Kevin Williams - Senior Scientist, bioMérieux
Take a minute to look outside. Hopefully you see some trees, grass, and dead leaves in the fall. What you likely don’t see but is everywhere as well are fungi and yeast on the plants and in the soil. This all drains into earth’s waters (lakes, seas, rivers, etc.) as foliage dies and is recycled. The waters also contain algae, lichens, and the ocean contains seaweed and plankton. All of these things mentioned (plants and fungi etc.) use β glucans and mannans and cellulose as cell wall forming molecules. These are ubiquitous molecules that predominate the earth much in the same way that gram negative bacterial endotoxin forms the outer layer of membrane for them and exist then as residue in soil and water.
Since it was discovered in 19811 that some glucans gave false positive activation of LAL (Factor G pathway) users have been advised by LAL manufacturers to use the “glucan blocking buffer” that they developed to overcome the effects. The suggestion has always been that the blocking is thoroughly effective. A second false positive revelation then included cellulosic materials from pharmaceutical drug filters2 . The former glucan is described as a β, 1,3, D-glucan and cellulosic residues are characterized as β, 1,4 D-glucans from drug manufacturing filters. LAL manufacturers are not incentivized to find false positive reactive substances for their products and no new LAL reactive substances have been identified since the 1980’s although older references include, besides β glucans and cellulose: polynucleotides, proteins3 and mannans4. Before discussing data generated on LAL-reactive substances, the structures of glucans, an overview of water systems, and LAL-rFC comparison studies are briefly overviewed.
Structures
A little background is in order in regard to the structure of glucans. Glucan structural variants affect molecular function in several ways including: (a) type of monomers which appear as α or β monomers (determined by the placement of the hydroxyl on carbon number 1 and whether it is on the same side or the other side of the number 6 carbon group as shown below) that are used to form the polymers (b) the polymer backbone type (1>3, 1>4, 1>6 etc.) and (c) the branching (location and composition) on the linear backbone. Plants and algae also contain cellulose, 1>3 and 1>4 β glucans as well as mannans.5 Mannans are polymers of mannose rather than glucose.
Comparison Studies and Pharmaceutical Water Systems
LAL-rFC comparison studies relevant to the pharmaceutical test environment (post filtration water, purified water, drugs, raw materials) have generally revealed very close correlation of results (Kikuchi7, Bolden8, Mozier9, Marius10, etc.). However, there is one study that demonstrated greater recovery using LAL (only one type of LAL was used-kinetic chromogenic) versus rFC but this water was pre-filtration water where one would expect β glucans, cellulosic residues, mannans etc. Industry has debated as to whether or not this was a fair comparison. The lack of specificity of LAL should detract from LAL rather than to be used in attempts to disqualify endotoxin specific reagents.
Modern Data on LAL Non-Endotoxin Reactivity
“Modern” data refers to the dated nature of most LAL non-endotoxin reactivity studies. The most recent was done by Roslansky and Novitsky in 1991 12. They studied the major types of LAL that still exist (chloroform extracted versus that using zwittergent) and found that chloroform extracted LALs are more sensitive to β glucans and cellulosic materials. LAL manufacturers rarely reference these studies and typically suggest users just “use the blocking buffer” to solve any glucan associated testing issues.
Glucan determining tests are also sometimes used to demonstrate that specific samples are free (or not) of glucans. However, these tests are specific to β-1,3 D-glucans and cannot detect β-1,4 glucans or mannans and their utility to detect degradants of complex natural plant and fungal constituents has never been demonstrated. A typical description is given here as an example of one such commercially available test online (product name redacted and underline added for emphasis):
The _______® assay kit is specific for (1→3)-β-D-glucan. The assay is based upon a modification of the Limulus Amebocyte Lysate (LAL) pathway. The ________® reagent is processed to eliminate Factor C, and is therefore specific for (1→3)-β-D-glucan. The reagent does not react to other polysaccharides, including β-glucans with different glycosidic linkages.
Other methods that detect a wider variety of fungal polysaccharides have been developed but not applied to natural water tests using LAL. 13
One has to wonder, given the horseshoe crab’s non-terrestrial existence in the sea and on the shore, just what is Factor G seeking to detect and prevent in the animal? There is some evidence that ancient algal blooms/marine fungi have been harmful to horseshoe crabs and could cover the carapace and invade the blood steam so the detection via reagents that employ the LAL Factor G pathway (minus Factor C) may be geared more toward algal/marine fungal type β-glucans and thus less sensitive to terrestrial derived β-glucans that would be expected to inhabit soil (plant and fungi) and therefore fresh water from soil runoff.
Three questions arise that will serve to confirm or deny the current paradigm in using β glucan blocking buffers:
- Can we really block all β-glucans by just using a β glucan blocking buffer (βGBB)?
- Do chrome and turb LAL methods give the same result in the presence of β-glucans?
- Are there other microbial polymer sugars that react with LAL?
The first question we approached in two different ways: (a) using a fairly clean natural water from two different retention ponds and testing it via LAL and rFC followed by treatment with (i) βGBB and (ii) βglucan and cellulosic enzymes that attack and break glycosidic bonds. For the second question we looked at simply testing purified sugars with both chromogenic and turbidimetric methods (all samples were negative via use of rFC). For the third question we used mannan which is not a β glucan and is a predominate sugar paired with β glucans in many fungi and plant cell wall structures. Two different mannans were used- one from plants and one from yeast.
1. Can we really block all β-glucans by just using a β glucan blocking buffer (βGBB)?
This test was repeated with nearly identical results using a second water source and has now been repeated with similar results in several independent labs. The original data was published in American Pharmaceutical Review15 and the method used is detailed there. The testing clearly shows that initial values tested with LAL are much higher than rFC and values are reduced with βGBB but not by much.
Using the enzymes served to reduce the values to around the values originally obtained with rFC thus demonstrating the false reactivity of LAL with non-endotoxin substances in the natural water.
A more recent study (this year) was performed using specific highly purified purchased sugars to try and identify which of the constituents that may be present in natural water were more (or less) reactive with LAL. Sample commercial sugars of the highest purity available and labeled <1 EU/mL and as tested by human TLR4-expressing HEK293 cells and as tested negative with rFC. It would be impossible to look at all the different sugar types or all the polymer sugar degradation variants in nature (even if they were all known) of course but a small sampling may give us an idea of non-endotoxin specific LAL reactivity.
Various highly purified sugars were purchased and reconstituted using purified water to 1 mg/mL with heat and vortexing for an extended period. Subsequently the solutions were diluted 1:100 or 1:1000 using purified water for testing. βGBB was purchased commercially and used as per package insert instructions.
As shown below the use of βGBB does generally significantly reduce the amount of false reactivity of various sugars with LAL, however, the significant residual amounts for most of the sugars, except curdlan, suggests that the use of βGBB is insufficient to produce results suitable for LAL-rFC comparison studies. These residual values will add systematically higher recovery for LAL relative to rFC.
Specific purified sugars show high reactivity with LAL (no reactivity with rFC, so not shown) and some reduction with βGBB, yet a significant residual amount of reactivity with all sugars except curdlan which was well blocked. Curdlan has served as the prototypical sugar for demonstrating βGBB utility.
2. Do chrome and turb LAL methods give the same result in the presence of β-glucans?
As seen above, chromogenic and turbidimetric methods have distinctly different reactions with non-endotoxin active sugars and the use of one LAL will not necessarily demonstrate that another LAL will behave similarly. As shown below, for zymosan and pustulan chromogenic gives much higher results than turbidimetric whereas for curdlan turbidimetric gives much higher results than chromogenic. This data suggests that when non-endotoxin reactants are present including β glucans, then LAL reagents cannot give a “gold standard” result by which rFC as an endotoxin specific reagent can be honestly compared.
3. Are there other microbial polymer sugars that react with LAL?
The answer to this question is already contained within the two previous graphs. Mannans, plant and yeast derived, both show significant LAL reactivity and no rFC reactivity. Oddly though, the mannans do seem to give very close reactivity with both turbidimetric and chromogenic when tested side by side.
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