RESEARCH

EVANS W 1

ACETIC

CH3C - S H Specialty Thiolating and Mild Acetylating Agent

, 0 RX-f CHaC'SH- -^CH,C'-SR - * *RSH

R-CH=CH, - f CH,C -SH->- CH,C -SCHiCHiR-** RCHICH^H

R N H Î + C H J Î - - SH .0

->CH,C'-NHR + H,S

OTHER THIOL ACIDS AVAILABLE THIOLBENZOIC DITHIOLTEREPHTHALIC

Samples and literature containing eight pages with over one-hundred references available on request.

C H e m e n c s , ΙΓΛΟ 90 Tokeneke Road

Darien, Connecticut 06820 Phone: 203-655-8741 Cable: EVANSCHEM TWX: 710-468-2148

Chemical blood substitutes tested in animals Perfluorinated hydrocarbon emulsion proves to be an efficient gas transport medium

Medical science is making steady prog­ress toward what might be the ultimate solution to the problem of tired blood—replacing the natural fluid with a chemical substitute. But although artificial blood is showing promising results in laboratory animals, humans are unlikely to benefit from it in the near future.

Dr. Robert P. Geyer of Harvard's school of public health told a meeting of the Federation of American Societies for Experimental Biology in Atlantic City last month that he had success­fully replaced up to 92% of the nat­ural blood in rats with a blood sub­stitute—an emulsion of fluorinated hydrocarbons and high-molecular-weight polyols. Late last year, Dr. Leland C. Clark and his colleagues at the University of Cincinnati reported similar replacement of blood in dogs with a fluorocarbon emulsion (C&EN, Dec. 15, 1969, page 51) .

Dr. Clark's group uses Allied Chem­ical's P-11D—a perfluorinated hydro­carbon—as the oxygen carrying agent. The fluorocarbon is emulsified with Wyandotte's F-68, a detergent with a molecular weight from 4000 to 8000 commercially used in such products as toothpaste. The fluorocarbon-deter­gent mixture is emulsified by ultra­sound and diluted with glucose-fortified Ringer's solution.

The substitute that Dr. Geyer used is similar to the one used by the Clark group. Dr. Geyer uses 3M's FC-47— a fully fluorinated tertiary butyl amine—as the oxygen transporting agent. Liquid fluorocarbons dissolve appreciable amounts of oxygen and carbon dioxide, Dr. Geyer points out, but do not "bind" gases as hemoglo­bin does. "The fluorocarbons dissolve blood gases strictly as a function of pressure," Dr. Geyer says. "There ap­pears to be no chelation or sequester­ing of gases."

Dr. Geyer uses block polymers— polyoxyethylene and polyoxypropyl-ene—to disperse the fluorocarbons in colloidal suspension. The polymers are nontoxic, according to Dr. Geyer, and bind water in a hydrophilic col­loid. "We have used the polymers in tissue culture systems and no

toxic effects show up," Dr. Geyer notes. The molecular weight of the polymer is high enough that the poly­mer stays in circulation.

The fluorocarbons are dispersed by high-pressure homogenization or soni-cation with polymers in an isotonic medium containing electrolytes, glu­cose, and amino acids.

Dr. Geyer thinks the particle size of the emulsified fluorocarbon is less than 0.01 micron. Earlier prepara­tions—with particle size about 0.5 mi­cron—were milky, but more recent preparations with smaller particles are essentially clear. "We don't know exactly how small the particles are," says Dr. Geyer, "but they are definitely in the true colloid range."

Nontoxic. Dr. Geyer administers his blood substitute to rats under an­esthesia via a tail vein catheter, si­multaneously replacing natural blood taken from the jugular vein. Follow­ing the transfusion, the rat is removed to a sealed jar, provided with food and water, and allowed to recover in an atmosphere of pure oxygen. Dr. Geyer observes the animal and evalu­ates its recovery on the basis of respiration pattern, mobility, respon­siveness to visual and auditory stimuli, and typical behavior patterns such as washing, urinating, and eating. "When the animals display their usual behavior, it can be assumed that there is no serious impairment of the vital organs or processes," Dr. Geyer points out.

The animals begin to wash or drink within five to 10 minutes after re­covering from the anesthesia, accord­ing to Dr. Geyer. "Of all the ma­terials and treatments used in these experiments, oxygen proved to be the most toxic," he comments. The ani­mal was allowed to remain in pure oxygen only 16 to 20 hours, after which the oxygen concentration was reduced stepwise in 10% increments. The animal was returned to atmospheric conditions (20% oxygen) in four to five days.

Dr. Geyer stresses that there is nearly always some serum present in the animals receiving blood substi­tutes. "The reason the animals live,"

30 C&EN MAY 18, 1970

φ ^ - Γ ν ^_«««1_

THUL·

BCD

says Dr. Geyer, "is that they regen­erate cells and plasma proteins rapidly."

Dr. Geyer has performed some ex­periments in which animals are con­tinuously infused with the blood sub­stitute, replacing all of the natural blood. These animals live up to eight hours with the blood substitute alone.

Dr. Clark's interest in perfluorinated hydrocarbons as oxygen carriers in artificial blood was stimulated by the discovery in 1965 that a mouse could live completely submerged in liquid fluorocarbon for as much as a day. "We just picked him out of the fluoro­carbon, turned him upside down, and drained him," says Fernando Becattini, one of Dr. Clark's coworkers. The group has since been working with the blood substitute in beagles and has, consequently, been able to moni­tor several physiological parameters.

Blood pressure. One of the most dramatic responses is an extreme low­ering of blood pressure when the animal receives a very small amount -as little as 0.05 cc./kg. body weight— of the blood substitute. Blood pres­sure may drop from a normal of 140 mm. Hg to 40 mm. Hg, according to Mr. Becattini.

The volume of fluorocarbon admin­istered does not seem to be important, according to Mr. Becattini. Dr. Clark speculates that the pressure drop might be due to an anaphyloid re­sponse. The pressure returns to nor­mal within 10 to 20 minutes and fur­ther administration of fluorocarbon is without effect.

The animal is apparently not "im­munized" permanently, however. A similar response may occur the second time an animal is transfused with ar­tificial blood.

Temperature remains normal and respiration is unaffected except during the period of the pressure drop. Both p C 0 2 and pH remain at normal levels and heart rate remains steady.

A marked increase in oxygen ten­sion of mixed venous blood is noted. A normal value of 50 to 60 mm. H g may be elevated to as much as 300 mm. Hg during infusion with the blood substitute.

Particle size is a major factor in determining how well the animal will do, according to Mr. Becattini. The smaller the fluorocarbon particle, the smaller the chance that it will be phagocytized by leucocytes. The par­ticles are inert and hence will not bind to plasma proteins or cells, but there are conflicting reports in the literature on clotting, according to Mr. Becat­tini.

The Clark group thinks the particles are spherical and in the range of 0.1 to 0.01 micron in diameter. Intense sonication or improved surfactants

could reduce the mean size by an or­der of magnitude, according to Dr. Clark, but even at the present size, the Cincinnati team has had no dif­ficulty with intravascular coagulation. Just what happens to the various com­ponents of the blood substitute when natural blood is regenerated by the animal is not well understood.

Metabolism. Dr. Geyer says the polymer component of the blood sub­stitute is excreted in the urine. The fluorocarbon component presents a more difficult analytical challenge. The spleen apparently removes some of the fluorocarbon from circulation, Dr. Geyer finds. ''Hopefully we will have 14C-labeled fluorocarbons for fur­ther metabolic studies soon," he adds.

Studies by Dr. Clark's group indi­cate that up to half of the fluorocar­bons removed from circulation eventu­ally accumulate in the liver. Some of the fluorocarbon is apparently removed by the reticuloendothelial system and ends up in the spleen.

According to Dr. Geyer, blood sub­stitutes should be widely applicable to in vitro organ perfusion as organ transplants become more common. Presently, donor organs must be per­fused with whole blood during the in­terval between removal from the do­nor to transplantation in the recipient.

Whole blood leaves much to be de­sired as a perfusion medium, Dr. Geyer notes. Red cells do not with­stand the shock of pumping well, he says, and proteins are denatured.

Humans. Despite encouraging re­sults with animal tests, use of the blood substitute in humans is appa­rently further in the future. "Al­though the ultimate goal is to be able to use blood substitutes in humans," Mr. Becattini says, "We need to take a couple of steps forward before hu­man use becomes a clear possibility." Among the unexplained phenomena encountered by Dr. Clark's group is the fact that the fluorocarbon particles apparently lose some of their oxygen-carrying capacity a few hours after administration. "The particles are there," says Mr. Becattini, "but we don't know why the oxygen capacity is decreased."

Dr. Geyer plans further experiments with whole organ perfusion, but warns that it would be "premature" to say that blood substitutes in their present form would be successful. There are, for example, complex nutritional re­quirements for isolated organs still to be worked out. Dr. Geyer also wants to know exactly what happens to the fluorocarbon component. As the search for artificial blood continues, scientists at the National Heart and Lung Institute announced the synthe­sis of hemoglobin last week (C&EN, May 11, page 22) .

New lIBaker

catalog lists

ΛΡ0 defined .solvents in seven

types

types of specially charac­terized solvents for specif­ic applications, including:

ULTREX® G r a d e . . . a new generation of ultra high purity solvents. Many have a guaran­teed 99.95% minimum assay by G. C.

Instra-Analyzed Grade . . . guaranteed 99 + % by G. C. Each bottle supplied with actual gas chromatogram, actual U. V. curve, actual I. R. curve and actual lot analysis.

Spectrophotometric Grade . . . a select group of 'Baker Analyzed'™ Reagents pro­vided with actual lot absorb-ance values at selected wave­lengths, values for infrared transmittance bands, and ac­tual lot analysis.

Plus . . . the broad range of 'Baker Analyzed' Reagent sol­vents, the most widely used laboratory products today.

For information on these solvents and more than 5,000 other chemicals, see our new Catalog 700.

For the products he needs most often . . . the profes­sional chooses J. T. Baker.

J. T. Baker Chemical Co. 222 Red School Lane C-1 Phillipsburg, N.J. 08865

MAY 18, 1970 C&EN 31

J.TBaker