CTS Endotoxin & Glucan Reference Information
WHAT IS ENDOTOXIN?
Endotoxin is a component of the cell wall in Gram negative bacteria.
Specifically, endotoxin is a major component of the outside portion of
the outer membrane of the Gram negative cell wall. Endotoxin is a very
strong pyrogen and is considered a bacterial toxin.
Endotoxin is a lipopolysaccharide (LPS). There are two major parts to
this molecule, the polysaccharide and lipid A. The polysaccharide is not
toxic. It is hydrophilic and faces outward from the cell membrane and
into the medium in which the bacteria is growing. It contains major antigens
of the bacteria and is the most important target of Gram-negative bacteria
for the immune response. Each species of Gram negative bacteria makes
its own unique LPS molecule. The polysaccharide portion of the molecule
is highly variable.
Lipid A is the toxic component of LPS. Lipid A is composed of a disaccharide
(glucosamine) containing phosphate groups and fatty acids. The fatty acid
portion of the molecule is very hydrophobic and faces inward toward the
middle of the outer membrane. This causes LPS to behave in a similar manner
as phospolipids, which make up most membranes. There is much less variability
among bacteria in the composition of lipid A and it is mainly found in
the fatty acid chains.
Why test for endotoxin? Endotoxin causes adverse reactions in humans.
If endotoxins are present in drugs, devices, or other items placed inside
the body, they can cause illness. Drugs taken orally do not need to be
tested for endotoxin. Bacteria do not need to be alive for endotoxin to
be toxic. In fact, endotoxin in live bacteria is less toxic than when
the bacteria are dead because endotoxin from dead cells is released, thereby
increasing the apparent dose.
Endotoxin is relatively heat stable. It will survive common heat-based
disinfection and sterilization procedures. Hence, sterile items are not
necessarily free from endotoxin.
A pyrogen is any compound that gives rise to
a pyrogenic reaction, which is characterized by fever. Endotoxin is the
most potent natural pyrogen and a potential health risk to humans. In
large doses, endotoxins can cause shock and death.
There are three classes of bacterial
toxins. Endotoxins are named “endo” because they are part of the cell
wall and not released in large quantities unless the cell is destroyed.
Endotoxins are made by only Gram-negative bacteria. Exotoxins are actively
secreted from bacteria while they are alive and growing. Enterotoxins
are secreted in the intestine and cause diarrhea and dysentery.
REGULATORY ASPECTS OF ENDOTOXIN TESTING
The FDA approves limits for exposure and testing methods for endotoxin.
The USP, in conjunction with the EP and JP, refine testing methods for
There are several major regulatory documents that describe how drugs,
devices, dialysate, water, and other substances are to be tested for endotoxin.
The limits apply for drugs and devices administered to humans or animals.
A limit listed for a compendial drug takes precedence over calculated
- limit for parenteral drugs: 5.0 EU/kg. Assume a 70 kg human adult,
unless administering drugs to children.
- limit for drugs being injected intrathecally: 0.2 EU/kg.
- limit for devices: 20 EU/device
- limit for devices being used intrathecally: 2.15 EU/device
WHAT IS GLUCAN?
Glucans are a class of polysaccharides. Specifically, they are chains
of glucose that are usually branched and sometimes cross-linked. They
are part of the cell wall in fungi (yeasts and molds), algae, some bacteria,
and plants, where they contribute mechanical strength and integrity to
EFFECTS OF GLUCAN
(1→3)-ß-D-Glucans are immunomodulators. Folk remedies from many
cultures use mushrooms to aid healing. It is believed that the healing
effect is attributable to the glucans in the mushrooms. There are many
types of (1→3)-ß-D-glucans, and physical and chemical properties
of the molecule affect its biological activities. (1→3)-ß-D-glucans
are known to bind to and activate macrophages, neutrophils, monocytes,
and NK cells. They are believed to bind to a variety of cells, eg, endothelial
cells and fibroblasts.
Among the biological effects caused by glucans is the production of cytokines,
which are important components in inflammation. Other activities associated
with glucans are nitric oxide synthesis, activation of the complement
cascade, and activation of lymphocytes and macrophages.
There is evidence from animal studies that glucan and endotoxin can act
synergistically to increase the inflammation response. In some studies,
this synergy has been used to treat cancer.
For manufacturers of drugs and devices, (1→3)-ß-D-glucans is considered
a contaminant. Although the FDA does not regulate this immunostimulatory
contaminant, it can be of concern because of two reasons. One, the presence
of (1→3)-ß-D-glucans can give a falsely higher reading in the LAL
assay for endotoxins. This could produce and Out-Of-Specification result.
Two, (1→3)-ß-D-glucans can contribute to adverse reactions in patients.
Common sources of glucans experienced by drug and device manufacturers
are filters made from cellulose materials, plant-derived raw materials,
cotton-containing enclosures, sugars, naturally-derived raw materials,
cellulose products (eg, sponges), etc.
Examples of products found to contain glucan contamination include blood
products (albumin, plasma protein, immunoglobulin preparations, coagulation
factors), oligonucleotide drugs, collagen products, saline preparations,
glucose preparations, and water for injection.
There are two approaches to testing for glucan. One, the subtraction
method, uses two assays for endotoxin, one of which has a blocking substance
to reduce the response of the assay to glucan in the sample. The difference
between the two results is proportional to the amount of glucan present.
Two, a direct measurement of (1→3)-ß-D-glucans. A direct measurement
assay is specific for (1→3)-ß-D-glucans. It is quantitative, specific,
and much more accurate than the subtraction method. The only available
versions of this assay are called Glucatell®
both are available only from Associates of Cape Cod, Inc. For more information
click on the links below.
Glucan Interference in Samples
buffer should be used when (1→3)-ß-D-glucan contamination is suspected to exist. ACC recommends using Glucashield®
when the sample contains or comes in contact with any of the possible sources of (1→3)-ß-D-glucan. A Glucatell®
assay should be performed first to confirm the presence of (1→3)-ß-D-glucan. However, the decision to have the Glucatell®
assay performed and the use of Glucashield®
buffer with any sample is the client's responsibility. For more information click on the links below.
COMMON TESTING PROBLEMS
Inhibition is characterized by a Positive Product Control (PPC) being
recovered at less than 50% in a chromogenic or turbidimetric assay or
a failure of the 2λ PPC to clot. Inhibition can also cause a result to
be lower than it should be. There are several possible causes for this:
- The pH. The LAL assay works best between pH 6.0 to
8.0. The further the pH of the testing solution is from this range,
the more inhibition will be seen. Check the pH of the sample to make
sure it is within the proper range. If the sample is close, addition
of Pyrosol, a buffer agent, will help bring the sample into the right
range. Other samples will require neutralization.
- Organic solvents. Organic solvents inhibit the assay
by inhibiting the activity of enzymes. Dilute the sample with water.
- Precipitates. Precipitates will adversely affect
the chromogenic assay because the light path will be blocked. Dilute
- Salt. If the concentration is too high, the enzymes
of the assay will be unable to work efficiently. Dilute the sample.
Enhancement is characterized by a Positive Product Control (PPC) being
recovered at more than 200% in a chromogenic or turbidimetric assay. Most
gel-clot assays cannot detect enhancement. In the Inhibition-Enhancement
assay, enhancement in a gel-clot assay is assumed when the 0.25λ Product
Standard clots. Enhancement can also cause results to be reported as higher
than they should be. There are several possible causes for this:
- Proteolytic enzymes. The LAL assay is a series of
serine protease enzymes. Serum, plasma, and other biological samples
may have enzymes that can cause the reaction to proceed, even when no
endotoxin is present. An example is Trypsin. Heat the sample to denature
the enzymes is an easy way to remove this interference.
- Glucans. (1→3)-ß-D-glucans may enhance the
assay by activating an alternative pathway in the LAL assay. Glucans
are biologically active compounds that can cause effects similar to
endotoxin. There are two ways to determine if glucans are causing enhancement.
One is to use an agent that blocks glucan. If the endotoxin result is
lower, then glucan was causing enhancement. Two is to use Glucatell™
which is specific for (1→3)-ß-D-glucans and can quantify the amount
of glucans present in the sample.
- Surfactants. Surfactants can reduce the size of endotoxin
aggregates, thereby making the endotoxin standard in the PPC tube appear
as if there were a higher level of endotoxin present. Conversely, too
much surfactant can inhibit the ability of enzymes to work efficiently,
which will appear as inhibition. Dilute the sample.