.1.1.26) from hog kidney and rat gastric mucosa in vitro with a molar I50 of about 10−7, 10−4, 10−3 and 10−3, respectively, for both enzymes. Brocresine, the alcohol metabolite and the acid metabolite inhibited rat gastric histidine decarboxylase after intraperitoneal administration of 200 mg kg , whereas the hippurate was only weakly inhibitory. All four compounds inhibited gastric acid secretion in the pylorus-ligated rat, but the acid and hippurate were only moderately inhibitory. The reaction of hemoglobin with brocresine to form methemoglobin readily explains the rapid disappearance of the inhibitory activity of the drug.

Purification of Intrinsic Factor.

Summary A highly purified intrinsic factor preparation (RAS fraction) was prepared in quantity from fresh hog duodenum by saline extraction, pH adjustment and ammonium sulfate precipitation. This fraction was active in pernicious anemia patients in relapse at daily oral dose of 5 mg. Ultracentrifugal separation could not completely separate the components of RAS fraction. RAS fraction was further purified on a DEAE-cellulose ion exchanger to give a fraction which was active in urinary excretion test at a dose of 3 mg. equivalent to a daily oral dose of 1 mg in pernicious anemia patients in relapse. Rechromatography of this active fraction resulted in no further increase in potency. Ultracentrifugal studies of the most active fraction obtained from rechromatography showed essentially 3 peaks with sedimentation constants of 1.0 S, 2.8 S and 11 S.

THE URINARY EXCRETION TEST IN THE DIAGNOSIS OF ADDISONIAN PERNICIOUS ANEMIA

Excerpt Addisonian pernicious anemia is a deficiency disease resulting from the lack of a specific gastric secretion which is essential for the optimal absorption in the intestine of orally ingeste...

Fenbufen and biphenylacetic acid inhibit platelet function and the arachidonate prostaglandin system

Abstract Biphenylacetic acid or BPAA ([1,1-biphenyl]-4-acetic acid) is a metabolite of the non-steroidal anti-inflammatory drug, fenbufen (γ-oxo[1,1-biphenyl]-4-butanoic acid). A 0.12 mM concentration of BPAA produces almost complete inhibition of arachidonate-induced aggregation in vitro comparable to the effect produced by similar concentration of other non-steroidal anti-inflammatory agents. Fenbufen does not inhibit arachidonate-induced aggregation in vitro . These data correlate with the ability of BPAA, but not fenbufen, to inhibit platelet prostaglandin synthetase in vitro . Respiratory distress and histologic evidence of platelet aggregates formed in the pulmonary microvasculature following arachidonate infusion are markedly inhibited by oral doses of both fenbufen and BPAA. The order of efficacy (at 10 mg/kg) for inhibition of respiratory distress is BPAA > fenbufen > aspirin. These data confirm other studies indicating metabolic conversion of fenbufen in vivo to the active metabolite, BPAA. Collagen-induced aggregation is reduced by in vitro addition of either BPAA or fenbufen and in platelet-rich plasma from rats receiving oral doses of these compounds. BPAA added in vitro is more potent than either aspirin or fenbufen as an inhibitor of collagen-induced aggregation. Following in vivo dosing at 10 mg/kg, BPAA is comparable to aspirin in efficacy as an inhibitor of collagen-induced aggregation, while the same dose of fenbufen is less effective. The ability of BPAA to inhibit platelet aggregation appears to be related to intererence with the arachidonate-thromboxane-prostaglandin system. Fenbufens inhibitory action may be dependent on conversion to BPAA or involve another mechanism. Other data suggest, however, that this mechanism is not related to inhibition of serotonin release or inhibition of phosphodiesterase activity in platelets.