Immunochemlstry 1975, Vol 12, pp 625-627
Pergamon Press
Pnnted in Great Britain
DESTRUCTION OF BACTERIAL E N D O T O X I N PYROGENICITY BY H Y D R O G E N PEROXIDE ARTHUR CHERKIN Psychobtology Research Laboratory, Veterans Administration Hospital, Sepulveda, Cahforma 91343, U S A and Departments of Anesthesiology and Psychiatry, University of Cahforma School of Me&cme, Los Angeles, Cahfornla 90024, U S A (Recewed 16 September 1974)
Abstract--The early development of oxypolygelatm as a plasma substltute was hampered by the presence m gelatin of pyrogemc bacterial endotoxms, which are complex hpopolysaceharldes known also as Boiwn anugens. Heat-sterdlzatmn reduced but did not ehmlnate the pyrogenlclty Campbell's observatton that gelatin solutions oxidized w~th hydrogen peroxide were nonpyrogemc prowded a safe, simple resolution of the problem The peroxide technique also destroyed the pyrogeniclty of potent crude bacterial endotoxms The procedure was successfully apphed to the large-scale production of oxypolygelatm for chmcal use and has since been extended to solutions of sodmm chloride and dextrose, and to other apphcat~ons
"Endotoxms possess an intrinsic fascination that is nothing less than fabulous They seem to have been endowed by Nature wtth wrtues and vices in the exact and glamorous proportions needed to render them irresistible to any investigator who comes to know them They intrigue the chemist The molecular basis for their biological action seems always on the verge of discovery but somehow .lust eludes detection." (Bennett. 1964) "No other natural product is known which would ehclt such a great variety of reactions as do endotoxlns when injected into the proper host" (Nowotny, 1969)
manzed by H~sslg and Stampfli (1969), as follows. It had long been recognized that death following severe hemorrhage was due not so much to loss of red cells as to loss of ctrculatlng intravascular fired. The plasma proteins, prlmardy serum albumin, norreally maintain the proper lntravascular fluid volume. But these proteins are so depleted following severe blood loss that crystallold solutions, such as lsotomc saline or Ringer's, leak from the intravascular space rapidly after rejection, hence the search for a welltolerated substance which could substitute for the One vice of bacterial endotoxlns which has exasper- missing serum albumin. ated many an investigator is their wanton way of getThe first substitute, an alkaline gelatin solution, ting into intravenous solutions and thence into exper- was tried clinically m 1915. During World War I it imental animals, where they upset the entire physiolo- was replaced by a 6~o solution of gum acacia (gum gical applecart. Perhaps the most conspicuous upset arabic) partly because the gelatin solution gelled at ~s in temperature regulation; indeed, it was the fever low room temperatures and had to be liquefied by reduced in animals by a wide variety of rejections warming before it could flow through the that respired the term 'pyrogen' for the then-unknown administration tubing; we shall return to the gelling causatwe agent. Endotoxlns are potent; the febrile re- problem later. During World War II, the Germans sponse can be elicited in rabbits by the intravenous developed a synthetic plasma substitute, polyvmylpyrinjection of as httle as 1 ng of endotoxm, or 1000 rolidone (PVP) with a weight average mol. wt of killed bacterial cells, per kg of body weight (Work, about 50,000. This PVP, and others of lower mol. 1971). The ubiquity of the gram-negative bacteria wt, have been in clinical use throughout the world which produce pyrogens, the small dose which suffices under such trade names as Penston (German) and to cause marked fever and other physiological upsets, Subtosan (French). Macrodex, a 6~o solution of dexand the widespread experimental and clinical use of tran later developed in Sweden, is also m world-wide the intravenous route, combine to make pyrogenlc use Dextran, a polyglucose formed as a by-product contamination an annoyance to the investigator who by bacterml synthesis during the refining of sucrose, is aware of this vice, a pitfall for the investigator who has a mol wt of about 70,000 is not, and a serious hazard to patients. This report After World War II the use of gelatin revived as relates a specific example of such an annoyance and the result of the molecular modificahon approach inhazard, how it was overcome by a serendipitous dis- troduced by an imposing Caltech group (Campbell, covery made by Dan Campbell 30 yr ago, and some Koepfll, Pauhng, Abrahamsen, Dandhker, Feigen, useful consequences since then. Lanni and LeRosen, 1951). At least three types of Campbell's discovery arose early during the Cal- modified gelatin are now in climcal use: oxypolygelatech development of oxypolygelatin as a 'blood sub- tin (OPG; Gelifundol), succlnylated "modified fluid stitute' intended to be particularly suitable for replac- gelatin" (MFG; Plasmagel; Phys~ogel), and gelatin ing severe blood loss in the wounded under battlefield peptides cross-hnked with hexamethylene di-isoconditions. A number of natural macromolecular sub- cyanate (Haemaccel). These preparations are consistances were in clinical use as plasma substitutes m dered to have the same therapeutic effect as dextran the early 1940's. Their early history and recent status, but offer the following advantages over dextran and pharmacologic inertness, detailed m Lundsgaard-Hansen et al (1969), is sum- polywnylpyrrohdone, 625
626
ARTHUR CHERKIN
absence of side effects in doses of several hters; no lmpaxrment o1 hemostasls; absense ol antigemclty, and conversion of the non-excreted portion to amino acids by the body's proteases (H~issig and Stampfll, 1969) More recently, the chemical moddicatlon of gelatin pioneered by Campbell et al. (1951) has also been extended to a condensation product of peroxidetreated gelatin with beef serum, polymerlzed with formaldehyde then cleaved (Pnstoupll and Ndd, 1972) and to a solution of gelatin depolymenzed by heating and then condensed with ethylene bls chloroformate (Watanabe, et al., 1972) We may now return to the period of World War II and the gelling problem. It was already known that 5?/o gelatin solutaon was safe and effective but the fact that ~t gelled at room temperature rendered ~t unsuitable for emergency and military field use. The Caltech group embarked upon a molecular engineering study of gelatin modification, to reduce the melting point of the infusion solution while preserving or improving its desirable properties. The final procedure had two steps: (l) condensation of gelatin with glyoxal, and (2) oxld~ation of the condensation product by hydrogen peroxide to form fragments of statable molecular size (Mn ~ 21,000), shape and properties. The pyrogen problem arose early during preliminary physiological experiments on various plasma expanders, carried out m collaboration with Drury and Mehl at the University of Southern California It was found that gelatin solutions caused a marked febrile response when injected intravenously into rabbits. The usual interpretation of such a febrile response is contanunatlon with pyrogens, potent endotoxms produced by gram-negative bacteria (Atkms, 1960; Wostenholme and Birch, 1971). These ublqmtous contammants of water are also often in any product such as gelatin, which comes into contact with pyrogenic water during the manufacturing process. The pyrogens under discussion are now known to be bacterial endotoxms, the complex, high mol. wt hpopolysaccharldes which form part of the plasma membrane layer of the cell wall of gram-negative bacteria (Ltidentz et al., 1971) Their complex molecular structure is presumably a virtue, as Ivan Bennett viewed it, in the eyes of investigators who embrace chemical challenge. The lipopolysaccharades occur as a complex with proteins and cephahn-type phosphohplds, representing the somatic O-antigen and the endotoxm of gram-negative bacterm. Since thousands of distinct serotypes have been identified, each producmg one specific hpopolysaccharide, there are thousands of pyrogens The Salmonella lipopolysaccharldes are long chain, phosphate-containing heteropolymers composed of three regions: I, O-specific chain, II, basal core; and III, Lipid A. In a Salmonella llpopolysaccharide, the O-specific chain may be built up of many sugar constituents, including glucose, galactose, glucosamlne, L-glycero-D-mannoheptose, and keto-deoxyoctonic acid, plus other hexoses, 6deoxy-hexoses, dldeoxy-hexoses, ammo-dxdeoxy-hexoses, and pentoses. (The trlvml names of these sugars cry out for incorporation into a Gdbert-and-Sulhvan patter song: fucose, rhamnose and tyvelose; paratose, abequose, and cohtose; glucosamme, mannosamlne, wosamme; and fucosamlne, rhamnosamme, thomosamine!j
The basal core (Region II) may comprise a pentasaccharide chain [N-acetyl-D-glucosamine; D-glucose; D-galactose; t-glycero-D-mannoheptose; 2-keto-3deoxy-D-mannooctonic acid (KDO)] attached to a heptose-KDO backbone containing L-glycero-D-mannoheptose, phosphate, and ethanolamlne The Lipid A (Region III) of Salmonella contains D-glucosamme (20~), esterphosphate phosphorus (~ 2~o), and fatty acids (~ 60~), the latter include laurlc, myrlstlc, palmltic, D-fl-hydroxymyristlc, and other acids, linked to hydroxy and amino groups of glucosamine A tentative structure for the whole hpopolysaccharlde of S typhonurium Is provided by Luderltz et al (1971, Fig. 24). Bacterial pyrogens are non-dmlyzable, markedly heat-resastant and extremely potent; for example, a purified llpopolysaccharide from Escherlchia coh causes fever in rabbit or man at a dose of less than l ng per kg of body weight (Atkms, 1960). Since pyrogens survive heat-sterilization which destroys bacteria and bacterial spores, a sterile intravenous solution may retain sufficient pyrogens to cause fever and chills as obvious symptoms and to induce myriad other pathological changes Characteristic endotoxlc reactions in addition to pyrogenlclty include: release of endogenous pyrogens, lmmunogenicity, inhibmon of antibody production, leukopema and leukocytosis, changes m blood clotting, metabolic changes, endocrmologlcal changes, release of hastamme, vascular effects, and shock (Nowotny, 1969). Severe pyrogenic reactions can be fatal. Mild pyrogemc reactions m man are self-hmmng and the threatening effects fade after several hours but they confuse interpretation of the patient's status and response to therapy during that time. Prior to the introduction of commercial pyrogen-tested intravenous solutions, febrile reactions with hospital-prepared intravenous solutions were so frequent as to limit their use severely. (A coincidence which interests me is that Caltech's magnificent Donald E. Baxter, M.D, Hall of the Humanities and Social Sciences, a hundred yards from Dan Campbell's lair m the Church Laboratories, owes Its existence largely to Dr. Baxter's success in solving the pyrogen problem when he pioneered the commercial preparation of Intravenous solutions in 1929. It was also an the laboratories founded by Dr Baxter that the preparation of oxypolygelatm solutions for clinical trial, the quantitative pyrogen studies to be described below, and some of the studies on gelatlon were carried out while I served there as Director of Research). It was obvaous that the pyrogens m gelatin solutions had to be removed or destroyed. Johnson and Nowotny (1964) cite numerous chemical procedures, published since 1859, to dlmimsh or destroy pyrogemcity by. heat trea*~ment; hydrolysis w.th mild acetic acid or mild alkah; treatment w~th oxidizing agents; acetylation; hydroxylaminolysis; or reductlve cleavage by lithium aluminum hydride. Some of the methods available in the 1940's and acceptable for treating solutions for clinical use were satisfactory for simple solutions of dextrose (D-glucose) or electrolytes but not for more complex solutions. For example, adsorptive filtration was inefficient for removing pyrogens from solutions of insulin (Francke and Rees, 1943) or protein hydrolysates (Zittle et al., 1945). But Campbell noticed that those gelatin solutions which
Destruction of Bacterml Endotoxln Pyrogeniclty
627
had received permanganate or hydrogen peroxlde but there was no opportunity to pursue this hne of treatment were not pyrogenic. It was this observation research at the time This may have been fortunate, which prowded the clue to an effective, elegantly sim- m view of the complexities of endotoxin structure unple resolution of the pyrogen problem. The hydrogen covered in the ensuing three decades. As pointed out peroxide procedure would be safe because the only by Johnson and Nowotny (1964), oxidation and the decomposluon products are water and oxygen. Thus, other chemical treatments known to destroy pyrogens the procedure would also be applicable to any solu- are all unspecific. They studied treatments of presumtion not containing lngre&ents attacked by hydrogen ably greater specificity, namely, transesterification by peroxide boron trifluorlde, deacylation by potassmm methyCampbell's observation was confirmed by a quanta- late, and &ssociation by py(idmmm formate All three taUve study of the effect of hydrogen peroxide upon agents reduced endotoxl/a pyrogemclty but the pyrogenic solutions of gelatin and of potent crude authors concluded that "the nature of the hnkage &sbacterml pyrogen preparations (Campbell and Cher- rupted by the three procedures, which leads to detoxlkin, 1945). Pyrogen content was assayed by the rise ficatlon, is as yet unknown". It may be significant m rectal temperature of rabb~ts measured 1, 2 and that all these procedures spht ester-bound carboxyhc 3 hr after m lect~on, compared to the basehne tempera- acids from model compounds and that the fatty acid ture measured w~thln 0-5 hr prior to injection. Any contents of the treated products were reduced (Work, increase of 0'6°C or more was regarded as a posltwe 1971), inasmuch as it has been asserted that the pyrotest for pyrogens. The dose was 10 ml of solution per gemc effect of a bacterial endotoxln is locahzed an kg of body weight, injected into the lateral ear vein the Lipid A moelty (Liaderltz et al., 1971; Galanos A 5~o gelatin solution, autoclaved at 116°C for et al., 1972) This claim, however, has been questioned 20 mln, caused temperature rises of 0'60 ~, 0"75° and (Mdner, Rudbach and Rlbl, 1971) 0.85°C m three rabbits, indicating that the pyrogemDespite the fact that it still remains to be shown city was clearly resistant to thermal destruction. In- how hydrogen peroxide attacks the pyrogemc moelty clusion of 0.04 M H202 prior to autoclaving abol- of bacterial endotoxins, the rehabfllty and utdlty of ished the febrile response completely (0-00°, 0.15 ~, Campbell's procedure remains firmly estabhshed. 0.20°C). In numerous pdot-plant runs later, we found that 0.02 M H202 resulted m consistently non-pyrogenic batches. At 100°C for 2 hr, the effect of 0"01 M H202 was borderline but 0 1 M H202 was fully effecREFERENCES tive. Similar results were obtained wxth the two potent crude preparations of pyrogen from Pseudomonas aeruomosa W~th one, heated for 2 hr at 100°C, the tem- Atklns E. (1960) Physiol Rev. 40, 580. Bennett I L (1964) cited by Work (1971). perature r~ses in three rabbits were 0 70 °, 1.50° and Campbell D H. and Cherkln A (1945) Scwnce 102, 535. 1.40°C; inclusion of 0.01 M H202 reduced the tem- Campbell D H., Koepfll J. B., Pauhng L., Abrahamsen perature changes to - 0 10°, - 0 I0 ° and 0"15°C. The N., Dandhker W, Felgen G A, Lanm F. and LeRosen second crude pyrogen was rendered nonpyrogenlc by A (1951)Tex Rep. Blol Med 9, 235 heating with 0.1 M H 2 0 : at 100°C for I hr. Galanos C, Rletschel E T, Liidentz O and Westphal O (1972) Eur J. Blochem 31,230 The results of the quantitative study led us to suggest that the hydrogen peroxide treatment m~ght be Gunther D. A (1969) U.S. Patent No. 3,440,157, cited m Chem Abs 71. 6463r (1969) of practical use m certain cases for rendering solutions nonpyrogemc. Subsequently, the effectiveness of Hasslg A. and Stampfll K (1969) Modified Gelatins as Plasma Substitutes, (Edited by Lundsgaard-Hansen P, peroxide treatment was confirmed by Taub and Hart Hhsslg A and Nitschmann Hs.), p 1 Karger. New York. (1948) for water and for sahne and dextrose solutions Johnson A G. and Nowotny A. (1964) J. Bact 87, and was adopted m Israel for the safe production 809 of such nonpyrogemc solutions from pyrogemc &s- Luderltz O, Westphal O., Staub A. M and Nlkaldo H. tilled water (Menczel, 1951). For these simple solu(1971) Mlcroblal Toxins, Vol IV, Bacterial Endotoxms tions, excess peroxide was removed by treatment with (Edited by Wembaum G, Kadls S and AjI S J), p 145. Academic Press, New York manganese dioxide or actwated charcoal The basic principle of oxidative destruction of pyrogens also Lundsgaard-Hansen P, H~isslg A and Nltschmann Hs (1969) Modified Gelatins as Plasma Substitutes Karger, appears in a process for removing pyrogenicity from New York water by electrolytic oxidation (Gunther, 1969). Menczel E. (1951) J Am pharm Assoc 40. 175 Sovak et al. (1972) treated gelatin wlth 0.28 M Mllner K C, Rudbach J A and Rlbl E (1971) Mwrobml H202 at 50°C to remove potentially present pyrToxins (Edited by Wembaum G., Ka&s S and mjl S ogens, as part of a procedure to purify gelatin for J ), Vol IV, p" 1. Academic Press, New York ad&tion to an angiographxc contrast medium; any Nowotny A (1969) Bact Rev. 33, 72 excess peroxide would be removed by the subsequent Pnstoupd T I. and Nlkl J (1972) Czechoslovak Patent No 145,552, cited in Chem Abs 78, 61266c (1973) alcohol precipitation and drying of the purified gelatin. It would appear that the peroxide step could be Sovak M. Lang J H and Sovak M (1972) Invest Radiol 7, 16 applied to advantage in the purification of other reagents which are not affected by hydrogen peroxide Taub A and Hart F (1948) J Am. pharm. Ass 37, 246 Watanabe T et al (1972) Japan Patent No. Kokai 72 Furthermore, peroxide treatment might prove useful 23,522, cited m Chem Abs 78, 20184b (1973) in certain cases, for distinguishing physiological re- Wolstenholme G. E W and Birch J (1971) Pyro#ens and sponses to pyrogenlc contamination from responses Fever Churchdl Lwmgstone, Edinburgh. to an injected substance itself. Work E (1971) Pyrogens and Fever (Edited by WolstenAn obvious question of interest to us was the holme G E W and Birch Jk P 23. Churchill Livingstone, Edinburgh mechanism of the peroxide effect upon pyrogemclty
Pergamon Press
Pnnted in Great Britain
DESTRUCTION OF BACTERIAL E N D O T O X I N PYROGENICITY BY H Y D R O G E N PEROXIDE ARTHUR CHERKIN Psychobtology Research Laboratory, Veterans Administration Hospital, Sepulveda, Cahforma 91343, U S A and Departments of Anesthesiology and Psychiatry, University of Cahforma School of Me&cme, Los Angeles, Cahfornla 90024, U S A (Recewed 16 September 1974)
Abstract--The early development of oxypolygelatm as a plasma substltute was hampered by the presence m gelatin of pyrogemc bacterial endotoxms, which are complex hpopolysaceharldes known also as Boiwn anugens. Heat-sterdlzatmn reduced but did not ehmlnate the pyrogenlclty Campbell's observatton that gelatin solutions oxidized w~th hydrogen peroxide were nonpyrogemc prowded a safe, simple resolution of the problem The peroxide technique also destroyed the pyrogeniclty of potent crude bacterial endotoxms The procedure was successfully apphed to the large-scale production of oxypolygelatm for chmcal use and has since been extended to solutions of sodmm chloride and dextrose, and to other apphcat~ons
"Endotoxms possess an intrinsic fascination that is nothing less than fabulous They seem to have been endowed by Nature wtth wrtues and vices in the exact and glamorous proportions needed to render them irresistible to any investigator who comes to know them They intrigue the chemist The molecular basis for their biological action seems always on the verge of discovery but somehow .lust eludes detection." (Bennett. 1964) "No other natural product is known which would ehclt such a great variety of reactions as do endotoxlns when injected into the proper host" (Nowotny, 1969)
manzed by H~sslg and Stampfli (1969), as follows. It had long been recognized that death following severe hemorrhage was due not so much to loss of red cells as to loss of ctrculatlng intravascular fired. The plasma proteins, prlmardy serum albumin, norreally maintain the proper lntravascular fluid volume. But these proteins are so depleted following severe blood loss that crystallold solutions, such as lsotomc saline or Ringer's, leak from the intravascular space rapidly after rejection, hence the search for a welltolerated substance which could substitute for the One vice of bacterial endotoxlns which has exasper- missing serum albumin. ated many an investigator is their wanton way of getThe first substitute, an alkaline gelatin solution, ting into intravenous solutions and thence into exper- was tried clinically m 1915. During World War I it imental animals, where they upset the entire physiolo- was replaced by a 6~o solution of gum acacia (gum gical applecart. Perhaps the most conspicuous upset arabic) partly because the gelatin solution gelled at ~s in temperature regulation; indeed, it was the fever low room temperatures and had to be liquefied by reduced in animals by a wide variety of rejections warming before it could flow through the that respired the term 'pyrogen' for the then-unknown administration tubing; we shall return to the gelling causatwe agent. Endotoxlns are potent; the febrile re- problem later. During World War II, the Germans sponse can be elicited in rabbits by the intravenous developed a synthetic plasma substitute, polyvmylpyrinjection of as httle as 1 ng of endotoxm, or 1000 rolidone (PVP) with a weight average mol. wt of killed bacterial cells, per kg of body weight (Work, about 50,000. This PVP, and others of lower mol. 1971). The ubiquity of the gram-negative bacteria wt, have been in clinical use throughout the world which produce pyrogens, the small dose which suffices under such trade names as Penston (German) and to cause marked fever and other physiological upsets, Subtosan (French). Macrodex, a 6~o solution of dexand the widespread experimental and clinical use of tran later developed in Sweden, is also m world-wide the intravenous route, combine to make pyrogenlc use Dextran, a polyglucose formed as a by-product contamination an annoyance to the investigator who by bacterml synthesis during the refining of sucrose, is aware of this vice, a pitfall for the investigator who has a mol wt of about 70,000 is not, and a serious hazard to patients. This report After World War II the use of gelatin revived as relates a specific example of such an annoyance and the result of the molecular modificahon approach inhazard, how it was overcome by a serendipitous dis- troduced by an imposing Caltech group (Campbell, covery made by Dan Campbell 30 yr ago, and some Koepfll, Pauhng, Abrahamsen, Dandhker, Feigen, useful consequences since then. Lanni and LeRosen, 1951). At least three types of Campbell's discovery arose early during the Cal- modified gelatin are now in climcal use: oxypolygelatech development of oxypolygelatin as a 'blood sub- tin (OPG; Gelifundol), succlnylated "modified fluid stitute' intended to be particularly suitable for replac- gelatin" (MFG; Plasmagel; Phys~ogel), and gelatin ing severe blood loss in the wounded under battlefield peptides cross-hnked with hexamethylene di-isoconditions. A number of natural macromolecular sub- cyanate (Haemaccel). These preparations are consistances were in clinical use as plasma substitutes m dered to have the same therapeutic effect as dextran the early 1940's. Their early history and recent status, but offer the following advantages over dextran and pharmacologic inertness, detailed m Lundsgaard-Hansen et al (1969), is sum- polywnylpyrrohdone, 625
626
ARTHUR CHERKIN
absence of side effects in doses of several hters; no lmpaxrment o1 hemostasls; absense ol antigemclty, and conversion of the non-excreted portion to amino acids by the body's proteases (H~issig and Stampfll, 1969) More recently, the chemical moddicatlon of gelatin pioneered by Campbell et al. (1951) has also been extended to a condensation product of peroxidetreated gelatin with beef serum, polymerlzed with formaldehyde then cleaved (Pnstoupll and Ndd, 1972) and to a solution of gelatin depolymenzed by heating and then condensed with ethylene bls chloroformate (Watanabe, et al., 1972) We may now return to the period of World War II and the gelling problem. It was already known that 5?/o gelatin solutaon was safe and effective but the fact that ~t gelled at room temperature rendered ~t unsuitable for emergency and military field use. The Caltech group embarked upon a molecular engineering study of gelatin modification, to reduce the melting point of the infusion solution while preserving or improving its desirable properties. The final procedure had two steps: (l) condensation of gelatin with glyoxal, and (2) oxld~ation of the condensation product by hydrogen peroxide to form fragments of statable molecular size (Mn ~ 21,000), shape and properties. The pyrogen problem arose early during preliminary physiological experiments on various plasma expanders, carried out m collaboration with Drury and Mehl at the University of Southern California It was found that gelatin solutions caused a marked febrile response when injected intravenously into rabbits. The usual interpretation of such a febrile response is contanunatlon with pyrogens, potent endotoxms produced by gram-negative bacteria (Atkms, 1960; Wostenholme and Birch, 1971). These ublqmtous contammants of water are also often in any product such as gelatin, which comes into contact with pyrogenic water during the manufacturing process. The pyrogens under discussion are now known to be bacterial endotoxms, the complex, high mol. wt hpopolysaccharldes which form part of the plasma membrane layer of the cell wall of gram-negative bacteria (Ltidentz et al., 1971) Their complex molecular structure is presumably a virtue, as Ivan Bennett viewed it, in the eyes of investigators who embrace chemical challenge. The lipopolysaccharades occur as a complex with proteins and cephahn-type phosphohplds, representing the somatic O-antigen and the endotoxm of gram-negative bacterm. Since thousands of distinct serotypes have been identified, each producmg one specific hpopolysaccharide, there are thousands of pyrogens The Salmonella lipopolysaccharldes are long chain, phosphate-containing heteropolymers composed of three regions: I, O-specific chain, II, basal core; and III, Lipid A. In a Salmonella llpopolysaccharide, the O-specific chain may be built up of many sugar constituents, including glucose, galactose, glucosamlne, L-glycero-D-mannoheptose, and keto-deoxyoctonic acid, plus other hexoses, 6deoxy-hexoses, dldeoxy-hexoses, ammo-dxdeoxy-hexoses, and pentoses. (The trlvml names of these sugars cry out for incorporation into a Gdbert-and-Sulhvan patter song: fucose, rhamnose and tyvelose; paratose, abequose, and cohtose; glucosamme, mannosamlne, wosamme; and fucosamlne, rhamnosamme, thomosamine!j
The basal core (Region II) may comprise a pentasaccharide chain [N-acetyl-D-glucosamine; D-glucose; D-galactose; t-glycero-D-mannoheptose; 2-keto-3deoxy-D-mannooctonic acid (KDO)] attached to a heptose-KDO backbone containing L-glycero-D-mannoheptose, phosphate, and ethanolamlne The Lipid A (Region III) of Salmonella contains D-glucosamme (20~), esterphosphate phosphorus (~ 2~o), and fatty acids (~ 60~), the latter include laurlc, myrlstlc, palmltic, D-fl-hydroxymyristlc, and other acids, linked to hydroxy and amino groups of glucosamine A tentative structure for the whole hpopolysaccharlde of S typhonurium Is provided by Luderltz et al (1971, Fig. 24). Bacterial pyrogens are non-dmlyzable, markedly heat-resastant and extremely potent; for example, a purified llpopolysaccharide from Escherlchia coh causes fever in rabbit or man at a dose of less than l ng per kg of body weight (Atkms, 1960). Since pyrogens survive heat-sterilization which destroys bacteria and bacterial spores, a sterile intravenous solution may retain sufficient pyrogens to cause fever and chills as obvious symptoms and to induce myriad other pathological changes Characteristic endotoxlc reactions in addition to pyrogenlclty include: release of endogenous pyrogens, lmmunogenicity, inhibmon of antibody production, leukopema and leukocytosis, changes m blood clotting, metabolic changes, endocrmologlcal changes, release of hastamme, vascular effects, and shock (Nowotny, 1969). Severe pyrogenic reactions can be fatal. Mild pyrogemc reactions m man are self-hmmng and the threatening effects fade after several hours but they confuse interpretation of the patient's status and response to therapy during that time. Prior to the introduction of commercial pyrogen-tested intravenous solutions, febrile reactions with hospital-prepared intravenous solutions were so frequent as to limit their use severely. (A coincidence which interests me is that Caltech's magnificent Donald E. Baxter, M.D, Hall of the Humanities and Social Sciences, a hundred yards from Dan Campbell's lair m the Church Laboratories, owes Its existence largely to Dr. Baxter's success in solving the pyrogen problem when he pioneered the commercial preparation of Intravenous solutions in 1929. It was also an the laboratories founded by Dr Baxter that the preparation of oxypolygelatm solutions for clinical trial, the quantitative pyrogen studies to be described below, and some of the studies on gelatlon were carried out while I served there as Director of Research). It was obvaous that the pyrogens m gelatin solutions had to be removed or destroyed. Johnson and Nowotny (1964) cite numerous chemical procedures, published since 1859, to dlmimsh or destroy pyrogemcity by. heat trea*~ment; hydrolysis w.th mild acetic acid or mild alkah; treatment w~th oxidizing agents; acetylation; hydroxylaminolysis; or reductlve cleavage by lithium aluminum hydride. Some of the methods available in the 1940's and acceptable for treating solutions for clinical use were satisfactory for simple solutions of dextrose (D-glucose) or electrolytes but not for more complex solutions. For example, adsorptive filtration was inefficient for removing pyrogens from solutions of insulin (Francke and Rees, 1943) or protein hydrolysates (Zittle et al., 1945). But Campbell noticed that those gelatin solutions which
Destruction of Bacterml Endotoxln Pyrogeniclty
627
had received permanganate or hydrogen peroxlde but there was no opportunity to pursue this hne of treatment were not pyrogenic. It was this observation research at the time This may have been fortunate, which prowded the clue to an effective, elegantly sim- m view of the complexities of endotoxin structure unple resolution of the pyrogen problem. The hydrogen covered in the ensuing three decades. As pointed out peroxide procedure would be safe because the only by Johnson and Nowotny (1964), oxidation and the decomposluon products are water and oxygen. Thus, other chemical treatments known to destroy pyrogens the procedure would also be applicable to any solu- are all unspecific. They studied treatments of presumtion not containing lngre&ents attacked by hydrogen ably greater specificity, namely, transesterification by peroxide boron trifluorlde, deacylation by potassmm methyCampbell's observation was confirmed by a quanta- late, and &ssociation by py(idmmm formate All three taUve study of the effect of hydrogen peroxide upon agents reduced endotoxl/a pyrogemclty but the pyrogenic solutions of gelatin and of potent crude authors concluded that "the nature of the hnkage &sbacterml pyrogen preparations (Campbell and Cher- rupted by the three procedures, which leads to detoxlkin, 1945). Pyrogen content was assayed by the rise ficatlon, is as yet unknown". It may be significant m rectal temperature of rabb~ts measured 1, 2 and that all these procedures spht ester-bound carboxyhc 3 hr after m lect~on, compared to the basehne tempera- acids from model compounds and that the fatty acid ture measured w~thln 0-5 hr prior to injection. Any contents of the treated products were reduced (Work, increase of 0'6°C or more was regarded as a posltwe 1971), inasmuch as it has been asserted that the pyrotest for pyrogens. The dose was 10 ml of solution per gemc effect of a bacterial endotoxln is locahzed an kg of body weight, injected into the lateral ear vein the Lipid A moelty (Liaderltz et al., 1971; Galanos A 5~o gelatin solution, autoclaved at 116°C for et al., 1972) This claim, however, has been questioned 20 mln, caused temperature rises of 0'60 ~, 0"75° and (Mdner, Rudbach and Rlbl, 1971) 0.85°C m three rabbits, indicating that the pyrogemDespite the fact that it still remains to be shown city was clearly resistant to thermal destruction. In- how hydrogen peroxide attacks the pyrogemc moelty clusion of 0.04 M H202 prior to autoclaving abol- of bacterial endotoxins, the rehabfllty and utdlty of ished the febrile response completely (0-00°, 0.15 ~, Campbell's procedure remains firmly estabhshed. 0.20°C). In numerous pdot-plant runs later, we found that 0.02 M H202 resulted m consistently non-pyrogenic batches. At 100°C for 2 hr, the effect of 0"01 M H202 was borderline but 0 1 M H202 was fully effecREFERENCES tive. Similar results were obtained wxth the two potent crude preparations of pyrogen from Pseudomonas aeruomosa W~th one, heated for 2 hr at 100°C, the tem- Atklns E. (1960) Physiol Rev. 40, 580. Bennett I L (1964) cited by Work (1971). perature r~ses in three rabbits were 0 70 °, 1.50° and Campbell D H. and Cherkln A (1945) Scwnce 102, 535. 1.40°C; inclusion of 0.01 M H202 reduced the tem- Campbell D H., Koepfll J. B., Pauhng L., Abrahamsen perature changes to - 0 10°, - 0 I0 ° and 0"15°C. The N., Dandhker W, Felgen G A, Lanm F. and LeRosen second crude pyrogen was rendered nonpyrogenlc by A (1951)Tex Rep. Blol Med 9, 235 heating with 0.1 M H 2 0 : at 100°C for I hr. Galanos C, Rletschel E T, Liidentz O and Westphal O (1972) Eur J. Blochem 31,230 The results of the quantitative study led us to suggest that the hydrogen peroxide treatment m~ght be Gunther D. A (1969) U.S. Patent No. 3,440,157, cited m Chem Abs 71. 6463r (1969) of practical use m certain cases for rendering solutions nonpyrogemc. Subsequently, the effectiveness of Hasslg A. and Stampfll K (1969) Modified Gelatins as Plasma Substitutes, (Edited by Lundsgaard-Hansen P, peroxide treatment was confirmed by Taub and Hart Hhsslg A and Nitschmann Hs.), p 1 Karger. New York. (1948) for water and for sahne and dextrose solutions Johnson A G. and Nowotny A. (1964) J. Bact 87, and was adopted m Israel for the safe production 809 of such nonpyrogemc solutions from pyrogemc &s- Luderltz O, Westphal O., Staub A. M and Nlkaldo H. tilled water (Menczel, 1951). For these simple solu(1971) Mlcroblal Toxins, Vol IV, Bacterial Endotoxms tions, excess peroxide was removed by treatment with (Edited by Wembaum G, Kadls S and AjI S J), p 145. Academic Press, New York manganese dioxide or actwated charcoal The basic principle of oxidative destruction of pyrogens also Lundsgaard-Hansen P, H~isslg A and Nltschmann Hs (1969) Modified Gelatins as Plasma Substitutes Karger, appears in a process for removing pyrogenicity from New York water by electrolytic oxidation (Gunther, 1969). Menczel E. (1951) J Am pharm Assoc 40. 175 Sovak et al. (1972) treated gelatin wlth 0.28 M Mllner K C, Rudbach J A and Rlbl E (1971) Mwrobml H202 at 50°C to remove potentially present pyrToxins (Edited by Wembaum G., Ka&s S and mjl S ogens, as part of a procedure to purify gelatin for J ), Vol IV, p" 1. Academic Press, New York ad&tion to an angiographxc contrast medium; any Nowotny A (1969) Bact Rev. 33, 72 excess peroxide would be removed by the subsequent Pnstoupd T I. and Nlkl J (1972) Czechoslovak Patent No 145,552, cited in Chem Abs 78, 61266c (1973) alcohol precipitation and drying of the purified gelatin. It would appear that the peroxide step could be Sovak M. Lang J H and Sovak M (1972) Invest Radiol 7, 16 applied to advantage in the purification of other reagents which are not affected by hydrogen peroxide Taub A and Hart F (1948) J Am. pharm. Ass 37, 246 Watanabe T et al (1972) Japan Patent No. Kokai 72 Furthermore, peroxide treatment might prove useful 23,522, cited m Chem Abs 78, 20184b (1973) in certain cases, for distinguishing physiological re- Wolstenholme G. E W and Birch J (1971) Pyro#ens and sponses to pyrogenlc contamination from responses Fever Churchdl Lwmgstone, Edinburgh. to an injected substance itself. Work E (1971) Pyrogens and Fever (Edited by WolstenAn obvious question of interest to us was the holme G E W and Birch Jk P 23. Churchill Livingstone, Edinburgh mechanism of the peroxide effect upon pyrogemclty
