Endotoxin, also known as lipopolysaccharide (LPS) is derived from the Gram-negative bacterial outer membrane and consists of three key components; the O antigen, core oligosaccharide, and the lipid-A molecule [1
]. These components may facilitate potent activation of innate immunity when triggered by low-grade endotoxemia, as demonstrated by the lipid-A molecule’s interaction with the toll-like receptor 4 (TLR4), mediating expression of pro-inflammatory cytokines [1
]. The potentiation of a pro-inflammatory state via low presence of endotoxins may progress to the onset of inflammatory diseases [2
]. The precise levels of endotoxin capable of damaging human health remains unclear, however modestly raised levels of endotoxins in human circulation have been increasingly linked in numerous studies to poor health and diseases such as; cardiovascular disease and atherosclerosis [3
], insulin resistance and type 2 diabetes [2
], and non-alcoholic fatty liver disease [5
Currently, there are three very different analytical techniques to assess human endotoxin levels; the Limulus amebocyte assay (LAL assay), the lipopolysaccharide binding protein (LBP) assay and the endotoxin activity assay (EAA™), which we have previously demonstrated is not suitable for use under conditions of metabolic endotoxemia (ME) [5
]. The LBPenzyme-linked immunosorbent assay (
ELISA) assay provides an indirect measurement of sub-acute endotoxin which has been designed specifically for biological samples and measures an acute-phase reactant protein, predominantly produced by the liver, in response to endotoxin exposure [6
]. The biological role of LBP is to deliver endotoxin to a co-receptor CD14, facilitating an interaction between endotoxin and TLR4, triggering a signaling cascade. This ultimately results in up-regulation and expression of pro-inflammatory cytokines [7
]. The LBP has been used extensively to detect low levels of ME associated with obesity (body mass index; BMI) [9
]. LBP in serum has also been used to detect endotoxemia in inflammatory bowel conditions, pancreatitis, cirrhosis, and sepsis (reviewed in [14
]). However, the use of an indirect measurement may not be ideal for analyzing rapid changes in LPS exposure, such as postprandial endotoxemia, as the LBP response to endotoxin exposure is delayed and the accuracy of the test is dependent on normal hepatic function for production of the measured binding protein [15
In contrast, there are three LAL assays which offer a direct measurement of endotoxin through initiating a blood clotting cascade, as Limulus polyphemus clots with Gram-negative bacteria during infection [16
]. The most commonly used LAL assay is a chromogenic test. Briefly, the conversion of a pro-enzyme to its active form is catalyzed by endotoxin, which then splits p-nitroaniline (pNA) from a colorless substrate, which is quantified by the time taken for the optical density (OD) to increase by 0.2 OD at 405 nm and is quantitatively proportional to the concentration of endotoxin in the sample (Lonza, Walkersville, MD, USA). The LAL assay’s ability to assess endotoxin under conditions of sepsis is undisputed, however the manufacturer cautions that the assay was “not meant for testing blood or blood products, nor are they meant to diagnose, treat, or mitigate any disease or condition such as endotoxemia in man or animals” [17
]. This is because inhibitors of the LAL assay present in complex biological samples, such as blood, interfere with endotoxin identification, particularly at low levels. Despite this, a number of research groups have quantified acute short-term changes in plasma endotoxin in healthy individuals before and after the ingestion of a fatty meal using the LAL assay [18
]. However, none of these research groups have verified the accuracy of the determinations under postprandial ME. Additionally, there is a lack of consistency in the methodology employed under conditions of ME, namely in the variable dilution rates and heat treatments required for adequate inhibition of chemical interferences such as β-glucan [26
], or protein modification in blood [27
]. Therefore, with significant research interest on the effect of low levels of endotoxin exposure on health [29
], there is a need to establish a gold standard assay to directly test for ME, thereby advancing research the field.
The aim of this study was to evaluate whether the LAL chromogenic assay was suitable to detect endotoxins under conditions of ME and if so, to then optimize the conditions of use and validate it against BMI, an established marker of ME and an established assay under conditions of fasting. This would provide a validated direct measure of ME which could be used to evaluate postprandial changes in ME during feeding studies.
Metabolic endotoxemia is increasingly reported in the literature, yet there is no gold standard method of analysis for low levels of circulating endotoxins. Human research studies using the LAL assay often poorly report the methodology employed; sample pre-treatment conditions are often not stated [36
], referenced to manufacturer’s instructions (which are non-existent for endotoxin levels observed under conditions of ME using a Lonza kit) [37
], or are scant [18
The key finding of this study was that existing published LAL sample inhibitor neutralization pre-treatments of 70 °C and 80 °C for both 15 and 30 min, vortexed for 2 min and subsequently diluted 1:50, 1:75, 1:100, and 1:200, or diluted 1:10, heat treated to 70 °C for 10 min, vortexed for one minute and ultrasonicated for 10 min or diluted using 0.5% pyrosperse and analyzed using the LAL chromogenic assay were not suitable to detect endotoxin levels in plasma, amongst overweight men typically exhibiting ME. There are two key findings which substantiate this. Firstly, ME has long been associated with obesity. In the current study, obesity (BMI) was significantly related to the indirect measure of endotoxemia (LBP) with a moderate effect size, yet there were no relationships between BMI and the direct measure of endotoxemia (LPS) using the LAL chromogenic assay under any of the sample pre-treatment conditions examined. The positive correlation between BMI and LBP concur with many other research groups [9
], including our own [34
], and in varied ethnic populations [11
], yet surprisingly many research groups reporting on ME measures, particularly those addressing methodology, fail to report on this relationship.
Secondly, the literature commonly reports both the LAL chromogenic assay [21
] and LBP ELISA assay for quantifying ME [21
], despite the mechanisms for detection being very different [7
]. As such, one would expect to observe a direct positive relationship between both measures in the fasting state. However, this was not the case in our study. Endotoxin levels determined using the LBP ELISA assay failed to correlate with any of the 17 variants of sample pre-treatment for analysis performed using the LAL assay. Interestingly, we have previously failed to find a relationship between LBP and LPS in 10 healthy overweight and obese males in the fasting state (r = −0.341, p
= 0.369; unpublished data) and Ghamim et al. who used both LAL and LBP assays to measure ME failed to detect a fasting relationship between the two measures of endotoxemia [21
There are several possible explanations for this lack of correlation; the most likely was deactivation of endogenous sample compounds capable of interfering with the clotting cascade was incomplete [39
]. The most common methods reported for ameliorating this inhibition or enhancement have been the combination of dilution and heating [25
], solvent extraction [39
], sonication [25
], and acid treatment [41
], or combinations thereof. While the most commonly reported heat treatments, dilutions, and the use of sonication were employed in this study, the lack of correlation between LBP and LPS suggests that they were not sufficient to deactivate the inhibitors. Secondly, LBP is produced principally by the liver in response to LPS exposure, making LBP levels dependent on hepatic function, unlike the LAL assay which is a direct measure of LPS activity. In an acute exposure to a bolus of LPS, LBP is consumed by binding to LPS and transported to responding immune cells-producing a paradoxical fall in LBP rather than increase. However, as all of the men in this study were heathy, not experiencing any acute event likely to produce a bolus exposure to LPS, this pattern is unlikely to explain the lack of correlation between LBP and LAL quantified endotoxemia.
Finally, ME mirrors the activity of LPS, which in the case of the LAL analysis is based on the structure of the lipid-A moiety and polysaccharide chain [45
]. We acknowledge that various LPS species exist with numerous activities according to their molecular structure, so it is possible that the increased levels of endotoxin may be the result of an increased number of LPS molecules, or a similar level of LPS molecules with increased activity due to different chemical structures [31
]. It has also been suggested that this discrepancy could be more fully understood by assessing the biochemical forms of LPS in parallel with the LAL assay at an optimum dilution [31
]. However, while this has been successful under conditions of sepsis [46
], correlations for LPS determined by the LAL analysis and biochemical methods of analysis (HPLC-MS/MS) are still lacking under conditions of ME [47
The strengths of this study include the quality measures taken to ensure the accuracy of the polynomial model; all correlation coefficients were greater than 0.980, and the agreement between replicates (%CV) was less than 10%. The activation of the LAL test is also easily detected by a fast response in the positive product control (PPC) well. The manufacturer specifies the reactivity of such PPC should lie within 50–200% and we report on compliance to this range and only included samples adhering to this range in the analysis. Additionally, the percentage coefficient of variation between two replicate analyses of the same sample was less than 10% as recommended by the manufacturer. Care was also taken to minimize cross contamination through the use of endotoxin free reagents (LAL water), pipette tips, glass storage containers, etc. [32
], and this was confirmed through the use of ‘reagent blanks’. The type of collection tube has been shown to affect the analysis; in this study, blood was collected in heparin tubes, centrifuged within 3 h of collection and then transferred to endotoxin free glass bottles and stored at −80 °C pending analysis. While others have reported heparin tubes to increase endotoxin levels [48
] and hydrophobic polymers to bind LPS [50
], in this case, limited storage for 3 h was not found to have an impact on the analysis with no detectable level above the limit of detection.
We acknowledge several potential weaknesses in our study. Laugerette et al. suggested sonication would disperse the aggregates of LPS into smaller and more uniform particles to enhance detection [31
]. The lack of correlation between this measure of LPS and LBP suggest the conditions in our study were insufficient to completely reduce this aggregation; (Digital Pulse Swept Power operating at 43 kHz ± 2 kHz sweep bandwidth with 20 Hz pulses). The study also failed to control for the possible presence of proteases and glucans; it is possible that proteases [50
] or glucans [26
], if present in sufficient quantities in the blood, could have inhibited the clotting cascade in the LAL assay. The recovery of LPS from serum after it has bound to lipoproteins is also challenging. While some suggest that re-extraction of plasma using chloroform could be used to release LPS from inhibitory proteins and lipoproteins [52
], others report as little as ~0.001% of the spiked biological activity is recovered using this method [53
], suggesting that even after additional preparative work, the majority of LPS in the circulation residing in lipoproteins was not detectable by the LAL assay.
The LAL method of analysis is often used and importantly reported in peer reviewed literature as a measure to detect endotoxin levels under conditions of ME, despite the manufacturer not endorsing its use under these conditions. However, 17 different pre-treatment methods employed prior to the use of the LAL chromogenic assay failed to show any relationships with BMI or LBP suggesting in its current form, it is unsuitable for use for detecting levels of endotoxin typically seen in ME. LBP is the best available surrogate marker of endotoxin exposure, but cannot be used for measuring acute changes in exposure over short periods of time (minutes/hours). Furthermore, we suggest future studies employing LAL chromogenic methodologies under conditions of ME report correlations with BMI and LBP (or an alternative method of analysis) as a quality measure.
As ME is increasingly emerging as a mediator in many chronic diseases, there is an urgent need for more research to establish a robust ‘gold standard’ analytical tool to determine endotoxin levels in the systemic system, particularly with feeding studies. There are a number of emerging analytical techniques involving biosensors [54
], electron microscopy, dynamic light scattering (DLS), fluorescence resonance energy transfer (FRET), and docking programs in the endotoxin-protein analysis (reviewed in [53
]) that may offer an alternative pending investigation under conditions of ME.