Probiotics can treat hepatic encephalopathy

Medical Hypotheses (2003) 61(2), 307 313 doi:10.1016/s0306-9877(03)00192-0 Probiotics can treat hepatic encephalopathy S. F. Solga Johns Hopkins Hospital, Baltimore, USA Summary Hepatic encephalopathy (HE) is a common and dreaded complication of liver disease. The effects of HE can range from minimal to life threatening. Even minimal HE causes major dysfunction in many aspects of daily living. The exact pathogenesis of HE remains unknown. However, the products of gut flora metabolism are universally recognized as critical. Present treatments for HE include the cathartic agent lactulose and poorly absorbable antibiotics. While effective, these treatments incur numerous side-effects and cost. Probiotics are viable bacteria given orally to improve health. Probiotics have multiple mechanisms of action that could disrupt the pathogenesis of HE and may make them superior to conventional treatment. BACKGROUND/SIGNIFICANCE Disease and pathogenesis Hepatic encephalopathy (HE) is a common and serious complication of chronic liver disease. This complex neuropychiatric syndrome has been defined as a disturbance in central nervous system function because of hepatic insufficiency (1). At least 50 70% of patients with cirrhosis will demonstrate abnormalities on pyschometric testing (2,3), and many will have significant functional impairment. Encephalopathy can occur in patients with both acute and chronic liver disease, and can be clinically overt or less apparent. Minimal encephalopathy is a term that describes patients with chronic liver disease who have no clinical symptoms of brain dysfunction, but perform substantially worse on pyschometric tests compared to healthy controls (4). An extensive body of research has consistently documented cognitive deficits in these patients, including impaired psychomotor speed, attention, and Received 3 September 2002 Accepted 11 November 2002 Correspondence to: Steven F. Solga MD, 600 North Wolfe Street, Blalock 4, Division of Gastroenterology, Johns Hopkins Hospital, Baltimore, MD 21205, USA. Phone: 410-502-7729; Fax: 410-955-2108; E-mail: solga@jhmi.edu visual perception. Predictably, such impairments lead to major difficulties in safely performing routine activities of life. Landmark work by Schomerus et al. (5) demonstrated that 60% of cirrhotics with minimal HE were unfit to drive, and an additional 25% were possibly unfit to drive. In agreement with this finding, other investigators have found impaired earning capacity, particularly amongst blue-collar workers requiring psychomotor skills in order to perform their jobs (6). Further, extensive work using a 136 part sickness impact profile (a generic, non-disease-specific quality of life questionnaire) found that minimal HE has major impact on all aspects of a patient s life (7). Clinically apparent encephalopathy has been subdivided into a semi-quantative grading scheme ranging from mild (grade I) to severe (grade II IV). According to the West Haven Criteria (8), grade I encephalopathy indicates a patient with trivial lack of awareness, euphoria or anxiety, shortened attention span, and impaired performance of addition. At times, clinicians may have difficulty distinguishing these patients from patients with minimal encephalopathy. The pathogenesis of HE is unknown, but is almost certainly multi-factorial. Gut-derived nitrogenous substances are universally acknowledged to play a major role. Specifically, ammonia is thought to be a critical factor in the pathogenesis. While ammonia is produced by many tissues, most results from the activity of urease 307

308 Solga producing gut flora and is released into the portal vein after absorption by the intestinal epithelium. Ammonia is converted into urea in the liver, carried to the kidneys, and then excreted into the urine. Normally a very efficient process, humans excrete over 20 pounds of urea a year (9) and first pass hepatic clearance of ammonia is around 80% (10). See Fig. 1. Further understanding of urease and ammonia is important to the pathophysiology of HE and potential treatments. Urease-producing bacteria exist in abundance in the gut of ureolytic animals (11). Ureolytic animals, including humans, excrete nitrogenous waste primarily via urea in the urine. Urease is a bacterial enzyme that catalyzes the hydrolysis of urea to carbamate and ammonia (12). Bacteria from many different genera produce urease, and its expression can be nitrogen regulated, urea inducible, or constitutive (13). Urease-producing bacteria are frequently gram negative Enterobacterceae. The potential therapeutic consequences of blocking urease activity were established decades ago by demonstrations that injection of antiurease antibodies reduced ammonia production and improved encephalopathy (14). Such immunization against urease, however, caused many side effects and was ultimately abandoned (15). Ammonia is a weak base with a pk a of 9.25 (16). Therefore, decreases in lumenal ph increases the ratio of ionized to unionized ammonia, and decreases passive non-inonic diffusion. As a result, less ammonia is absorbed into the portal blood and more is excreted in feces (17). Further, lower lumenal ph itself reduces the degradation of nitrogenous compounds (proteins and amino acids) and production of ammonia (18). The physiologic balance of ammonia production and clearance is disrupted on multiple levels in patients with cirrhosis, resulting in HE. An extensive body of evidence reports that cirrhotics harbor more gut urease-active bacteria than controls (19), and that this leads directly to increased intestinal hydrolysis of urea and absorption of nitrogenous products (20). Altered small intestinal dysmotility frequently accompanies cirrhosis (21) and likely exacerbates this problem. Further, increased portal ammonia results in markedly increased systemic ammonia because of: (1) impaired hepatic processing of ammonia and (2) the shunting of portal blood away the liver. Finally, ammonia crosses the blood brain barrier more readily patients with HE (22), where it acts on impaired astroctyes and results in a cascade of pathopysiologic neurochemical events (23). See Fig. 2. Other gut-derived toxins may also play a role in the pathogenesis of HE (24). For example, intestinal flora may produce benzodiazepine-like substances (25) or mercaptans (26) which can be additive or synergistic to the effects of ammonia. The importance of gut-derived products for HE is further supported by the efficacy of complete surgical exclusion (e.g., total colectomy) in the treatment of refractory HE (27). Standard treatment options Presently, lactulose and poorly absorbable antibiotics are the mainstay of treatment for HE. Lactulose is a nonabsorbable, synthetic disaccharide that has multiple effects on gut flora and, therefore, several potential mechanisms of action. Its most obvious effect is as a laxative; however, laxatives alone (e.g., water enemas) (28) are ineffective for HE. Additional putative mechanisms for the efficacy of lactulose may include: 1. Decreasing ammonia production by decreasing urease activity and increasing assimilation of nitrogenous products by bacteria; 2. Acidifying the colon contents resulting in a decrease in ammonia absorption into the gut; and 3. Decreasing toxic C4 C6 short chain fatty acid production by enhancing the production of non-toxic acetate (29). Finally, lactulose may function as a prebiotic in the treatment of hepatic encephalopathy (30). A prebiotic is defined as a non-digestible food ingredient that Fig. 1 Normal physiology. (A) Urease producing gut flora cleave urea in an enzymatic process resulting in net ammonia production. (B) Portal blood is then processed in the liver where most of it is cleared, allowing for normal brain function, (C). Medical Hypotheses (2003) 61(2), 307 313

Probiotics can treat hepatic encephalopathy 309 Fig. 2 Pathophysiology in cirrhosis. Intestinal dysmotility (A) exacerbates overgrowth of urease þ bacterial (B) and increased absorption of nitrogenous products (C) into the portal blood. Shunting (D) and impaired hepatic processing (E) result in increased systemic exposure to an impaired blood brain barrier (F) and astrocyte dysfunction (G) results. beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health (31). Specifically, lactulose significantly increases concentrations of bifidobacteria and lactobacilli, and may have therapeutic effect through the mechanisms of these flora (32,33). Non-absorbable antibiotics, principally neomycin and metronidazole, are also effective, presumably by killing gram negative and anaerobic urease producing bacteria. Further treatments can include dietary protein restriction (34), ornithine salts (35), and benzoate (36), although the latter are rarely used in practice. While these treatments are effective, they impose side effects, toxicities, and cost. Lactulose has an unpleasant taste and causes flatulence and diarrhea. Due to unpredictable dose responses, the diarrhea can be severe and result in hypertonic dehydration with hypernatremia with subsequent hyperosmolarity and altered mental status (37). Neomycin causes auditory loss, renal failure, diarrhea, and staphylococcal superinfection. Metronidazole neurotoxicity can be severe in cirrhotics (38). Antibiotics alter flora and result in bacterial resistance. Even dietary recommendations come with disadvantages; compliance is low, and an overly negative protein balance lead to loss of muscle mass and susceptibility to infections (39). As a result of these concerns, clinicians and patients at times under-appreciate and under-treat HE, and often overlook minimal HE. Clearly, safe, well-tolerated, inexpensive alternatives are needed. Rationale for probiotics Probiotics may have multiple beneficial effects in the treatment of minimal HE. In principle, probiotics may exhibit efficacy in the treatment of hepatic encephalopathy by: 1. Decreasing total ammonia in the portal blood by: (a) decreasing bacterial urease activity, (b) decreasing ammonia absorption by decreasing ph, (c) decreasing intestinal permeability, (d) improving nutritional status of gut epithelium. 2. Decreasing inflammation and oxidative stress in the hepatocyte leading to increased hepatic clearance of ammonia and other toxins. 3. Decreasing uptake of other toxins. These processes may be additive or synergistic in treating minimal HE. First, by altering gut flora composition, selected viable, non-pathogenic bacteria can directly decrease ammonia production and absorption. This can be accomplished by changes in gut metabolism and ph, gut permeability, and the nutritional status of gut epithelium. As noted above, urease is a critical enzyme of bacterial lumenal metabolism that results in ammonia production and increased ph. Increased ammonia generation and higher ph accelerate ammonia absorption into the portal blood; decreased ph result in decreased ammonia absorption. Probiotics may alter this process by competitive inhibition with urease-producing bacteria and increasing lumenal bacteria concentration. Experimental evidence (presented below) have proven these mechanisms in humans. The exact mechanism by which probiotics have been shown to decrease fecal urease activity and ph are uncertain, but probiotics have been demonstrated to result in reduced concentrations of many bacteria (40), particulary gram negatives that produce urease. Further, probiotics improve human intestinal permeability in experimental models (41). In addition, some have proposed that probiotics may Medical Hypotheses (2003) 61(2), 307 313

310 Solga enhance intestinal epithelial viability by providing essential nutritional support (e.g., medium chain fatty acids) that inhibits apoptosis of lumenal epithelial cells (42). Thus, there are numerous possible mechanisms by which probiotics could decrease the absorption of ammonia into the portal blood. Second, an extensive body of research has demonstrated that gut-derived inflammatory signaling adversely effects the hepatocyte itself, and that therapy directed against gut flora (e.g., probiotics) can limit or reverse this damage. These observations were first made inflammation (i.e., NfK-B and TNFa) in the livers of mice that were fed oral probiotics (48). Third, probiotics might inhibit the uptake of toxins other than ammonia that have not yet been identified. This notion is supported by research in patients with end stage renal disease on hemodialysis. These patients often have altered mental status due in part to gut-derived toxins, such as phenol and indican, that not cleared by dialysis. Trials of lactic acid probiotics in humans with end stage renal disease to alter the gut flora and consequently reduce such toxins have demonstrated efficacy Summary of putative mechanisms Lactulose Antibiotics Probiotics Alter flora, + N4 þ production + Intra-luminal ph, + N4 þ absorption Alter short chain fatty acid production + Intestinal permeability + Inflammatory signaling, mitochondrialoxidative stress in hepatocyte p p p p p p p p p p + Absorption of other toxins in rodent studies that identified a pathogenic role for intestinal bacteria in alcohol-induced liver disease. When ethanol-fed rats are given neomycin (to partially decontaminate the gut) polymyxin (43) (to bind lipopolysacchride (LPS) and reduce its translocation from the intestinal lumen into the mesenteric blood) or lactobacillus (44) (to modify intestinal flora), they are protected from alcohol-induced liver damage. This protective effect is the result of reduced hepatic exposure to intestinal products, such as LPS, that promote the release of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFa), from hepatic macrophages. Similar mechanisms are now acknowledged to be important for the pathogenesis of both alcohol-related and non-alcohol related fatty liver disease (45,46), and a growing body of evidence suggests that the same mechanism may also contribute to liver damage caused by other hepatotoxins. Accordingly, there may be a common mechanism (namely, LPS-induced hepatotoxicity) that explains how diverse insults lead to liver damage (47). Such damage, in turn, disrupts normal hepatocyte function and leads to mitochrondial oxidative stress. Ultimately, the hepatocyte is impaired, and the clearance of toxins (including ammonia) is reduced. Treatment with probiotics may be ideal because they may protect against inflammation and hepatocyte damage from intestinal flora due to numerous mechanisms. As noted above, this has already been demonstrated in rodent models of alcohol liver disease. Recent work on a murine model of non-alcoholic fatty liver disease also supports this concept. These investigators found improvement in numerous molecular markers of (49,50). As noted above, some of the efficacy lactulose may indeed derive from its action as a prebiotic encouraging the growth of the same lactic acid bacteria used in probiotics. Probiotics are inexpensive, safe, and have no known negative long-term effects. This hypothesis is especially timely given that the expanding list of positive effects of probiotics are delineated by various laboratories (51,52). Further, probiotics are a natural therapy and, as such, are widely accepted by the public. Indeed, they are sometimes considered part of complementary or alternative medicine (CAM). Studies consistently demonstrate extensive use of CAM by patients, including those with liver disease (53). PRELIMINARY DATA The study of hepatic encephalopathy has been greatly hindered by the lack of properly designed therapeutic trials (54). According to a recent consensus statement, criticisms that apply, to some degree, to all trials include the large spectrum of clinical conditions summarized under the [term hepatic encephalopathy], the definition of study endpoints, the treatment of control groups, and the methods used to quantify therapeutic effects (55). Unfortunately, no useful animal models exist to study minimal hepatic encephalopathy. Accordingly, preliminary data must nevertheless come from relevant human trials and consideration of the relevant mechanisms of action. All four published studies on the effect of probiotics on hepatic encephalopathy have demonstrated efficacy (56 59). These trials employed high doses of non- Medical Hypotheses (2003) 61(2), 307 313

Probiotics can treat hepatic encephalopathy 311 urease-producing bacteria, either Lactobacillus acidophilus or Enterococcus faecium SF68. Because these studies did not employ highly concentrated, viable bacteria, they required frequent dosing and/or ingestion of a large quantity of fluid (up to a liter). Further, the mechanisms of action of these probiotic strains in liver disease or hepatic encephalopathy are uncertain, and have not been thoroughly studied with this interest in mind. Finally, these studies were small, lacked a placebo controlled design and firm, well-established endpoints. Nevertheless, their success demonstrates a certain proof of principle that warrants further attention. One possible probiotic compound that might be ideally suited to HE is the highly concentrated combination probiotic, VSL#3.This product contains 5 10 11 cfu/g of viable, lyophilized bifidobacteria (Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve), lactobacilli (L. acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp. Lactobacillus bulgaricus, and Lactobacillus plantarium) and a mixture of Streptococcus thermophilus strains. Viability has been proven by stool collection (60). Potential advantage for its application to HE include: 1. VSL#3 has been shown to reduce stool urease activity in humans. 2. VSL#3 has been shown to reduce stool ph in humans. 3. VSL#3 alters production of short chain fatty acids in humans. 4. VSL#3 improves intestinal permeability and decrease inflammatory signals in murine and human colonic cell culture models. First, VSL#3 has been proven to reduce stool urease activity. In a clinical trial (61), 10 patients with irritable bowel syndrome or functional diarrhea were given VSL#3, and urease activity was measured at study entry, 20 days after VSL#3 administration, and 10 days after discontinuation. The investigators found a greater than 50% reduction during VSL#3 administration, and a subsequent return toward baseline levels upon discontinuation. Second, VSL#3 is proven to reduce stool ph (60). Stool specimens were studied in 20 patients with ulcerative colitis who were intolerant of or allergic to 5-aminosalicylic acid in order to determine the impact on fecal composition by VSl#3. Stool composition of component bacteria all increased significantly. Of particular interest is that the stool ph dropped significantly (p < 0:005) and remained stable throughout the treatment. Since uptake of nitrogenous compounds is favored by a higher ph and diminished by a lower ph, this effect could have a major impact on ammonia generation in patients with cirrhosis. Further, VSL#3 may reduce short chain fatty acids, including butyrate in particular. In vitro culture of human ileostomy effluent inoculated with VSL#3 demonstrated a decrease in short chain fatty acids and butyrate compared to control (62). VSL#3 also improves intestinal permeability and decreases inflammatory signaling in murine colitis models (the interleukin-10 knockout mouse) and human colonic cell cultures (T84 monolayers) (63). Oral VSL#3 for four weeks lead to decreases in mucosal secretion of the pro-inflammatory cytokines TNFa and interferon c and increased resistance to samonella invasion. Finally, as noted previously, an attribute shared by all probiotics is their intrinsic safety and tolerability. CONCLUSIONS Hepatic encephalopathy is a serious and common complication of liver disease. While the exact pathogenesis remains uncertain, nitrogenous products of gut flora metabolism certainly play a critical role. 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