Aschauer H, Grob A, Hildebrandt J, Schuetze E, Stuetz P
Sandoz Forschungsinstitut, Vienna, Austria.
J Biol Chem. 1990 Jun 5;265(16):9159-64.
Lipid X, an early precursor in the biosynthesis of lipid A has been reported to directly induce cytokine release in macrophages but also to inhibit endotoxin-induced tumor necrosis factor (TNF) induction. In this report we provide evidence that these conflicting results could be due to contaminants present in different batches of lipid X used. Thus, in an apparently pure batch of crystalline lipid X as obtained by a published procedure (Macher, I. (1987) Carbohydr. Res. 262, 79-84) small amounts of N,O-acylated disaccharide-1-phosphates could be identified. Their isolation was achieved by gel filtration on Sephadex LH-20 and further analysis of fractions showing elevated limulus amebocyte lysate values by thin layer chromatography and reverse-phase high performance liquid chromatography (HPLC) in combination with bioassays. Identification of immunostimulatory by-products was possible by testing HPLC-fractions for TNF-induction in bone marrow-derived mouse macrophages. Applying these procedures a disaccharide-1-phosphate, containing four 3(R)-hydroxymyristic acids at positions 2, 3, 2', 3', was identified as the main immunostimulatory side product. Two isomeric hydrolysis products of this compound with only three 3(R)-hydroxymyristic acid moieties attached to the disaccharide-1-phosphate were also identified. Surprisingly, these compounds behave quite differently in the TNF induction test. The disaccharide-1-phosphate, acylated at positions 2, 2', 3', is a very potent inducer of TNF-release whereas the corresponding isomer containing the 3(R)-hydroxymyristic acids in positions 2, 3, 2', does not induce TNF release, but strongly inhibits TNF release as induced by the former compound. Thus, contamination of "pure" lipid X with immunostimulatory or immunoinhibitory impurities may explain the divergent pharmacological profiles which were attributed to synthetic lipid X.
脂质X是脂质A生物合成中的早期前体，据报道它可直接诱导巨噬细胞释放细胞因子，同时还能抑制内毒素诱导的肿瘤坏死因子（TNF）生成。在本报告中，我们提供的证据表明，这些相互矛盾的结果可能是由于所用不同批次脂质X中存在污染物所致。因此，按照已发表的方法（Macher, I. (1987) Carbohydr. Res. 262, 79 - 84）获得的一批看似纯净的结晶脂质X中，可鉴定出少量的N,O - 酰化二糖 - 1 - 磷酸酯。通过在Sephadex LH - 20上进行凝胶过滤实现其分离，并通过薄层色谱和反相高效液相色谱（HPLC）结合生物测定法对显示鲎试剂值升高的馏分进行进一步分析。通过检测HPLC馏分对骨髓来源的小鼠巨噬细胞中TNF生成的影响，有可能鉴定出免疫刺激副产物。应用这些方法，一种在2、3、2'、3'位含有四个3(R) - 羟基肉豆蔻酸的二糖 - 1 - 磷酸酯被鉴定为主要的免疫刺激副产物。还鉴定出了该化合物的两种异构体水解产物，它们的二糖 - 1 - 磷酸酯上仅连接有三个3(R) - 羟基肉豆蔻酸部分。令人惊讶的是，这些化合物在TNF生成试验中的表现差异很大。在2、2'、3'位酰化的二糖 - 1 - 磷酸酯是TNF释放的非常有效的诱导剂，而在2、3、2'位含有3(R) - 羟基肉豆蔻酸的相应异构体不诱导TNF释放，但强烈抑制前一种化合物诱导的TNF释放。因此，“纯净”脂质X被免疫刺激或免疫抑制杂质污染可能解释了归因于合成脂质X的不同药理学特征。
