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How do animals contain and dispose of bacterial endotoxins (lipopolysaccharides)?

How do animals contain and dispose of bacterial endotoxins (lipopolysaccharides)?


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Gram negative bacteria naturally release endotoxins which are lipopolysaccharide molecules. These molecules are toxic to many eukaryote cells, including macrophages. My question is how animal organisms locate, isolate, contain these toxic molecules. For example, if a lipopolysaccharide attaches to a macrophage, thus neutralizing it, does this actually kill the macrophage or just render it inert? In any case, the ineffective macrophage must now be removed as waste. How does that occur?

In the Wikipedia article on endotoxin it reads "Normal human blood serum contains anti-LOS antibodies… " without further describing them. I find this statement strange because normally when I think of antibodies, I think of them attacking cells or organisms, not individual molecules like lipopolysaccharides. What are these anti-LOS antibodies and how do they function?


There are too many questions embeded here to cover in a single answer. I'll summarize and answer what I interpret to be the "central" question.

How do animals A) locate, B) isolate, and C) contain/eliminate bacterial endotoxins?

LPS in the blood stream is rapidly bound by LPS-binding protein (LBP) and localized to the liver for detoxification and excretion (1).

A) LBP recognizes and binds the Lipid-A region of the LPS molecule (2),

B) At this point LPS is released from bacterial cell membrane and sequestered into complexes with high-density lipoprotein (HDL). This is also the stage at which free LPS can activate CD14+ monocytes, triggering an acute inflammation response (3). However, additional HDL reduces monocyte activation, suggesting that these complexes inactivate LPS by "isolating" it from CD14 (4).

C) Finally, the LPS-HDL complex is taken up by the liver, either into Kupffer Cells (~25%) or liver sinusoidal endothelial cells (~75%), where enzymatic inactivation of LPS occurs either by dephosphorylation of the phosphates or deacetylation of the primary acyl components of lipid A (5)

Given the hydrophobic characteristics of LPS lipid-A and the fact that it's processed in the liver, I suspect that LPS and its metabolites are eliminated from the body via excretion into the bile ducts, but I haven't found a reference in the literature to confirm that suspicion.

References:

  1. Wassenaar and Zimmermann, Eur J Microbiol Immunol. 2018 Sep 28; 8(3): 63-69.
  2. Tobias et al., J Biol Chem. 1989 Jun 25;264(18):10867-71.
  3. Vesy et al., Infect Immun. 2000 May; 68(5): 2410-2417.
  4. Flegel et al, Infect Immun. 1989 Jul; 57(7): 2237-2245.
  5. Yao et al. J Immunol. 2016 Sep 15; 197(6): 2390-2399.

What are the Functions of Lipids, Proteins, and Lipopolysaccharides on the Cell Membrane?

The cell membrane is an important barrier that separates the internal environment of a cell from the external environment. It separates the internal cell environment from the extracellular matrix in multicellular animals.

While it's commonly referred to as a phospholipid bilayer structure, the cell membrane also consists of several other components including proteins and carbohydrate groups.


Some infectious disease texts recognize three clinical forms of salmonellosis: (1) gastroenteritis, (2) septicemia, and (3) enteric fevers. This chapter focuses on the two extremes of the clinical spectrum—gastroenteritis and enteric fever. The septicemic form of salmonella infection can be an intermediate stage of infection in which the patient is not experiencing intestinal symptoms and the bacteria cannot be isolated from fecal specimens. The severity of the infection and whether it remains localized in the intestine or disseminates to the bloodstream may depend on the resistance of the patient and the virulence of the Salmonella isolate.

The incubation period for Salmonella gastroenteritis (food poisoning) depends on the dose of bacteria. Symptoms usually begin 6 to 48 hours after ingestion of contaminated food or water and usually take the form of nausea, vomiting, diarrhea, and abdominal pain. Myalgia and headache are common however, the cardinal manifestation is diarrhea. Fever (38ଌ to 39ଌ) and chills are also common. At least two-thirds of patients complain of abdominal cramps. The duration of fever and diarrhea varies, but is usually 2 to 7 days.

Enteric fevers are severe systemic forms of salmonellosis. The best studied enteric fever is typhoid fever, the form caused by S typhi, but any species of Salmonella may cause this type of disease. The symptoms begin after an incubation period of 10 to 14 days. Enteric fevers may be preceded by gastroenteritis, which usually resolves before the onset of systemic disease. The symptoms of enteric fevers are nonspecific and include fever, anorexia, headache, myalgias, and constipation. Enteric fevers are severe infections and may be fatal if antibiotics are not promptly administered.


HISTORY OF ENDOTOXIN

Studies about the occurrence of fever after intravenous administration of certain solutions are dated before the 19th Century. In 1894, Sanarelli showed that liquid cultures of Eberth bacillus, free from microorganisms, could produce an intoxication accompanied by fever when injected in animals, sometimes even lethal (15). By the end of the 19th Century, the designation &ldquoinjection fever&rdquo was generally used to express the fever reactions observed after intravenous administration of several solutions. The administration of pharmaceuticals via intravenous route in the 20th Century increased the number of such accidents, leading several researchers to develop series of evaluating works about this subject.

In 1912, Hort and Penfold created the name &ldquopyrogenic&rdquo to designate the &ldquowaters&rdquo which, when injected, cause &ldquohyperthermia&rdquo. Such designation was retaken further, in 1923, by Florance Seibert, who called pyrogenic the &ldquohyperthermizing&rdquo substances, which contained either dead bacteria &ndash intact or disintegrated, pathogenic or not &ndash or more often the bacterial metabolic products, such as the denaturized protein, endotoxins or exotoxins (16, 17).

The term pyrogen became popular after frequent use by Seibert, and for that reason often its creation is attributed to him (17). Seibert and co-workers continued the research started by Hort and Penfold, isolating a living Gram-negative microorganism from distillated water, which was able to produce pyrogens (15). The authors designated this microorganism as Pyrogenic bacterium, realizing that it was not a new bacterium since several varieties of microorganisms could produce pyrogens (16).

The major impulse given to increase the knowledge about pyrogens occurred between 1925 and 1945. In particular, Co-Tui, helped by Schrift, deserves special credits for showing that Gram-negative bacteria are the most dangerous producers of pyrogens. (18). It does not mean that Gram-positive bacteria cannot generate such molecules however, they do in a lower level. In fact, Gram-positive bacteria, when destroyed by heat, produce almost no pyrogen, since in such bacteria exotoxins of proteic origin are generally formed, thus being easily denaturalized by heat. On the other hand, Gram-negative bacteria usually generate endotoxins composed mainly of lipopolysaccharides and, therefore, are more heat resistant than the first ones.

According to Westphal (1945), the pyrogens, which shall really be feared in the pharmaceutical preparations, correspond to the endotoxins of Gram-negative bacteria, and such lipopolysaccharide complexes are found in the outer layer of the bacterial cell wall (19). Essentially, pyrogens are originated in the microorganisms from the Enterobactereaceae family and are thought to be the main contaminant of an injectable solution prepared without the proper disinfecting and sterilizing processes. About two decades later, a collaborative study was developed by the US National Institutes of Health and 14 pharmaceutical industries to establish an animal system which would be adequate to evaluate the &ldquopyrogenicity&rdquo of solutions. Such study culminated in the development of the first official pyrogen test in rabbits, which was incorporated in USP XII, in 1942. In parallel, other efforts to purify and characterize the endotoxins have taken place, and isolated pyrogenics were obtained by several researchers (17, 19)

Shear and Turner (1943) were the first researchers to use the term lipopolysaccharide to name the endotoxin extract, a term that describes the nature of the endotoxin, and that has been adopted by the scientific community (20). In 1954, Westphal and collaborators detailed the use of water-phenol systems for the production of purified lipopolysaccharides (LPS), free of proteins, from several Enterobacteriaceae. (15, 21, 22). Consequently, in recent years great progress has been made in understanding the molecular organization and mechanisms underlying the detrimental and beneficial activities of endotoxins (8, 23).

ENDOTOXIN: CHEMICAL AND PHYSICAL PROPERTIES

Endotoxins, also called lipopolysaccharides (LPS), are a major component of the outer membrane of Gram-negative bacteria (Figure 1). They are composed of a hydrophilic polysaccharide moiety, which is covalently linked to a hydrophobic lipid moiety (Lipid A) (Figure 2) (3, 6, 24). LPS from most species is composed of three distinct regions: the O-antigen region, a core oligosaccharide and Lipid A (LipA) (Figure 2).

Figure 1: Molecular model of the inner and outer membranes of E. coli K-12 according to Raetz et al., 1991 (24). Geometric form: ovals and rectangles represent sugar residues, as indicated, whereas circles represent polar head groups of various lipids. Abbreviation: PPEtn (ethanolamine pyrophosphate) LPS (lipopolysaccharide) Kdo (2-keto-3-deoxyoctonic acid).

Figure 2: Chemical structure of endotoxin from E. coli O111:B4 according to Ohno and Morrison 1989 (25). (Hep) L-glycerol-D-manno-heptose (Gal) galactose (Glc) glucose (KDO) 2-keto-3-deoxyoctonic acid (NGa) N-acetyl-galactosamine (NGc) N-acetyl-glucosamine.

The lipid A is the most conserved part of endotoxin (8, 26) and is responsible for most of the biological activities of endotoxin, i.e. its toxicity. Endotoxin is composed of b-1,6-linked D-glucosamine residues, covalently linked to 3-hidroxy-acyl substituents with 12-16 carbon atoms via amide and ester bonds. These can be further esterified with saturated fatty acids. This hydrophobic part of endotoxin adopts an ordered hexagonal arrangement, resulting in a more rigid structure compared to the rest of the molecule (8, 9). Strains lacking lipid A or endotoxin are not known. The core oligosaccharide has a conserved structure with an inner 3‑deoxy-D-manno-2-octulosonic acid (KDO) - heptose region and an outer hexose region. In E. coli species, five different core types are known, and Salmonella species share only one core structure. The core region close to lipid A and lipid A itself are partially phosphorylated (pK1=1.3, pK2 = 8.2 of phosphate groups at lipid A), thus endotoxin molecules exhibit a net negative charge in common protein solutions (8, 27). The O-antigen is generally composed of a sequence of identical oligosaccharides (with three to eight monosaccharides each), which are strain specific and determinative for the serological identity of the respective bacterium (8).

The molar mass of an endotoxin monomer varies from 10 to 20 kDa, owing to the variability of the oligosaccharide chain even extreme masses of 2.5 (O-antigen-deficient) and 70 (very long O-antigen) kDa can be found. It is well known that endotoxins form various supra-molecular aggregates in aqueous solutions because of their amphipathic structures. These aggregates result from non-polar interactions between lipid chains as well as of bridges generated among phosphate groups by divalent cations (1). The aggregate structures have been studied by numerous techniques such as electron microscopy, X-ray diffraction, FT-IR spectroscopy, and NMR. Results from these studies have shown that, in aqueous solutions, endotoxins can self assemble in a variety of shapes, such as lamella, cubic, and hexagonal inverted arrangements, with diameters up to 0.1 mm and 1000 kDa, and high stability depending on the solution characteristics (pH, ions, surfactants, etc) (28, 29). It is proposed that proteins may also shift equilibrium by releasing endotoxin monomers from aggregates (6, 8). According to molecular dynamics, the three-dimensional structure of endotoxin, especially the long surface antigen, is much more flexible than the globular structure of proteins (8).

Endotoxins are shed in large amount upon cell death as well as during growth and division. They are highly heat-stable and are not destroyed under regular sterilizing conditions. Endotoxin can be inactivated when exposed at temperature of 250º C for more than 30 minutes or 180º C for more than 3 hours (28, 30). Acids or alkalis of at least 0.1 M strength can also be used to destroy endotoxin in laboratory scale (17).

MECHANISM OF ENDOTOXIN ACTION

Endotoxin elicits a wide variety of pathophysiological effects, such as endotoxin shock, tissue injury, and death (3). Endotoxins do not act directly against cells or organs but through activation of immune system, especially the monocytes and macrophages, thereby enhancing immune responses. These cells release mediators, such as tumour necrosis factor, several interleukins, prostaglandins, colony stimulating factor, platelet activating factor and free radicals (31, 32). The mediators have potent biological activity and are responsible for the side effects upon endotoxin exposure. These include alterations in the structure and function of organs and cells, changes in metabolic functions, increased body temperature, activation of the coagulation cascade, modification of hemodynamics and induction of shock. Many attempts have been made to prevent or treat the deleterious effects of endotoxins on immune cells, such as the use of anti-endotoxin antibodies, and endotoxin partial structures for blocking endotoxin receptor antagonists. Nevertheless, the interaction of endotoxins with immune cells is not only mediated by specific receptors. Cell priming may also occur by non-specific intercalation of endotoxin molecules into the membranes of the target cells (33).

Finally, it should be mentioned that endotoxins may also have beneficial effects. They have been used in artificial fever therapy, to destroy tumors and to improve, non-specifically, the immune defense. The uncertainty about its role for the human health was once described by Bennett (34). On the other hand, any superfluous endotoxin exposure must be strictly avoided to prevent complications. This is especially true for intravenously-administered medicines.

TECHINIQUES OF ENDOTOXIN DETERMIN -ATION

The commonly used FDA-approved techniques for endotoxin detection are the rabbit pyrogen test and Limulus Amoebocyte Lysate (LAL) assay (35, 36). The rabbit pyrogen test, developed in the 1920s, involves measuring the rise in temperature of rabbits after intravenous injection of a test solution. Due to its high cost and long turnaround time, the use of the rabbit pyrogen test has diminished, and is now only applied in combination with the LAL test to analyze biological compounds in the earlier development phase of parenteral devices. Today the most popular endotoxin detection systems are based on LAL, which is derived from the blood of horseshoe crab, Limulus polyphemus, and clots upon exposure to endotoxin. The simplest form of LAL assay is the LAL gel-clot assay. When LAL assay is combined with a dilution of the sample containing endotoxin, a gel will be formed proportionally to the endotoxin sensitivity of the given assay. The endotoxin concentration is approximated by continuing to use an assay of less sensitivity until a negative reaction (no observable clot) is obtained. This procedure can require several hours (5, 36). The concentration of 0.5 EU/mL was defined as the threshold between pyrogenic and non-pyrogenic samples (17, 36).

In addition to the gel-clot technique, manufacturers have also developed two other techniques: turbidimetric LAL technique and the chromogenic LAL technique. These newer techniques are kinetic based, which means they can provide the concentration of endotoxin by extracting the real-time responses of the LAL assay. Turbidimetric LAL assay contains enough coagulogen to form turbidity when cleaved by the clotting enzyme, but not enough to form a clot (37). The LAL turbidimetric assay, when compared to the LAL gel-clot assay, gives a more quantitative measurement of endotoxin over a range of concentrations (0.01 EU/mL to 100.0 EU/mL.). This assay is based on the turbidity increase due to protein coagulation related to endotoxin concentration in the sample. The optical densities of various test-sample dilutions are measured and correlated to endotoxin concentration helped by a standard curve obtained from samples with known amounts of endotoxin (38). A kinetic chromogenic substrate assay differs from gel-clot and turbidimetric reactions because the coagulogen is partially or completely replaced by a chromogenic substrate (39). When hydrolyzed by the pre-clotting enzyme, the chromogenic substrate releases a yellow-colored substance known as p-nitroaniline. The time required to attain the yellow substance is related to the endotoxin concentration (40). However, kinetic turbidimetric and chromogenic tests, although more accurate and faster than the gel-clot, can not be used for fluids with inherent turbidity such as blood and yellow-tinted liquids, e.g. urine, and their performance may be compromised by any precipitation from solution (37). Therefore, different methods for detection of endotoxin in different samples have been studied (37, 41).

INTRACTIONS OF ENDOTOXINS WITH PROTEINS

A number of biomolecules show interactions with endotoxins, such as lipopolysaccharide-binding protein (LBP), bactericidal/permeability-increasing protein (BPI), amyloid P component, cationic protein (42, 43), or the enzyme employed in the biological endotoxin assay (anti-LPS) factor from Limulus amebocyte lysate (LAL) (44). These proteins are directly involved in the reaction of many different species upon administration of endotoxin (45, 46). Molecular recognition can be assumed as interactions with anti-endotoxin antibodies and proteineous endotoxin receptors (e.g. CD14, CD16, CD18) (47). Other proteins interact with endotoxins even having no strong links to a biological mechanism, such as lysozyme (25) and lactoferrin (48), which are basic proteins (pI>7), electrostatic interactions can be assumed as the main driving force. Regardless of the mechanism that proves to be most significant, these interactions result in hiding endotoxin molecules, and consequently these molecules are not removed in the removal procedures. A typical example is described by Karplus et al. (49).

However, other mechanisms must exist as interactions with neutral hemoglobin (50) and even acidic proteins (pI<7) are known, taking place also at low ionic strength. It is still controversially discussed how these interactions occur. Generally, hydrophobic interactions with proteins are conceivable. However, there is no strong evidence that it drives the interaction mechanism. It is more probable that competition of protein-bound carboxylic groups and endotoxin-bound phosphoric acid groups for Ca2+ may result in dynamically stable calcium bridges between proteins and endotoxins (8).
The fact that LPS forms micellar aggregates that are considered the biologically active forms of LPS (51) could indicate that multiple proteins interact with LPS molecules. Ma et al. (2006) (52) suggested an alternative aggregation form, where the self-assembly of lipophorin particles, a protein that serves as pro-coagulant (53-55), into globular structures are the result of oligomeric interactions. This may provide cage-like coagulation products, where the lipid moiety forms a protective layer that separates the toxin from interaction with the surrounding environment.

Due to protein&ndashendotoxin interactions, endotoxin removal from protein solutions requires techniques that are able to generate strong interactions with endotoxins, such as affinity chromatography. Alternatively, a specific dissociation of protein&ndashendotoxin complexes may improve the availability of endotoxin molecules for removal. In view of the large variety of products, it is not possible to develop one general method for endotoxins removal from all products.

TECHNIQUES OF ENDOTOXIN REMOVAL

The question about how endotoxin removal can be carried out in an economical way has attracted the attention of many investigators and has been &ndash although not published &ndash the reason for process rearrangements in many cases. However, this issue has not yet been resolved satisfactorily. The discussion of relevant aspects of endotoxin removal from biological preparations and a critical review of the existing approaches are mandatory in order to develop more refined methods in the future.

In the pharmaceutical industry several alternative routes are known to generate products with low-endotoxin levels. However, their diversity indicates a dilemma in endotoxin removal. Several procedures were developed for pharmacoproteins, taking advantage of the characteristics of the production process, tailored to suit specific product requirements. Therefore, each procedure addresses the problem in a completely different way none of them turns out to be broadly applicable. Anionic-exchange chromatography, for example, is potentially useful for the decontamination of positively-charged proteins, such as urokinase (56). However, decontamination of negatively-charged proteins would be accompanied by a substantial loss of the product due to adsorption (27, 57). For small proteins, such as myoglobin (Mr

18000 Da), ultrafiltration can be useful to remove large endotoxin aggregates. With large proteins, such as immunoglobulins (Mr

150000 Da) ultrafiltration would not be effective. In addition, ultrafiltration would fail if interactions between endotoxins and proteins cause endotoxin monomers to permeate with proteins pick-a-pick through the membrane.

Endotoxins can be considered to be temperature and pH stable, rendering their removal as one of the most difficult tasks in downstream processes during protein purification (58, 59). The removal of endotoxins becomes more challenging when associated with labile biomolecules, such as proteins (60). A number of approaches are typically utilized to reduce endotoxin contamination of protein preparations, including ion-exchange chromatography (61, 62), affinity adsorbents, such as immobilized L-histidine, poly-L-lysine, poly(&gamma-methyl L-glutamate), and polymyxin B (57, 63, 64), gel filtration chromatography, ultrafiltration, sucrose gradient centrifugation (65), and Triton X-114 phase separation (66, 67). The success of these techniques in separating LPS from proteins is strongly dependent on the properties of the target protein (9).

Two important factors influencing the success of any approach are the affinity of the endotoxin and protein antigen for the chromatography support or media used and the affinity of the endotoxin for the protein antigen. A third factor is how the affinity of the endotoxin for the protein can be modified by factors such as temperature, pH, detergents (surfactants), solvents and denaturants (4).

Usually, the procedures employed for endotoxin removal are unsatisfactory regarding selectivity, adsorption capacity and recovery of the protein. In the selective removal of endotoxin from protein-free solutions, it is easy to remove endotoxins by ultrafiltration taking advantage of the different sizes of the endotoxin and water, or by non-selective adsorption with hydrophobic adsorbent (68) or an anion-exchanger (69). For selective removal of endotoxin from protein solutions, it is necessary to know what is the form of the endotoxins in protein solutions.

Hirayama and Sakata (2002) (6) assumed that endotoxin aggregates form supermolecular assemblies with phosphate groups as the head group and exhibits a negative net charge because of its phosphate groups that originate from lipid A (6). These characteristics suggest that ionic interaction plays an important role in the binding between the cationic adsorbent and phosphate groups of the endotoxins. When hydrophobic adsorbents are used in protein solutions, it is suggested that there is also hydrophobic binding between the adsorbent and the lipophilic groups of endotoxins. These binding processes depend on the properties of proteins (net charge, hydrophobicity) and the solution conditions (pH, ionic strength).

Some commonly used techniques for removing endotoxin contaminants are ultrafiltration (70) and ion exchange chromatography (71). Ultrafiltration, although effective in removing endotoxins from water, is an inefficient method in the presence of proteins, which can be damaged by physical forces (72). Anion exchangers, which take advantage of the negative net charge of endotoxins, have been extensively used for endotoxin adsorption. However, when negatively charged proteins need to be decontaminated, they may co-adsorb onto the matrix and cause a significant loss of biological material. Also, net-positively charged proteins form complexes with endotoxins, causing the proteins to drag endotoxin along the column and consequently minimizing the endotoxin removal efficiency (57).

Alkanediols were shown to be effective agents for the separation of LPS from LPS-protein complexes during chromatography with ionic supports. Their effectiveness in reducing the protein complexation with LPS is dependent on (I) the size of the alkanediol, (II) the isomeric form of the alkanediol, (III) the length of the alkanediol wash, (IV) the concentration of alkanediol, and (V) the type of ionic support used, cationic or anionic. Alkanediol are non-flammable and as such are safer alternatives when compared to alcohols (ethanol or isopropanol) which have also been used to remove LPS from protein-LPS complexes (9). LPS removal is more efficient on cationic exchangers than on anionic exchangers.

In order to remove endotoxin from recombinant protein preparations, the protein solution may be passed through a column that contains polymyxin B immobilized on Sepharose 4B, in the hope that contaminating endotoxin binds to the gel. Similarly, histidine immobilized on Sepharose 4B also has the capability to capture endotoxin from protein solutions (63). Polymyxin B affinity chromatography is effective in reducing endotoxin in solutions (73). Polymyxin B, a peptide antibiotic, has a very high binding affinity for the lipid A moiety of most endotoxins (74). Karplus et al. (1987) (49) reported an improved method of polymyxin B affinity chromatography in which endotoxin could be absorbed effectively after dissociation of the endotoxin from the proteins by a nonionic detergent, octyl-&beta-D-glucopyranoside.

The methods mentioned above are reasonably effective for removal of endotoxins from protein solutions with relatively high protein recoveries. However, these affinity phases cannot be cleaned with standard depyrogenation conditions of strong sodium hydroxide in ethanol (75). Anspach and Hillbeck (1995) (57) revealed that these supports suffer from considerable efficiency decrease in the presence of proteins. Hence, they are not in general applicable for the above mentioned problem (57).

Membrane-based chromatography has been successfully employed for preparative separations predominantly for protein separations (76-83). Nevertheless, universal adoption of this technology has not taken place because membrane chromatography is limited by the binding capacity, which is small when compared to that of bead-based columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity (78). Although bead-based chromatography is still predominant and affective for product elution operations, it has several inherent disadvantages for trace-impurity removal or polishing applications. Furthermore, the adsorptive binding capacity of bead-based columns used in this application is typically 3-4 orders of magnitude larger than required because columns are normally sized to achieve a desired flow rate rather than capacity. Since membrane-based systems have a distinct flow rate advantage and sufficient capacity for binding trace levels of impurities and contaminants, membrane adsorbers are ideally suited for this application. Work has been done recently using membrane chromatography to remove DNA, host cell protein (HCP) and endotoxin with reasonable success (8, 84-86).

Jann et al., (1975) (87) reported that slab-polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) can be used for the separation of bacterial LPS. The authors showed that LPS molecular structures could be assigned to the separated LPS bands by correlating the electrophoretic banding pattern, as detected with periodic acid-Schiff stain, with the chromatography profile generated by gel permeation of chemically characterized carbohydrate moieties released from the LPS. While LPS obtained from rough (R) mutant bacteria, which contained a short oligosaccharides chain, exhibited only a fast-moving band, the LPS from wild-type smooth (S) strains, which had a core oligosaccharide substituted with various sizes of the O-specific polysaccharide chain, showed both fast and slow-migrating bands. On the other hand, LPS from the semirough (SR)-type bacteria containing a core oligosaccharide and truncated O-chain were detected as a fast-moving band migrating somewhat slower than R-type LPS bands. In spite of this great advance in the separation and analysis of intact LPS, the limited sensitivity of detection that resulted in the visualization of few broad and diffused LPS bands hindered the uncovering of further molecular intricacies of LPS (88). LPS from smooth and rough strains may be dispersed by surfactants such as sodium dodecyl sulfate (89, 90), Triton X-100 (91), and sodium deoxycholate (92-94). Upon removal of excess surfactant by dialysis, a more homogeneous population of particles with average molecular weight of about 5x105 to 1x106 Da is formed. Such observations suggest that hydrophobic interactions between subunits of LPS are important determinants of particle size (92).

Several methods have been used to separate the different subclasses of LPS from individual strains, with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (87-95) and gel filtration (96) being perhaps the most successful. These methods are, however, hampered by the tendency of LPS to aggregate and by the difficulty in detecting and identifying each distinct subclass (97). Agarose-gel electrophoresis has been used for various purposes, such as to separate polysaccharides extracted from tissues, organs, and biological fluids of invertebrates and vertebrates (97-100). Furthermore, densiometric band analysis enables one to obtain quantitative evaluation of single polysaccharide species in mixtures (97).

Although common purification protocols may reduce the endotoxin content below the threshold level, an absolute guarantee cannot be given. It may happen that a batch of the final products is accidentally contaminated and fails the quality control. This product has to be discarded reprocessing is not ruled out specifically but is a costly alternative.

TWO-PHASE MICELLAR SYSTEM

In recent years, the interest in the use of two-phase aqueous micellar systems for the purification or concentration of biological molecules, such as proteins and viruses has been growing (101-103). In these systems an aqueous surfactant solution, under the appropriate solution conditions, spontaneously separates into two predominantly aqueous, yet immiscible, liquid phases, one of which has a greater concentration of micelles than the other (101). The difference between the physicochemical environments in the micelle-rich phase and in the micelle-poor phase forms the basis of an effective separation and makes two-phase aqueous micellar systems a convenient and potentially useful method for the separation, purification, and concentration of biomaterials (101).

In their simplest realization, these systems exploit excluded-volume interactions between nonionic surfactant micelles and biomolecules. Specifically, in the phase-separated system one of the coexisting phases is rich in micelles while the order is poor in micelles (Figure 3) (104, 105). As a result, the stronger excluded-volume interactions between the nonionic surfactant micelles and the biomolecules in the micelle-rich phase drive the biomolecules preferentially into the micelle-poor phase based on their sizes (106).

Particularly for endotoxin removal, above the critical micelle concentration (CMC) of surfactants, endotoxins are accommodated in the micellar structure by non-polar interactions of alkyl chains of lipid A and the surfactant tail groups and are consequently separated from the water phase (micelle-poor phase). Surfactants of the Triton series show a miscibility gap in aqueous solutions. Above a critical temperature, the so-called cloud point, micelles aggregate to droplets with very low water content, by that forming a new phase. Endotoxins remain in the surfactant-rich phase. Through centrifugation or further increase in temperature the two-phases separate with the surfactant-rich phase being the bottom phase (66, 105, 107). If necessary, this process is repeated until the remaining endotoxin concentration is below the threshold limit. The cloud point of Triton X-114 is at 22°C, which is advantageous when purifying proteins.

Figure 3: Schematic illustration of a Triton X-114 micellar solution phase separation, upon temperature increase. Each of the resulting coexisting phases contains cylindrical micelles but at different micellar concentrations. Note also that, on average, the cylindrical micelles in the micelle-rich (bottom) phase are larger than those in the micelle-poor (top) phase.

Using Triton X-114, Adam et al. (1995) (108) showed a 100-fold endotoxin reduction in two steps with a final endotoxin content of 30 EU mg-1 and 50% loss in bioactivity of the exopolysaccharide. In addition, about 100-fold endotoxin reduction was shown by Cotten et al. (1994) (109), from plasmid DNA preparation with a final endotoxin content of 0.1 EU in 6 &mug DNA.

A comparison of affinity adsorption and Triton X-114 two-phase extraction for the decontamination of the recombinant proteins cardiac troponin I, myoglobin and creatine kinase isoenzymes is described by Liu et al. (1997) (67). They concluded that phase separation was the most effective method, reducing the endotoxin content by 98-99% with remaining amounts of 2.5-25 EU mg-1, depending on the protein. However, Cotton et al. (1994) (109) observed slightly better removal efficiency with a polymyxin B sorbent.

Aida and Pabst (1990) (66) reported a method to reduce endotoxin in protein solutions using Triton X-114, in which the surfactant aids in dissociation of endotoxin from the protein, while also providing a convenient phase separation capability for removing the dissociated endotoxin. According to these same authors, phase separation using Triton X-114 was effective in reducing endotoxin from solutions of three different proteins (cytochrome c, albumin and catalase). The first cycle of phase separation reduced endotoxin contamination by 1000-fold. Further cycles of phase separation resulted in complete removal of endotoxin. The endotoxin was found in the detergent phase, and the upper aqueous phase contained the desired biomolecule. In addition to decontamination of endotoxin from protein preparation like recombinant products or monoclonal antibodies, phase separating using Triton X-114 should be useful for the removal of lipids from albumin or lipoproteins. Considering that a certain amount of surfactant always remains in the protein solution, which needs to be removed by additional adsorptions or gel filtration processes, this process leads to 10-20% product loss (66). It has been proposed that the detergent dissociates the endotoxin molecule from the protein and separates the dissociated molecule by phase separation using the physical characteristics of Triton X-114 (66). Liu et al. (1997) (67) demonstrated that the Triton X-114 phase separation was then further applied to other recombinant protein preparations. By performing three cycles of Triton X-114 phase separation, endotoxin levels in all recombinant proteins derived from E. coli were reduced by as much as 99% of the original amount. Furthermore, the immunoactivity, physical integrity, and the biological activity of the protein remained unchanged after the phase separation process. The phase separation can be repeated multiple times until endotoxin in the aqueous phase reaches a satisfactory level. In addition to its simplicity, this procedure is cost effective, especially in large scale.

Fiske et al. (2001) (4) examined a number of approaches to reduce the level of endotoxin, such as the use of the zwitterionic surfactants Zwittergent 3-12 (Z3-12) and Zwittergent 3-14 (Z3󈚲) for the dissociation of endotoxin from the purified UspA2 protein and the subsequent separation of endotoxin from UspA2 using either ion-exchange or gel filtration chromatography. UspA2 protein is a potential vaccine candidate for preventing otitis media and other diseases caused by Moraxella catarrhalis (110). The approach that was proved successful for the dissociation of endotoxin from UspA2 was the replacement of the Triton X-100 by a zwitterionic surfactant. The inability of Triton X-100 to dissociate the endotoxin-UspA2 complex, despite success of both Z3-12 and Z3-14 may reside in the charge characteristics of the surfactants. Triton X-100 is a non-ionic surfactant containing no charged moieties while the Zwittergents contain zwitterionic head groups with both negatively and positively charged moieties. Most zwitterionic surfactants are effectively neutral however, in some cases strong polarization exists (111). The charge characteristics of Z3󈚰 and Z3-14 and the interaction of the surfactant with either the endotoxin and/or the protein may aid in the dissociation of the endotoxin from protein (in this case UspA2). Structural differences between the surfactants may also play a role in effective dissociation of endotoxin and protein. Whatever the mechanism, the use of the Zwittergent surfactant was proved to be quite suitable for the removal of LPS from UspA2 without disrupting the immunogenic properties of the protein. Prior to endotoxin reduction, the UspA2 preparations contained as much as 158 EU/Kg. However, following chromatography in the presence of Z3-12 Fiske et al. (2001) (4) achieved levels of approximately 0.0072 EU/Kg. The endotoxin removal process has been successfully implemented following GMP, to produce UspA2 subunit vaccine for clinical trials.

The levels of endotoxin appear to be much higher in recombinant proteins derived from soluble or cytoplasmatic fractions than in proteins derived from insoluble or inclusion bodies. This is consistent with the belief that lipopolysaccharides present in the cell wall are solubilized during the cell lysis procedure. Schnaitman (112) demonstrated that treatment of E. coli with the combination of Triton X-114, EDTA, and lysozyme resulted in solubilization of all lipopolysaccharide from the cell wall.

Reichelt et al. (2006) (59) tested whether the removal of endotoxin could be achieved during chromatography purification with the use of Triton X-114 in the washing steps. The application of 0.1% Triton X-114 in the washing steps was successful at reducing endotoxins during histidine and GST (resin GST sepharose) fusion protein purification, whereas washing steps lacking surfactant were ineffective in eliminating endotoxins. In contrast to purified materials employing the standard protocol which contained from 2500 to 34000 EU mg-1, purified recombinant proteins treated with Triton X-114 contained concentrations as low as 0.2 to 4 EU mg-1 (less than 1% of initial endotoxin content). Residual endotoxins in solubilized inclusion bodies can reach levels of 8 x 106 EU mL-1 despite the fact that endotoxin levels were found to be higher in recombinant proteins which are isolated from soluble fractions (113).

Endotoxins have been shown to form complexes with proteins of different isoelectric points (8) where electrostatic interactions are thought to be the main driving forces. As a result, the removal of endotoxins from basic proteins should prove to be more difficult than from acidic proteins (114). Reichelt et al. (2006) (59) studied whether the use of Triton X-114 in washing steps could eliminate endotoxins from proteins with a pI above 8.5. They found that washing with Triton X-114 coupled with affinity chromatography effectively removed endotoxins from negatively-charged proteins (SyCRP and NdhR). The minimal endotoxin concentration achieved was lower than 0.2 EU mg-1 protein recovery and yield were close to 100% (59).

Temperature-induced phase separation with Triton X-114 is a recent and powerful technique which efficiently separates hydrophobic and hydrophilic membrane proteins at room temperature, without denaturation (107, 115). This method was also successfully applied to the removal of endotoxin from proteins and enzymes, while retaining their normal functions (66). Because of an amphipathic character, LPS was also significantly removed from Klebsiella sp I-714 EPS (extracellular polysaccharides termed exopolysaccharides) after two extractions steps in 2% Triton X-114, with only a twofold decrease in bioactivity (108). According to the same authors, the Triton X-114 partitioning technique is fast, efficient, nondegradative, and allows a high level of detoxification of the Klebsiella sp. I-714 EPS. The separation of endotoxin and exopolysaccharides from Klebsiella sp. I-714 is difficult to achieve with techniques other than two-phase extraction. In addition, this method has also been successfully employed for the purification of an endotoxin-contaminated negatively-charged EPS from Pseudomonas solanacearum (108).

The detergents, even though they were also very effective at reducing the LPS levels, are relatively expensive, would add significant cost to a manufacturing process, and may affect the bioactivity of the protein of interest. Alternative chemicals are desired that could safely and cost effectively be used in place of the alcohols or detergents as washing agents for the separation of LPS from proteins during chromatographic unit operations. Ideally, these chemicals would be relatively inexpensive, chemically well defined, present minimal safety issues, and have minimal impact on the bioactivity of the protein in question when implemented into a process (9).

PERSPECTIVES

Taking into consideration the properties of two-phase aqueous micellar systems to remove biomolecules, this research group had some promising results, using Triton X-114 to remove endotoxins present in fermented culture of E. coli cells during the production of protein. According to the literature ([7], [58], [65], [107]), phase separation using Triton X-114 was effective in reducing endotoxin from solutions containing biomolecules, however the optimized conditions are still uncertain and requires further investigation.

ACKNOWLEDGMENT

The authors are grateful for the financial support from CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico and FAPESP &ndash Fundação de Apoio a Pesquisa do Estado de São Paulo.


Biosynthesis and Export of Bacterial Lipopolysaccharides

Lipopolysaccharide molecules represent a unique family of glycolipids based on a highly conserved lipid moiety known as lipid A. These molecules are produced by most gram-negative bacteria, in which they play important roles in the integrity of the outer-membrane permeability barrier and participate extensively in host–pathogen interplay. Few bacteria contain lipopolysaccharide molecules composed only of lipid A. In most forms, lipid A is glycosylated by addition of the core oligosaccharide that, in some bacteria, provides an attachment site for a long-chain O-antigenic polysaccharide. The complexity of lipopolysaccharide structures is reflected in the processes used for their biosynthesis and export. Rapid growth and cell division depend on the bacterial cell's capacity to synthesize and export lipopolysaccharide efficiently and in large amounts. We review recent advances in those processes, emphasizing the reactions that are essential for viability.


Lipopolysaccharide Endotoxins

AbstractBacterial lipopolysaccharides (LPS) typically consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating “core” oligosaccharide, and a distal polysaccharide (or O-antigen). Recent genomic data have facilitated study of LPS assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and have established the importance of lateral gene transfer in generating structural diversity of O-antigens. Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics. Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants, indicating that eukaryotic lipid A-like molecules may exist. Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment, and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection.


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Structure and Composition

  1. A phospholipid called Lipid A embeds in a lipopolysaccharide layer in the outer leaflet. Also known as endotoxin, it is responsible for toxic effects (fever and shock). Generally, it is not released until the death of a cell.
    Exception: Neisseria meningitidis, which over-produces outer membrane fragments.
  2. A core polysaccharide of five sugars linked through ketodeoxy-octonate (KDO) to lipid A.
  3. O antigen: An outer polysaccharide consisting of up to 25 repeating units of 3-5 sugars. These are hydrophilic in nature. O antigen is highly varied among species.
    Example: E.coli O157:H7 which causes food poisoning and hemolytic uremic syndrome. O antigens are used to identify certain organisms in microbiology laboratories. O antigens are toxic and account for some of the virulence of certain gram-negative bacteria.

Note: LPS is heat stable and not strongly immunogenic so it cannot be converted to a toxoid.


6 MECHANISTIC BASIS OF BACTERIAL CONTAMINATION IN THE MENSTRUAL BLOOD

The authors have proposed two mechanisms that were involved in the E. coli contamination of the menstrual blood: (1) higher prostaglandin E2 (PGE2) levels in the MF and PF of women with endometriosis was involved in the bacterial growth of E. coli in a bacterial culture system 39 and that this effect of PGE2 on bacteria might be contributed to by its direct growth-promoting effect on E. coli or by its indirect immunosuppression effect on peripheral blood lymphocytes 39 and (2) the decreased expression of antimicrobial peptides, such as human β-defensin (HBD) and/or secretory leukocyte protease inhibitor (SLPI), in the endometrium. Usually, HBD and SLPI are expressed by the epithelial layers of the vagina, ectocervix, endocervix, endometrium, and fallopian tubes. 40 These antimicrobial peptides are normally regulated by cyclic estrogen 40 and their expression pattern might be down-regulated after estradiol (E2)/progesterone withdrawal during menstruation. The decreased expression of the antimicrobial peptides in the intrauterine or intravaginal luminal epithelium during the menstrual phase could be involved in the bacterial contamination of menstrual blood in women with endometriosis.


The Elephant in the Pet Food: Endotoxins

Think your pet is safe just because Salmonella or E. coli bacteria is killed by the cooking process of pet food? Think again. The elephant ignored in pet food: endotoxins produced by dead bacteria. This is one of the most concerning things I’ve learned about pet food.

Think your pet is safe just because Salmonella or E. coli bacteria is killed by the cooking process of pet food? Think again. The elephant ignored in pet food: endotoxins produced by dead bacteria. This is one of the most concerning things I’ve learned about pet food.

The medical dictionary of theFreeDictionary.com states: Endotoxins are ‘toxins’ that are released on bacterial death. Endotoxins are produced by Gram-negative bacteria. Examples of gram-negative bacteria: Salmonella and E. coli. Endotoxins are “moderately toxic fatal to animals in large doses.”

Endotoxins are sort of a defense mechanism for bacteria (gram-negative bacteria) you kill me, I’m going to release a toxin. When bacteria such as Salmonella or E. coli are killed through heat or other ‘kill steps’, they release a toxin (endotoxins). Endotoxins cannot be destroyed through heat or acid or any ‘kill step’ recommended by FDA. FDA would like consumers to believe that the only bacterial risk in pet food is live bacteria. But live bacteria in not the only concern. Endotoxins produced upon bacterial death causes another whole set of problems.

Endotoxins are also a concern for human food, but not nearly to the extent of pet food. The amount of endotoxins in food (pet food ingredients or human food) directly relates to the amount of bacteria present before the ‘kill step’ (cooking). In recent testing, Consumer Reports examined 300 packages of ground beef (for human consumption), every single sample tested positive for bacteria that signified fecal contamination. Ten percent of the samples had a strain of Staphylococcus aureus bacteria – an endotoxin producing bacteria.

If human food meat has a high risk of bacteria contamination, we can assume pet grade/feed grade meat has a dramatically increased risk of bacteria contamination, considering the inferior quality of ingredients FDA allows into pet food. Most pet foods are made with pet grade ingredients, also known as feed grade or inedible ingredients. As example of pet grade, feed grade or inedible ingredients is FDA Compliance Policy CPG 690.300 which allows pet food to be made from “animals which have died otherwise than by slaughter”.

This is example of animals that have died other than by slaughter…animals that have died in the field. These animals can lay in the field for days, once picked up by the renderer they can again lay for days before processing.

From a 2003 USDA document on animal carcass disposal : “Because raw materials in an advanced stage of decay result in poor-quality end products, carcasses should be processed as soon as possible if storage prior to rendering is necessary, carcasses should be refrigerated or otherwise preserved to retard decay. The cooking step of the rendering process kills most bacteria, but does not eliminate endotoxins produced by some bacteria during the decay of carcass tissue. These toxins can cause disease, and pet food manufacturers do not test their products for endotoxins.”

Another example of animals that have died other than by slaughter: Spent laying hens (hens no longer producing eggs) and male chicks are ground whole (including feathers, beaks, feet and intestines). FDA and all other pet food regulatory authorities have no concern with poultry feces being included in a pet food (which is full of dangerous bacteria), their only concern is the kill step to destroy live bacteria. No attention is paid to what the dead bacteria releases – endotoxins.

FDA tells the public occasionally meat from animals that may have died otherwise than by slaughter” is used in pet food. “Occasionally” is not even close to accurate. From the same 2003 USDA document quoted above, about three billion pounds of carcasses” from animal mortalities need to be disposed of annually. The disposal method of choice: rendering (after rendering, ingredients are sold to pet food/animal feed manufacturers). Three billion pounds is not occasionally (and this was estimate 12 years ago – we can easily increase that number to four billion pounds or higher – annually…again, not ‘occasionally’). As well, this three billion pounds does not include perhaps an equal or greater billions of pounds of spent laying hens rendered whole (feathers, feet, beaks and intestines included) each year that heads directly to pet food.

Another concern in pet foods is lack of requirement to transport and warehouse ingredients under refrigeration and in clean conditions (actions to prevent the growth of bacteria). Consider the commonly used pet food ingredient chicken meal or lamb meal (any meal ingredient). This ingredient is ground meat, bones and internal organs – the end ingredient (meal) has been cooked. Thousands of pounds of these meat meal ingredients are commonly delivered to pet food manufacturing in a dump truck. That’s right…a dump truck. No refrigeration, no clean conditions…a dump truck. The image to the left is a meat meal ingredient being delivered to a pet food manufacturer.

So…in pet food, there is a significant risk for high bacterial contamination of ingredients. Add in more risk for bacterial contamination via transportation/warehousing and the end result can be a massive bacteria load on pet food ingredients. Kill step taken (cooking of the pet food and/or ingredients) equals a potential for massive amounts of endotoxins in many pet foods.

FDA does insist on some type of “kill step” to destroy live bacteria, but FDA neglects to warn the public or even consider the risks of endotoxins from dead bacteria. Existing pet food regulations almost encourage an environment for massive bacterial growth (prior to kill step) allowing an environment for massive levels of endotoxins in finished pet foods/treats.

What are the risks of endotoxins?

Endotoxins cause inflammation inflammation is the foundation of disease. Endotoxins have been linked to obesity, diabetes.

“Endotoxin entering the body is carried to the liver where it is inactivated. Increased endotoxin levels can damage the liver. Moreover, when the amount of endotoxin reaching the liver is normal, the presence of another potential toxin can interact with endotoxin to damage the liver. The other substances are not necessarily toxins. They include vitamin A, copper and iron, and many drugs. Thus, any level of endotoxin can damage the liver. Exposure to endotoxin should be minimized as much as possible.”

Endotoxemia: the presence of endotoxins in the blood. Endotoxins consumed through food sources are most often absorbed into the blood stream through the intestinal lining.

Dogs: “No clinical signs have been found that are pathognomonic for endotoxemia. Animals show either signs associated with infection (e.g., purulent vaginal discharge with pyometra, coughing with pneumonia, mastitis) or localizing signs such as inactivity and inappetence. Tachycardia, tachypnea, and fever are the clinical hallmarks of SIRS. If a dog is progressing towards severe sepsis or septic shock, signs associated with the GI tract (the canine Shock organ system) can develop, including vomiting or diarrhea, or both.”

Cats: “Endotoxemia is rarely reported and poorly described in cats. In a retrospective case series of cats with confirmed severe sepsis (9 of 29 cats had confirmed Escherichia coli or Pseudomonas spp. infections endotoxin was not assayed), clinical signs included lethargy, pale mucous membranes, weak femoral pulses, tachypnea, hypothermia or fever, diffuse pain on abdominal palpation, bradycardia, and icterus.”

What can you do to protect your pet?

The most important thing is to provide your pet a food made with ingredients that had minimal bacterial growth in their raw state (prior to manufacturing of the pet food). Talk to your pet food manufacturer – ask them how they prevent bacteria growth. Ask if all raw material (raw ingredients) are transported and warehoused under refrigeration (optimal would be ingredients transported and warehoused according to human food safety standards). The only way to prevent endotoxin contamination in a pet food is to prevent bacteria growth on the ingredients.

Human grade ingredients – ingredients that are USDA inspected and approved for human consumption, and transported and warehoused under proper refrigeration is vital. Yes, human grade meats do contain endotoxin producing bacteria. However pet grade or feed grade meats are most often a lesser quality and are commonly not transported under refrigeration. Assumed risk of pet grade ingredients is dramatically higher than human grade ingredients.

Supplement your pet’s diet with a quality probiotic. It is key to have a healthy balance of bacteria in your pet’s gut. From a study published in World Journal of Gastroenterology, ‘Probiotics and gut health: A special focus on liver diseases’ : “Newer evidence suggests that probiotics have the potential to reduce the risk of developing inflammatory bowel diseases and intestinal bacterial overgrowth after gut surgery. In liver health, the main benefits of probiotics might occur through preventing the production and/or uptake of lipopolysaccharides (endotoxins) in the gut, and therefore reducing levels of low-grade inflammation.”

Supplement your pet’s diet with fish oil or cod liver oil (omega-3 fatty acids). A 2013 study on pigs found that fish oil and cod liver oil supplements reduced endotoxin levels in the blood by 50%. Foods that provide natural sources of omega-3 are flaxseeds, sardines, and salmon. Pet food consumers can add these foods (human grade) to their pets diet to help control the effects of endotoxins they could be consuming.

Please consult your veterinarian for dosage of probiotics, fish oil and/or cod liver oil, and all food supplements.

If you suspect your pet has symptoms of endotoxemia, please ask your veterinarian to test for it (level of endotoxins in the blood). If you find high levels of endotoxins in the blood of your pet, you might want to consider having the pet food tested for endotoxin levels ask your veterinarian to suggest a trusted lab. Also – please report any diagnosis of endotoxemia to FDA (send FDA your pet’s laboratory results). You can do that here: http://www.fda.gov/AnimalVeterinary/SafetyHealth/ReportaProblem/ucm182403.htm

How prevalent are endotoxins in commercial pet foods?

Pet food is manufactured by grinding the ingredients – similar to the process of grinding hamburger. In human food, hamburger meat has been tested to contain up to 100 million (live) bacteria per hamburger. The result after cooking, a massive load of endotoxins remain in that hamburger. It is believed the reason that hamburger meat (ground meat) contains such high levels of (live) bacteria is due to using meat and fat trimmings from multiple animals, grinding the meat co-mingles all the the bacteria.

A similar system is used in pet food – meat and fat trimmings from multiple animals are ground prior to ingredient and/or pet food manufacturing. But…and this is a big but…pet food most commonly uses a lesser quality meat and fat trimmings than human food. So… if we know human food hamburger has been tested containing 100 million bacteria per hamburger (all USDA inspected and approved meat, required to be handled and transported under strict regulation meant to control bacterial growth), we can assume that many pet food meats (billions of pounds non-USDA inspected and not required to be transported under any regulation to control bacterial growth) contain significantly higher bacteria per serving. That significantly higher bacterial load – after the ‘kill step’ – results in significantly higher levels of endotoxins in the pet food.

An example of how prevalent endotoxins in pet food could be, we look back at our Pet Food Test Results published earlier this year. Our testing found 10 of 12 pet foods contained gram-negative bacteria that upon ‘kill-step’ would produce endotoxins.

Pet foods tested found to contain one or more types of gram-negative (endotoxin producing) bacteria:

Royal Canin Veterinary Diet Renal LP Modified in Gravy Can Cat Food
Fancy Feast Grilled Chicken Variety Pack Purina Canned Cat Food
Science Diet Adult Hairball Control Minced Chicken Entree Cat Food Can
Meow Mix Tender Centers Salmon & Turkey Flavors Dry
Friskies Grillers Cat Food Dry
Wellness Complete Health Deboned Chicken, Chicken Meal & Rice Adult Cat Food Dry
Hill’s Prescription Diet C/D Urinary Tract Health Can
Blue Freedom Grain Free Chicken Recipe Adult Dog Food Dry
Beneful Original Dog Food Dry
Ol’ Roy Dog Food Soft & Moist Beef

And another concern. Synergy: the interaction of elements that when combined produce a total effect that is greater than the sum of the individual elements.

There can be a synergistic effect of endotoxins with other toxins of pet foods and with some ingredients. Mycotoxins are stated to “aggravate exposure to endotoxins”. In a pet food industry/animal feed industry publication , regarding poultry and endotoxin exposure risk, it states that mycotoxins “increase intestinal permeability” to absorb endotoxins.

Recent science (2014) found that propylene glycol – commonly used in pet foods – “increased the mortality rate in sepsis induced by the bacterial endotoxin lipopolysaccharide in mice.”

Of the pet foods we tested, the following list of pet foods included gram-negative bacteria that will produce endotoxins, contained levels of mycotoxins and/or the ingredient propylene glycol…

Synergy – Mycotoxins
Royal Canin Veterinary Diet Renal LP Modified in Gravy Can Cat Food
Fancy Feast Grilled Chicken Variety Pack Purina Canned Cat Food
Meow Mix Tender Centers Salmon & Turkey Flavors Dry
Wellness Complete Health Deboned Chicken, Chicken Meal & Rice Adult Cat Food Dry
Hill’s Prescription Diet C/D Urinary Tract Health Can
Blue Freedom Grain Free Chicken Recipe Adult Dog Food Dry
Beneful Original Dog Food Dry

Synergy – Propylene Glycol
Beneful Original Dog Food Dry

Hypothesis: a supposition or proposed explanation made on the basis of limited evidence as a starting point for further investigation.

Let’s look again at the symptoms of endotoxemia (the presence of endotoxins in the blood).

Dog Symptoms: “No clinical signs have been found that are pathognomonic for endotoxemia. (pathognomonic: specifically characteristic or indicative of a particular disease or condition) Animals show either signs associated with infection (e.g., purulent vaginal discharge with pyometra, coughing with pneumonia, mastitis) or localizing signs such as inactivity and inappetence. Tachycardia (abnormally rapid heart rate), tachypnea (abnormally rapid breathing), and fever are the clinical hallmarks of SIRS. If a dog is progressing towards severe sepsis or septic shock, signs associated with the GI tract (the canine Shock organ system) can develop, including vomiting or diarrhea, or both.”

And now we look at pet illness symptoms reported by pet owners linked to pet foods. Taken from consumer complaints reported on Consumer Affairs.com …

“Just came back from the Vet with my sick Dachshund. Started giving her Beneful dog food 4 days ago. She suddenly stopped eating and drinking, and started vomiting. After 24 hrs I took her to the Vet because her gums were so pale.”

“My dog ate Beneful and recently started getting very sick. Incontinent of urine and feces, blood in his loose stools, vomiting, and declining appetite.”

Cat Symptoms: “Endotoxemia is rarely reported and poorly described in cats. In a retrospective case series of cats with confirmed severe sepsis (9 of 29 cats had confirmed Escherichia coli or Pseudomonas spp. infections endotoxin was not assayed), clinical signs included lethargy, pale mucous membranes, weak femoral pulses, tachypnea (abnormally rapid breathing), hypothermia (abnormally low body temperature) or fever, diffuse pain on abdominal palpation, bradycardia (abnormally slow heart action), and icterus (jaundice).”

Taken from consumer complaints reported on Consumer Affairs.com …

“On January 9, I fed my cat Fancy Feast cat food, and within hours, he was lethargic, had a high fever, diarrhea and nausea.”

“My 8 year old, healthy male cat Oliver passed away last night. He was fine all day, but got sick in the evening after eating a can of Fancy Feast Delights Chicken and Cheese in gravy. He threw up about 3 times. The vomit was watery and there was no signs of anything else in it other than the canned food. He also hid under the bed and was panting with a hard stomach, we thought it was just an upset stomach. We woke up the next morning and he was gone.”

My hypothesis: With FDA approval for pet foods to use the most inferior quality ingredients imaginable, certain to be overgrown with dangerous bacteria, and certain to produce an overwhelming level of endotoxins once cooked – endotoxin contamination of pet foods is more than a probable risk. Symptoms reported by consumers are almost an identical match to clinical symptoms of endotoxemia. Are endotoxins the unknown cause to so many pet deaths and illnesses? It certainly is an issue that needs to be investigated.

The following letter was sent to FDA…

Dr. Bernadette Dunham
Dr. Dan McChesney
Dr. William Burkholder

Association for Truth in Pet Food (ATPF) – on behalf of our pet food consumer members – is requesting FDA/CVM to investigate endotoxin levels in pet foods and to establish a safe maximum level for cats and dogs to consume (on a daily basis).

There is a multitude of science discussing the dangers of endotoxins. Science links endotoxins to obesity and diabetes. Science links endotoxins to inflammation and gastrointestinal disease.

Mycotoxins are stated to “aggravate exposure to endotoxins”. In a pet food industry/animal feed industry publication, regarding poultry and endotoxin exposure risk, it states that mycotoxins “increase intestinal permeability” to absorb endotoxins. (Source: http://www.wattagnet.com/articles/23248-poultry-and-endotoxin-exposure-risk)

Recent science (2014) found that propylene glycol – commonly used in pet foods – “increased the mortality rate in sepsis induced by the bacterial endotoxin lipopolysaccharide in mice.” (Source: http://www.ncbi.nlm.nih.gov/pubmed/24975968)

Veterinarian and Professor Emeritus, University of California, Davis, School of Veterinary Medicine Dr. Donald R. Strombeck states “Endotoxin entering the body is carried to the liver where it is inactivated. Increased endotoxin levels can damage the liver. Moreover, when the amount of endotoxin reaching the liver is normal, the presence of another potential toxin can interact with endotoxin to damage the liver. The other substances are not necessarily toxins. They include vitamin A, copper and iron, and many drugs. Thus, any level of endotoxin can damage the liver. Exposure to endotoxin should be minimized as much as possible.” (Source: http://dogcathomeprepareddiet.com/commercial_pet_food_contaminatio.html)

How many other common toxins of pet food or common pet food ingredients collectively work to increase the absorption of endotoxins into the blood?

Considering the source of feed grade/pet grade ingredients, we can safely assume the bacterial load on commonly used pet food ingredients is massive compared to food grade ingredients. FDA encourages a ‘kill step’ to prevent the risks from live bacteria to pets consuming the food and/or humans handling the food. However FDA does not offer consumers protection from the dangerous endotoxins left after the ‘kill step’. ATPF would like to know why FDA has not addressed this serious concern with pet food?

Science tells us that high levels of endotoxins can be deadly to animals. What is the no observed adverse effect level of endotoxins for cats and dogs consumed in food?

It is truly concerning to read the dog and cat symptoms of endotoxemia from veterinary publications. From Infectious Diseases of the Dog and Cat (3 rd Edition), Chapter 38 Endotoxemia:

Dogs: “Animals show either signs associated with infection (e.g., purulent vaginal discharge with pyometra, coughing with pneumonia, mastitis) or localizing signs such as inactivity and inappetence. Tachycardia, tachypnea, and fever are the clinical hallmarks of SIRS. If a dog is progressing towards severe sepsis or septic shock, signs associated with the GI tract (the canine Shock organ system) can develop, including vomiting or diarrhea, or both.”

Cats: “Endotoxemia is rarely reported and poorly described in cats. In a retrospective case series of cats with confirmed severe sepsis (9 of 29 cats had confirmed Escherichia coli or Pseudomonas spp. infections endotoxin was not assayed), clinical signs included lethargy, pale mucous membranes, weak femoral pulses, tachypnea, hypothermia or fever, diffuse pain on abdominal palpation, bradycardia, and icterus.”

ATPF asks FDA/CVM to compare these symptoms with many of the consumer reported pet food adverse events received. We suspect you will find many consumer reported symptoms match – almost identically – with the clinical symptoms of endotoxemia. Endotoxins levels in the blood of sick pets should be investigated, recorded and compared with the endotoxins levels found in suspect pet foods.

ATPF believes this is a serious concern that deserves immediate attention and investigation by FDA/CVM. Prevention of bacterial contamination in pet food ingredients must be of significant importance to control the level of endotoxins in pet foods. A ‘kill step’ is not the only concern. We await your report on this issue.

Susan Thixton
On behalf of pet food consumer members
Association for Truth in Pet Food

Should FDA respond, it will be shared with all.

I do believe this is a serious situation. The more I read and researched this issue, the worse it got. The FDA/CVM website states : “CVM had no evidence of human or animal disease associated with the feeding of properly rendered and handled animal feed ingredients despite the use of tissues from diseased animals or animals that have died otherwise than by slaughter.” If FDA/CVM is unaware of animal disease associated with rendered feed ingredients…perhaps it is because they have never looked.

The only thing we can do – until FDA establishes a maximum level of endotoxins in pet food that will protect our pets – is to become proactive. Repeating from above…

What can you do to protect your pet?

The most important thing is to provide your pet a food made with ingredients that had minimal bacterial growth in their raw state (prior to manufacturing of the pet food). Talk to your pet food manufacturer – ask them how they prevent bacteria growth.

Human grade ingredients – ingredients that are USDA inspected and approved for human consumption, and transported and warehoused under proper refrigeration is vital. Yes, human grade meats do contain endotoxin producing bacteria. However pet grade or feed grade meats are most often a much lesser quality and have not been transported under refrigeration.

Supplement your pet’s diet with a quality probiotic. It is key to have a healthy balance of bacteria in your pet’s gut. Supplement your pet’s diet with fish oil or cod liver oil (omega-3 fatty acids). Pet food consumers can add (human grade) foods that provide natural sources of omega-3 such as flaxseeds, sardines, and salmon. Please consult your veterinarian for dosage of probiotics, fish oil and/or cod liver oil.

If you suspect your pet has symptoms of endotoxemia, please ask your veterinarian to test for it (level of endotoxins in the blood). If you find high levels of endotoxins in the blood of your pet, you might want to consider having the pet food tested for endotoxin levels ask your veterinarian to suggest a trusted lab. Also – please report any diagnosis of endotoxemia to FDA (send FDA your pet’s laboratory results). You can do that here: http://www.fda.gov/AnimalVeterinary/SafetyHealth/ReportaProblem/ucm182403.htm

For a brief overview of this post, Click Here to view (and share) an info-graphic.

Wishing you and your pet(s) the best,

Susan Thixton
Pet Food Safety Advocate
Author Buyer Beware, Co-Author Dinner PAWsible
TruthaboutPetFood.com
Association for Truth in Pet Food

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The 2015 List

Susan’s List of trusted pet foods. Click Here


Watch the video: ΖΩΑ ΚΑΙ ΣΥΝΑΙΣΘΗΜΑΤΑ36. (October 2022).