By: Dr. Harriet Burge and Dave Gallup

Endotoxin
Endotoxins are bacterial products that are ubiquitous in the environment and, although they do take part in some disease processes, exposure at some level is probably essential for the normal development of the human (and animal) immune systems.

Click here for more information about your immune system. The immune system is a host of defence system.This is made up of several biological structures and processes in the organism. It helps to protect one from diseases. Even a unicellular organism, say for example a bacteria, has an elementary immune system, this is present in the enzyme form.

The nature of endotoxin
Bacteria produce two general types of toxins: exotoxins and endotoxins. Exotoxins are soluble proteins that are excreted from the bacterial cell. Anthrax, tetanus, botulism, and toxic shock syndrome are a few of the diseases caused by bacterial exotoxins. Endotoxins, on the other hand, are lipopolysaccharides (LPS) that are part of the bacterial cell structure. The toxin is minimally soluble and effects occur directly from contact with the bacteria. Endotoxins are produced by Gram negative bacteria such Escherischia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, and many others. All Gram negative bacteria have lipopolysaccharides as part of the cell wall. However, the lipopolysaccharides are not always toxic and those that are vary in potency.

Health Effects
Endotoxins are potent inflammatory agents, stimulating the release of many chemicals that activate the immune system. In high concentrations, such as would occur during a Gram negative bacterial infection, fever, inflammation, coagulation of platelets, hemorrhage, and shock may result. At the much lower concentrations encountered during inhalation exposures, inflammation is the primary outcome.

In some work environments in which organic material is handled or large amounts of water are used (e.g., swine confinement, fiberglass manufacturing) work-related respiratory illnesses may occur. These illnesses are characterized by fever, chills, muscle aches, shortness of breath, and cough. These symptoms may be worse on Monday and may gradually abate during the week, only to return following a weekend of no exposure. In residential and office environments, effects are less clear. Some studies relate dust or air levels of endotoxin to respiratory inflammation.

In residential environments, endotoxin exposure appears to be related to a decrease in the risk of developing asthma. This effect is called the hygiene hypothesis, which considers that the reduction in exposure to endotoxin caused by increasing standards for home hygiene has led in part to the rising incidence of asthma.

Sampling and measurement technologies

Analytical methods

The most commonly used analytical method for endotoxin detection and measurement is the Limulus amoebocyte assay. This assay measures endotoxin in units of toxic activity as measured by the ability to clot the blood of Limulus (the horseshoe crab). Results from this assay are highly dependent on the lot of Limulus lysate being used, and the assay is affected by many factors, including materials that may be present in a sample. In general, this means that data from different laboratories cannot be directly compared and, even within a laboratory, data from different days or different batches of chemicals are difficult to compare. The ACGIH Bioaerosols Committee has proposed a relative standard for airborne endotoxin based on the Limulus assay that requires outdoor measurements for comparison. The comparisons are made on data produced by the same assay using the same lot of reagents.

Total lipopolysaccharides can be measured using gas chromatography. This method is stable and interlaboratory comparisons can be made. However, the method measures both toxic and non-toxic lipopolysaccharides. Since the non-toxic forms may be immunostimulants, this approach may actually be the most relevant in the long run. LPS is measured in concentration of the actual compound (e.g., nanograms/gram of dust). Endotoxin is measured in units of potency-endotoxin units (EU). In some literature, endotoxin concentrations are listed as nanograms (ng) or micrograms (ug)/ unit of measure. In these cases, either GCMS has been used to measure total LPS, or the authors are referring to units of the reference LPS standard used in the assay. However, because the type of lipopolysaccharide in the unknown sample may differ from the standard, EU is the preferred unit for Limulus assay data.

Sampling methods
Samples for endotoxin analysis can be collected from air or dust. For air samples, endotoxin-free membrane filters can be used. For dust, a variety of vacuum-based methods have been used with variable results. It is important to use the method by which any reference data that is to be used for comparison was collected.

Distribution in the environment

Outdoor air
Endotoxin is always present in outdoor air. Measured levels have ranged from 0.01-more than 5 EU/m³ of outdoor, with geometric means generally in the 0.02-0.5EU/m³. Endotoxin concentrations in outdoor air near farming activities can be much higher, reaching levels in excess of 1,000 EU/m³. In residential and other supposedly “clean” environments, measured levels of airborne endotoxin are rare. Concentration ranges for the few available studies are in the range 0.5-2,500 EU/m³, with the higher part of the range possibly indicating unusually high levels. Most residential measures are dust concentrations, which range from 8,000-250,000 EU/g of dust. Repeated measurements of endotoxin in residential dust tend to be correlated. The presence of a dog in the house tends to be strongly correlated with endotoxin concentrations in dust.

Most of the data on endotoxin exposure comes from occupational environments where organic material or water is used in the manufacturing process. In these types of environments, extremely high endotoxin concentrations have been measured. A summary of measured levels are listed in Table 1.

Air Samples	EU/m³ of air
Outdoors	0.01-5
Residential
Office	.5-3; 350-460; 2500
Aircraft	1.5
Outdoor Feedlots (USA)	26-83
Soybean harvest	460-4438
Farms	2534-3175
Machining fluid mist	16-234
Fiberglass manufacturing	139000-278000

Table 1

Role in environmental investigations
In occupational environments where organic material is handled, or water is a part of the manufacturing process, airborne endotoxin measures are important and air samples should be collected in both the occupational environment and in outdoor air. There is a large amount of literature on such occupational exposures, and reference to the medical literature is an important component of interpretation. The National Library of Medicine is a free and very useful resource: www.pubmed.org.

There is little indication that endotoxin levels in residential and office environments are likely to be related to ongoing problems. Measuring endotoxin in house dust has been done often enough that there is reference data that can be used for interpretation of whether levels are low, moderate, or high. How the data is interpreted with respect to health is less clear.

Lipopolysaccharides from BuyReagents.com 

Liquid ( aqueous solution )
Lipopolysaccharide from E.coli O157	1ml	400,000 EU/ml	01772-41	-20ºC
Lipopolysaccharide(LPS) from Salmonella serover paratyphi-A(O antigen 2+)	1ml	400,000 EU/ml	02453-91	-20ºC
Lipopolysaccharide(LPS) from Salmonella serover paratyphi-B(O antigen 4+)	1ml	400,000 EU/ml	02454-81	-20ºC
Lipopolysaccharide(LPS) from E.coli O2	1ml	400,000 EU/ml	02452-01	-20ºC
Lipopolysaccharide(LPS) from E.coli O18	1ml	400,000 EU/ml	02451-11	-20ºC
Lipopolysaccharide(LPS) from E.coli O145	1ml	400,000 EU/ml	02450-21	-20ºC
Lipoarabinomannan (LAM) from M.TB Aoyama-B	1ml	1mg/ml	02449-61	-20ºC
Lyophilized powder
Lipopolysaccharide (LPS) from E.coli O2	10mg	500,000 EU/mg	20386-34	2-8ºC
Lipopolysaccharide (LPS) from E.coli O18	10mg	500,000 EU/mg	20387-24	2-8ºC
Lipopolysaccharide (LPS) from E.coli O145	10mg	500,000 EU/mg	20388-14	2-8ºC
Lipopolysaccharide (LPS) from E.coli O157	10mg	500,000 EU/mg	20389-04	2-8ºC
Lipopolysaccharide (LPS) from E.coli O55:B5	100mg	500,000 EU/mg	20382-74	2-8ºC
Lipopolysaccharide (LPS) from E.coli O111:B4	100mg	500,000 EU/mg	20383-64	2-8ºC
Lipopolysaccharide (LPS) from E.coli O127:B8	100mg	500,000 EU/mg	20384-54	2-8ºC
Lipopolysaccharide (LPS) from E.coli O128:B12	100mg	500,000 EU/mg	20385-44	2-8ºC