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Lipopolysaccharide (LPS) is a bacterial endotoxin and a component of the bacterial cell wall (Nowotny 1969; Elin and Wolff 1976) and induce potent pathophysiological effects in mammals. LPS is an amphipathic molecule consisting of a polysaccharide chain and a fatty acid-containing lipid A component (Shands et al. 1967; Westphal 2009), which represents the active centre responsible for the endotoxic properties of LPS, such as lethality, fever, induction of cytokine production and coagulation of horseshoe crab haemolymph (Lüderitz et al. 1982). LPS bioactivities are thought to be greatly affected by the particle size or state of the LPS complex in aqueous media (Ribi et al. 1962, 1966; Roberson and Cromartie 1962; Milner et al. 1963; McIntire et al. 1967; Tarmina et al. 1968; Komuro et al. 1987). The lipid A component of LPS consists of two conjugated glucosamine residues attached to a variable number of long chain fatty acids and 1 phosphoryl group on each carbohydrate (Rietschel et al. 1984). This distinctive structure is necessary for the bioactivity of LPS, and omission of 1 phosphoryl group significantly reduces this bioactivity, with the monosaccharide structure being the least active (Rietschel et al. 1994).
The limulus amebocyte lysate (LAL) test was introduced by Levin and Bang (1968) and is based on an endotoxin-induced coagulation reaction in amebocyte lysates (Levin and Bang 1964). The bacterial endotoxins test (BET) is a simple, highly sensitive assay for determining endotoxin levels and is listed in the American, European and Japanese Pharmacopoeia for use in detecting endotoxin contamination in parenteral drugs (US Pharmacopeia 2012; European pharmacopoeia 2010; The Japanese Pharmacopoeia 2011). An endotoxin-induced coagulation reaction is triggered by binding of endotoxins to factor C present in LAL, and both the ionic bond contributed by the negative charge of the phosphoryl group or hydrophobic interactions with the fatty acid of lipid A are important for this coagulation.
Some substances interfere with the BET, preventing accurate determination of endotoxin levels present in a drug sample (Twohy et al. 1984; Ogawa et al. 1993). However, few studies have investigated the mechanisms through which such substances interfere with the BET due to the large size and microheterogeneity of LPS, especially in length and composition of its terminal glycan chains. We previously reported that some substances, including iron sulfate, strongly affect the LAL reaction and act on endotoxins directly, altering the state of endotoxin complexes in solution; such interference could be minimized by preparing sample solutions in saline, D-PBS, BES, or Tris buffers instead of water (Fujita et al. 2011).
We now report a qualitative and structural analysis of lipid A after inactivation by the addition of iron sulfate. We sought to elucidate the mechanisms through which iron sulfate interferes with lipid A function because this substance strongly and directly acts on endotoxins (Fujita et al. 2011). To this end, we analysed the negative charge, hydrophobic interactions, complex formation and structures of inactivated lipid A by size-exclusion high-performance liquid chromatography (HPLC), anion-exchange HPLC and reverse-phase liquid chromatography/mass spectrometry (LC/MS), respectively. Moreover, to investigate the involvement of the phosphoryl group of lipid A on the interfering action of iron sulfate, we compared the sensitivity of lipid A to iron sulfate with that of monophosphoryl lipid A (MPLA) prepared by acid hydrolysis of lipid A. Finally, we used an enzyme-linked immunosorbent assay (ELISA)-based binding assay to verify the iron sulfate–dependent binding activity of lipid A with factor C, a protease present in LAL that participates in the coagulation reaction and interacts with lipid A (Tan et al. 2000).
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- Materials and methods
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In the present study, we sought to determine the mechanism through which iron sulfate interferes with lipid A activity and to develop BET methods to suppress this interference. Iron sulfate acts strongly and directly on endotoxins and is contained in various items and environments, such as injection needles; thus, this substance is one of the most influential interfering factors for the detection of endotoxin. We clearly demonstrated that iron sulfate-induced cleavage of lipid A at the glycosidic bond and hydrophilic/hydrophobic imbalance, which in turn reduced LAL coagulation activity. Our study also suggested that, because access of interfering factors to the lipid A phosphoryl group and the resulting cleavage of lipid A was a key event in interference, prevention of these actions would be highly effective in suppressing this interference event.
Principle factors interfering with the BET include suboptimal pH conditions, aggregation or adsorption of endotoxins, unsuitable cation concentrations, enzyme or protein modification, β-glucan and samples containing endotoxin (Cooper 1990; Duner 1993; Cooper et al. 1997; Williams 2007). Furthermore, we reported that some substances, such as metal ions, antibiotics, and anticancer drugs, directly affect endotoxins and produce a strong interfering action (Fujita et al. 2011). Despite these previous reports concerning interference with the LAL coagulation activity of LPS, the detailed mechanisms of these interference events remained unclear.
In this study, direct evidence for the interfering action of iron sulfate on lipid A was provided by the following experimental observations: (i) reverse-phase LC/MS showed cleavage at the glycosidic bond of lipid A containing iron sulfate, and such cleaved fragments were eluted faster than intact lipid A due to lack the capacity to participate in hydrophobic interactions (data not shown); and (ii) an increase in the peak area of corresponding lipid A complexes was observed at the same time as the onset of LAL coagulation, and this peak could be disrupted by iron sulfate, suggesting that the lipid A structure, its hydrophobic interactions, and formation of complexes were important factors for lipid A activity. In addition, lipid A containing 0·5 mmol l−1 iron sulfate, which has no coagulation activity, accounted for about 32% of intact lipid A in reverse-phase LC/MS, and this finding suggested that the activity of intact lipid A was suppressed by the fragments of lipid A cleaved at the glycosidic bond.
In contrast, the negative charge of lipid A was not affected by the presence of iron sulfate. The phosphoryl group and the negative charge it carries are important for lipid A bioactivity. Indeed, MPLA, which lacks this phosphoryl group, had weaker activity than lipid A. In LAL tests with lipid A and MPLA, lipid A had higher sensitivity to iron sulfate than did MPLA, supporting the role of the phosphoryl group in iron sulfate sensitivity. However, anion-exchange HPLC analysis indicated that the negative charge of lipid A in the presence of iron sulfate was unaltered in spite of the disappearance of peak A corresponding lipid A in size-exclusion HPLC; these findings indicated that the negative charge was sustained in lipid A containing iron sulfate. Therefore, the observed disruption of lipid A complex and interference with LAL coagulation activity were likely not due to changes in the lipid A negative charge status. Furthermore, reverse-phase LC/MS demonstrated that MPLA was infrequently cleaved at the glycosidic bond by iron sulfate. From these results, we hypothesize that the lipid A phosphoryl group has an important role in LAL coagulation activity and is involved in the interference event. For example, the Fe2+ ion may be attracted to lipid A due to its negative charge, and the glycosidic bond of lipid A will then be cleaved by the attracted Fe2+ ion; thus, iron sulfate would not be required to act on the phosphoryl group directly such as dephosphorylation. In addition, the interfering action of iron sulfate in the BET was minimized by using buffers (Fujita et al. 2011), and the attraction of iron sulfate to lipid A could likely be similarly inhibited by the high ionic strength supplied by buffers.
Lipid A-factor C binding activity was previously analysed by ELISA and surface plasmon resonance (Tan et al. 2000; Ariki et al. 2004), and these studies demonstrated that this binding event was necessary to trigger the gelation reaction. In the ELISA-based binding assay performed here, absorbance corresponding to factor C binding to lipid A was observed and was consistent with previous studies. However, iron sulfate–inactivated lipid A also had a similar degree of factor C binding. These results suggested that, in the presence of iron sulfate, lipid A retained its factor C binding activity, despite the inactivation of LAL coagulation, implying that lipid A-factor C binding alone was insufficient to trigger the LAL coagulation reaction. Moreover, the formation of the lipid A complex is thought to be necessary for some additional actions on factor C.
The most important factor for LAL coagulation activity is likely the formation of lipid A complexes resulting from its amphipathic characteristics, because disruption of lipid A complexes coincided with the reduction in LAL coagulation activity. In the results of size-exclusion HPLC, two peaks were observed for sonicated lipid A; however, only peak A was produced by the sonication of lipid A and disappeared by the addition of iron sulfate in a concentration-dependent manner. In addition, such production and disappearance of peak A coincided with a change in the LAL coagulation activity of lipid A. Therefore, we speculate that it is important for LAL coagulation activity of lipid A to form a particular size of complex corresponding with peak A. LPS forms a micelle complex in solution because of these amphipathic properties, and the amphipathicity of lipid A allows it to form micelle-like complexes in solution that are maintained by a balance between hydrophilic moieties, including the glucosamine residue, phosphoryl group, and hydrophobic subunits (i.e., fatty acids). In addition, a previous study reported that lipid IVA, a bioactive precursor of lipid A, also forms micelles or vesicles (Hofer et al. 1991). In this study, cleavage at the glycosidic bond was observed for inactivated lipid A, and the disappearance of the relevant peak in size-exclusion HPLC suggested that the amphipathic complex was disrupted by a hydrophilic/hydrophobic imbalance induced by cleavage at this glycosidic bond. Therefore, we speculate that cleavage at this glycosidic bond may be the result of direct action of iron sulfate on lipid A, generating the resulting lipid A lacking the ability to form hydrophobic interactions and thereby disrupting complexes (Fig. 7).
Figure 7. Proposed mechanism of iron sulfate–induced inactivation of lipid A limulus amebocyte lysate coagulation activity. ( ) Normal lipid A; ( ) Cleaved lipid A.
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In conclusion, the results of the present study indicate that inactivation of the LAL coagulation reaction was induced by the disruption of lipid A complexes resulting from cleavage at the glycosidic bond and subsequent hydrophilic/hydrophobic imbalance. We used ciprofloxacin and epirubicin as other interfering factors in reverse-phase LC/MS, and cleavage at the glycosidic bond was also observed, similar to that found with iron sulfate. However, despite strong suppression of LAL coagulation activity by the addition of ciprofloxacin and epirubicin, the peak representing lipid A fragments cleaved at the glycosidic bond were only increased several fold over that of normal lipid A. Cleavage at the glycosidic bond may be an important event for interference, but ciprofloxacin and epirubicin may exert other effects on lipid A. Moreover, other substances that also act directly on lipid A, such as aluminium, copper, quinolone antibacterial agents and anticancer agents (Fujita et al. 2011), would be predicted to cleave lipid A in a manner similar to that of iron sulfate or ciprofloxacin and epirubicin, because such substances produce an irreversible interfering effect on lipid A that cannot be decreased through filtration or rinsing of LPS-substance mixed solutions. Thus, our data provide important insights into the mechanisms of lipid A complex formation and activity and have implications in the optimization and further development of the BET.