In vitro synthesis of nitroxide free radicals by hog liver microsomes.

The in vitro biooxidation of 4-hydroxy-2,2,6,6-tetramethylpiperidine (TEMP), 4-hydroxy-2,2,4,4-tetramethyl-1,3-oxazolidine (TEMO) and diphenylamine (DPA) by hog liver microsomes to their respective nitroxide free radicals, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 2,2,4,4-tetramethyl-1,3-oxazolidine-1-oxyl (TEMOO), and diphenylnitroxide (DPNO) has been investigated. For extending the life span of the liver microsomes, a calcium alginate immobilization procedure was used. The biooxidation rates of the above amines to their respective nitroxide metabolites were measured by means of oxygen uptake at 37 degrees C and pH 7.4. N-octylamine was found to be an activator in the biooxidation of the amines. The formation of the nitroxide radicals was identified by E.S.R. spectroscopy.

Non-conventional treatment of hepatic failures.

It is our intention to present a short review of various approaches to the non-conventional treatment of hepatic failures of the fulminant type. Our review is directed to the scientist, technologist, and clinician with a budding interest in the hepatic assist area. We shall discuss parabiosis, liver transplants, and various extracorporeal devices including hemoperfusion, hemodialysis, and enzymic detoxification systems. We feel that the present technological approaches to the treatment of hepatic failure are very primitive at this stage. Some of the recent advances are very encouraging, and it is our opinion that these approaches show great promise in the long term.

Experimental considerations for the centrifugal separation of blood cell components.

Processing techniques using recycle and staging for blood cell collection lead to numerous conceptual design configurations. From a practical viewpoint, many of these schemes can become incongruous. Experimental fractional recoveries with up to six stages are presented, and material balance considerations and process comparisons are made. Isolation of specific leukocyte types, and cell viability effects are studied.

The effect of recycle on the continuous centrifugal processing of blood cells.

Recycle configurations are presented for continuous-flow blood cell separation systems. A sample design calculation is presented for a single-stage unit with recycle. Increases in blood-cell collection efficiencies may be obtained with recycle, and various design configurations are proposed.

Stagewise separation to improve continuous centrifugal blood cell separators.

Staging is a technique used to improve blood cell collection efficiencies. In this paper, fractional cell recoveries are presented as functions of hematocrit as well as flow distribution. Material balances are presented, and the effect of increasing the number of stages studied in relation to increases in white blood cell collection efficiencies.

Sedimentation theory and practical considerations for the design of centrifugal blood cell processes.

This is the first of a five-part series discussing the basic conceptualization and evaluation of techniques for centrifugal separation of blood cells. In this paper, the basic concepts of sedimentation theory will be discussed. The next two papers will discuss the effects of plasma recycle and stagewise separations, respectively, as applied to continuous-flow and semicontinuous-flow processing systems. The subsequent paper will underline the differences between the theoretical approach and our experimental efforts, and the final paper will consider potential future process configurations and areas of expected improvement. A series of calculations is presented for the prediction of sedimentation velocities for red and white cells. The equations are simplified to investigate the regions of high red cell concentration, and are not valid for regions of concentrated platelets or white cells. The intent is to present a sample calculational design approach.

The synthesis of poly-L-lysine-succinyl-NADP: an analogue for NADP/H.

Poly-L-lysine-succinyl-NADP, an analogue for nicotinamide adenine dinucleotide phosphate (NADP/H), has been synthesized by linking NADP/H to poly-L-lysine hydrobromide. This analogue (PL-SNP) was assayed with N, N dimethylaniline (DMA) in the presence of hepatic microsomal oxidase. A regeneration system of G-6-p and G-6-PD was used for reducing the PL-SNP+ to PL-SNPH. This reaction scheme with PL-SNP was found to exhibit up to 100% of the initial activity of pure NADP+ based on polarographic studies. Reaction rate of production for dimethylaniline oxide (DMA-0) was determined by oxygen consumption and by oxide formation. One of the potential uses of this analogue is as a cofactor in a membrane/liver enzyme detoxification system.

The removal of 14C labeled endotoxin by activated charcoal.

Endotoxin shock due to Gram-negative enteric bacteria is of major medical concern with an estimated 100,000 fatalities in the United States per year. An effective therapy for endotoxin shock, particularly in combination with significant liver damage, has not been available to date. Since activated charcoal is known as a universal sorbent, the use of activated charcoal in a hemoperfusion apparatus to remove endotoxin has interesting possibilities. Current assays for endotoxin are inadequate. The Limulus Amoebocyte Lysate (LAL) assay was found to give nonreproducible results within our range of requirements for accuracy. We, therefore, grew Salmonella typhimurium in 14C-labeled glucose to obtain 14C labeled endotoxin. Radiolabeled endotoxin was used to measure the rate of adsorption on activated charcoal. The rates of removal of endotoxin from normal saline, plasma, and whole blood will be presented in graphical form for use in design calculations. This work provides a foundation for encouraging in vivo hemoperfusion experimentation now underway at the University of Oklahoma and the Veteran's Administration Hospital in Oklahoma City.

A mathematical simulation of the AIDS patient and extracorporeal detoxification.

A simple numerical simulation of AIDS patient detoxification by a hypothetical extracorporeal device for the removal of viruses, infected white cells, and syncytia has been designed. The mathematical model accounts for healthy blood white cells attacking and destroying the viruses, while at the same time the viruses attack and infect certain white cells. The infected white cells serve as a site for viral growth; eventually the cells lyse, releasing a large number of viruses into the blood stream. The healthy white cells and infected white cells combine to form syncytia, where the virus multiplies, and finally the syncytium ruptures releasing all the virus. This model can be used to predict concentrations over a specified period for the patient. This is a mathematical model to be used as a research and design tool only.

Mechanism of squalene biosynthesis: evidence against the involvement of free nerolidyl pyrophosphate.

Several mechanisms that utilize farnesyl pyrophosphate and nerolidyl pyrophosphate as condensing substrates have been postulated for the asymmetric condensation reaction in squalene biosynthesis. Although there is ample evidence that farnesyl pyrophosphate is a substrate for this reaction, there has been no information concerning the role of nerolidyl pyrophosphate. We have made the following observations that demonstrate that nerolidyl pyrophosphate cannot be a free intermediate in squalene biosynthesis. (a) There is no significant interconversion of farnesyl pyrophosphate and nerolidyl pyrophosphate in a squalene-synthesizing system from yeast. (b) Nerolidyl-1-(3)H(2) pyrophosphate is not converted to squalene in the presence or absence of farnesyl pyrophosphate. (c) The addition of unlabeled nerolidyl pyrophosphate to incubation mixtures does not alter the relative loss of alpha-hydrogens from farnesyl pyrophosphate during its conversion to squalene. The synthesis of nerolidyl-1-(3)H(2) pyrophosphate is described. Chromatographic methods for the separation of pyrophosphate esters of triprenols and terpenols are included.

Microsomal mixed-function amine oxidase. Oxidation products of piperazine-substituted phenothiazine drugs.

Oxidation products of fluphenazide, thioproperazine, and trifluoperazine obtained in reactions catalyzed by homogeneous preparations of the microsomal mixed-function amine oxidase have been isolated and identified. Approximately 0.5 g of metabolite of each piperazine-substituted phenothiazine drug was prepared in reactors containing, as catalyst, the purified oxidase covalently attached to glass beads. Nuclear magnetic resonance spectra of the isolated products indicated that with all three substrates the enzyme preferentially catalyzes N-oxidation of the piperazine nitrogen furthest from the phenothiazine nitrogen atom. The enzyme-catalyzed oxidation is quite specific and oxidation of the sulfur or nitrogen atoms in the phenothiazine ring could not be detected. Concentrations of piperazine-substituted phenothiazines required to half-saturate the amine oxidase were in the micromolar range and at pH 8.3 and 37 degrees C, all those tested were oxidized at approximately 2 mumol/min/mg of enzyme. Kinetic constants for the piperazine-substituted phenothiazines were very similar to those obtained with phenothiazines containing a dimethylaminopropyl sidechain.

A model enzymic extracorporeal detoxification system.

Preliminary studies at the University of Oklahoma have incorporated the use of a continuous, seal-less blood centrifuge as an extracorporeal detoxification unit to aid in the removal of foreign chemicals from the blood. Detoxification is performed by immobilized enzymes in conjunction with a cofactor (NADPH) bound to a water-soluble macromolecule. A drug enters the device with the plasma and then passes across a semipermeable membrane which serves to retain the cofactor. At this point, a combination of the drug, the cofactor and the enzyme react to form the drug-oxide. The oxide then passes back through the membrane into the blood and back into the body. Concurrently, the macro-NADP+ is reduced by G-6-P and G-6-PD in the cofactor regeneration portion of the device. To facilitate detoxification, the centrifuge is employed to provide plasma rich in toxins, but void of potentially interfering blood components such as platelets and whole blood cells. These components tend to dilute the toxins or adhere to the interfacing membrane, decreasing the permeability of these toxins into the detoxification unit. It is felt that the centrifuge-detoxification combination will provide a potentially efficient hepatic assist device.

Endogenous anticoagulation during extracorporeal perfusion: generation of a heparinlike inhibitor.

Studies were done to define the coagulation defect that develops in hemodynamically stable anesthetized dogs perfused on our arteriovenous extracorporeal system without added heparin. After 45 min, the dogs developed whole blood clotting times (WBCT) greater than 24 h. There was an associated decrease in ADP-induced platelet aggregation and a drop in factor V, VIII, and X levels of 75.8, 33.5, and 46.8%, respectively. Despite an increase in fibrinogen degradation products, there was no significant change in fibrinogen level or platelet count. An inhibitor of thrombin and factor Xa clotting of plasma appeared that was "heparinlike", because it stimulated the inactivation of factor Xa by antithrombin III (ATIII) but not by O-methyl isoureamodified ATIII. Thrombin inhibition by ATIII was also stimulated. The inhibitor was heat stable, adsorbed by BaSO4, and neutralized by protamine. Infusion of protamine sulfate into two perfused dogs neutralized the inhibitor and brought the WBCT from greater than 24 h to less than control. Six dogs developed inhibitor levels equivalent to 0.98 to 6.15 U/ml heparin. Five eviscerated dogs in which the hepatic artery was ligated developed peak plasma inhibitor levels of 3.2 +/- 1.0 U/ml. Thus, the endogenous heparinlike inhibitor is a major contributor to the anticoagulated state induced with our perfusion system and may have an extrahepatic origin.