Toxicity purified lipid

Figure 1 shows the scheme for the preparation of purified lipid A from endotoxin. S. typhimurium G30/C21 was extracted by the method of Galanos t aK (24) and submitted to one of two different conditions of hydrolysis (a) 0.1 N HC1 [in methanol-water (1 1, v/v)], 100 °C, 45 min, to yield the crude monophosphoryl lipid A (nontoxic), and (b) 0.02 M sodium acetate, pH 4.5, 100 °C for 30 min (two cycles) to yield the crude diphosphoryl lipid A (toxic). The 0.1 N HC1 hydrolysis product was fractionated on a Sephadex LH-20 column (23). Each of these fractions was then separated by preparative thin layer chromatography (TLC) on silica gel H (500 ym), with the solvent system chloroform-methanol-waterconcentrated ammonium hydroxide (50 25 4 2, v/v) as previously described (23) to yield TLC fractions 1-7 and 1-9 respectively. 

Toxicity and Tumor Regression Activity of Purified Lipid A... 

Table XI. Toxicity and Tumor Regression Activity of Purified Lipid A Obtained from S. typhimurium G30/C21 ...

Le Page, KN., Cheeseman, K.H., Osman, N. and Slater, T.F. (1988). Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the toxicity of carbon tetrachloride. Cell Biochem. Function 6, 87-99. 

Crude and three diethyl ether extracted, acetone treated, fractions were isolated from large-scale cultures of Gambierdiscus toxicus. Crude extracts at. 04 mg/ml inhibited the histamine contraction response in smooth muscle of the guinea pig ileum. Three semi-purified fractions at 5 ng/ml, effectively inhibited the guinea pig ileum preparation. Two of these fractions followed Michaelis-Menten kinetics for a competitive inhibition. The third fraction inhibited in a non-reversible manner. This study has established the presence of three lipid extracted toxins in toxicus, outlined a method for their assay in small quantities, and identified at least two of the effects of these toxic extracts in animals. 

While purified LPS displays potent immunostimulatory properties, it also induces various toxic side effects (Table 10.24), the most prominent of which is pyrogenicity. These effects render application of LPS as an adjuvant unacceptable. Both its immunostimulatory and toxic properties are mainly associated with the lipid A portion of the molecule. Attempts have been made to chemically or otherwise alter the lipid A portion in order to ameliorate the observed toxicity. 

When purified endotoxin was treated with 0.1 N HC1 for 30 min at 100 °C, the resulting nondialyzable residue was also free of KDO but was nontoxic (CELD50, >10 yg) and nonpyrogenic (FI401 20 yg) (Table IV). We have designated this product nontoxic lipid A (Detox) and demonstrated its low toxicity in four animal species (Table II). The nontoxic lipid A in combination with P3 and ACP (or CWS) retained a degree of tumor regressive potency (80% cures) similar to that observed with the toxic lipid A (88% cures) and the purified endoxtoxin (81% cures). 

Results of chemical analysis showed that the glucosamine and total fatty acid contents of the KDO depleted, toxic lipid A and the nontoxic lipid A were essentially the same but that the nontoxic lipid A was significantly lower in the phosphorus content (Table V). The molar ratio of glucosamine phosphorus fatty acids was 2 2 4 for the toxic lipid A and 2 1 4 for the nontoxic lipid A. The relative molar distribution of normal fatty acids (lauric, myristic, and palmitic acids) and the 3-hydroxymyristic acid did not indicate a correlation between the content of these components and toxicity. The nontoxic lipid A possessed as high a tumor regression activity when combined with CWS as did the purified... 

We showed the existence of a toxic and a nontoxic lipid A fraction in the acid hydrolyzed endotoxin preparation (23). These two fractions were separated and the composition was determined on the purified components so that we could relate specific structural features of lipid A to both toxicity and tumor regression activity (23). 

The results of the biological tests carried out on the purified monophosphoryl lipid A fractions are shown in Table XI. The chick embryo lethality test showed that both TLC-1 and -3 were nontoxic, whereas TLC-5 exhibited some toxicity. These results indicate that there might be two levels of toxicity based on structure. The presence or absence of the sugar 1-phosphate group (and possibly some other unknown group) would control the upper... 

Purified mineral oils have been used medicinally and in foods. Subchronic toxicity studies of selected mixtures of mineral oil hydrocarbons (composed primarily of branched chain alkanes or cyclic alkanes) in F344 rats have identified the liver and the mesenteric lymph nodes as potential targets of toxicity for these mineral oils. The TPHCWG (1997c) derived chronic RfDs for low and high molecular weight mineral oils based on the hepatic effects (lipid granulomas) seen in these studies. 

MPL (monophosphoryl lipid A) Although the effectiveness of LPS as an immunomodulator has long been known, the pharmacologic use of purified LPS (or lipid A) as an adjuvant is precluded by its toxicity. LPS is highly... 

Nevertheless, LAM has been studied often, as it is the most abundant surface carbohydrate of M. tuberculosis. The purified LAM-oligosacchar-ides, deprived of lipid-associated toxicity, and covalently linked to different proteins induce both cell-mediated and humoral responses in mice, rabbits and guinea-pigs. Immunized mice and guinea-pigs were protected against experimental infection with virulent tubercle bacilli to the same degree as immunization with the control live attenuated BCG... 

Unfractionated saponins caused moderate to severe local inflammation following s.c. injection into mouse footpad. One strategy for reducing systemic toxicity and local reactogenicity of complex mixtures, such as Quil A, is to mix the saponin with a lipid carrier such as ISCOM, or to select purified saponins with low toxicity, such as QS-21 (3). In general, most adjuvant formulations are combinations of immunostimulants and vehicle but QS-21 (3) is typically used in the absence of a... 

Briefly, the most satisfactory isolation procedure was as follows 1) extraction of minced skins twice with 70% aqueous methanol, followed by evaporation in vacuo to yield a dry tan residue 2) trituration with 0.9% NaCl, adjustment of pH to 2 with HCl, followed by extraction of lipids into chloroform 3) adjustment of pH to 8.5 with aqueous NH3, followed by extraction of toxic principles into chloroform 4) after evaporation to dryness in vacuo the active principles were purified by thin-layer chromatography on silica gel with chloroform-methanol (6 1). Two iodoplatinate-positive alkaloid spots were detected the higher Rf material accounted for virtually all of the toxicity. In view of subsequent studies this higher Rf... 

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