Cholesterol lowering with bilesalt hydrolase-active probioticbacteria, mechanism of action,clinical evidence, and futuredirection for heart healthapplicationsMitchell L Jones, Catherine Tomaro-Duchesneau, Christopher J Martoni &Satya Prakash††McGill University, Faculty of Medicine, Departments of Biomedical Engineering, Physiology, and
Artificial Cells and Organs Research Center, Biomedical Technology and Cell Therapy Research
Laboratory, Montreal, Quebec, Canada
Introduction: Cardiovascular diseases (CVD) are the leading cause of global
mortality and morbidity. Current CVD treatment methods include dietary
intervention, statins, fibrates, niacin, cholesterol absorption inhibitors, and
bile acid sequestrants. These formulations have limitations and, thus,
additional treatment modalities are needed. Probiotic bacteria, especially
bile salt hydrolase (BSH)-active probiotic bacteria, have demonstrated
cholesterol-lowering efficacy in randomized controlled trials.
Areas covered: This review describes the current treatments for CVD and the
need for additional therapeutics. Gut microbiota etiology of CVD, cholesterol
metabolism, and the role of probiotic formulations as therapeutics for the
treatment and prevention of CVD are described. Specifically, we review
studies using BSH-active bacteria as cholesterol-lowering agents with empha-
sis on their cholesterol-lowering mechanisms of action. Potential limitations
and future directions are also highlighted.
Expert opinion: Numerous clinical studies have concluded that BSH-active
probiotic bacteria, or products containing them, are efficient in lowering
total and low-density lipoprotein cholesterol. However, the mechanisms of
action of BSH-active probiotic bacteria need to be further supported. There
is also the need for a meta-analysis to provide better information regarding
the therapeutic use of BSH-active probiotic bacteria. The future of
BSH-active probiotic bacteria most likely lies as a combination therapy with
already existing treatment options.
Keywords: bile salt hydrolase, cardiovascular disease, gut microbiota, heart health,
hypercholesterolemia, hyperlipidemia, mechanism of action, microbiome, probiotic
Expert Opin. Biol. Ther. (2013) 13(5):631-642
1. Introduction
1.1 Overview of hyperlipidemia and cardiovascular diseaseCardiovascular disease (CVD), responsible for an estimated 16.7 milliondeaths worldwide, is the leading cause of global mortality and morbidity [1].In Canada, 32.1% of all deaths in 2004 were due to CVD [2]. In 2007, 1.3 millionCanadians were diagnosed with CVD by a health professional [2]. AmongCanadians 75 years and older, 20.1% of women and 26.9% of men reported having
CVD [2]. Economically, the direct and indirect costs of CVDhave reached $22.2 billion in Canada and $403.1 billion inthe United States [2]. A total of 162 million prescriptions werefilled in Canada for the treatment of CVD [2]. Coronaryartery disease (CAD), the most common form of CVD,is now the leading cause of death globally, as reported bythe World Health Organization (WHO), accounting for7.25 million deaths a year [3]. According to the present trends,one in two healthy males and one in three healthy females willdevelop CAD in their lifetime in the United States [4].Among confirmed independent risk factors, clinical
and epidemiological evidence have established a clear linkbetween elevated serum cholesterol and CAD [5-7]. Serumtotal cholesterol (TC) levels are correlated with CAD riskover a broad range of cholesterol values and in many popu-lations of the world [7]. Furthermore, evidence demonstratesa log-linear relationship between increasing low-densitylipoprotein cholesterol (LDL-C) concentration and arelative risk for CAD [8]. In addition, clinical trials forcholesterol-lowering therapies have generally confirmedthis relationship, showing an almost identical pattern ofassociation [9].This review highlights the need for additional cholesterol-
lowering therapeutics. We introduce the use of probioticformulations for the treatment and prevention of CVD. Wefocus on bile salt hydrolase (BSH)-active bacteria of the gutmicrobiota, and their use as cholesterol-lowering agents. Thecurrent evidence from in vitro, pre-clinical and clinicaldata is presented. A discussion on the cholesterol-loweringmechanisms of action of BSH-active probiotic bacteria isundertaken. Potential limitations and future directions arethen highlighted.
1.2 Current treatment modalities and limitationsThe primary target for the management of lipid levels innational and international guidelines remains LDL-C [10-12].Lower target cholesterol levels have also been recommendedfor patients with CAD, patients with very high CAD riskequivalents, and asymptomatic primary prevention patientswith multiple risk factors [11,13]. Dietary intervention is thefirst line of treatment; however, the effects are insufficientfor most individuals. Previous studies have shown that com-plete elimination of dietary cholesterol and limiting fat con-tent to < 10% of the daily caloric intake, results in only a4% regression of atherosclerotic plaques after 5 years, whencombined with stress management and aerobic exercise [14].
Several pharmacological agents are currently available forthe treatment of elevated LDL-C. These include 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins),fibric acids (fibrates), nicotinic acids (niacin), cholesterolabsorption inhibitors, and bile acid sequestrants. Due to along history of safety and efficacy, statins are the corner-stone of lipid-lowering therapy for reducing the levels ofLDL-C [15]. While statin therapy has been shown to signifi-cantly reduce the incidence of first or recurrent CAD-relatedevents, the majority of patients, especially those in the highestcategory of risk (individuals with the lowest LDL-C targets),fail to reach their goal on statin monotherapy [16-19]. Thesefindings are supported by the Lipid Treatment AssessmentProject (L-TAP), which found that 38% of patients treatedfor dyslipidemia, for a minimum of 3 months, attained theirNational Cholesterol Education Program (NCEP) LDL-Cgoals of which, only 18% with CAD reached these goals [19].Moreover, because of intolerance and safety concerns, patientsfrequently do not begin on or are titrated to an appropriatestatin dose [17,20]. Furthermore, the efficacy of statins islimited if stringent goals for serum LDL-C levels are notachieved in patients receiving statins alone or if side-effectsdevelop that require dose reduction or discontinuation [10,19].Studies have demonstrated that a doubling of the statin doseresulted in only a 6 -- 8% LDL-C reduction using currentlyavailable treatments [21,22]. Statins have also been found tobe less effective in lowering other lipid markers, such astriglycerides and lipoprotein(a), that are associated with therisk of atherosclerotic vascular disease [23,24]. Further, non-compliance to cholesterol-lowering medications was reportedin several clinical trials for primary and secondary prevention,with the risk of discontinuation shown to increase continu-ously over the treatment period, reaching 30% after 5 years [25].Thus, a wide gap between target and practice currently exists,and additional treatment modalities for LDL-C shouldbe explored.
1.3 Need for additional cholesterol-lowering
therapeuticsIn order to achieve a greater LDL-C reduction and, in turn,increase the percentage of patients attaining their NCEPLDL-C goal, it may be necessary to combine statins with
Article highlights.
. CVD, responsible for an estimated 16.7 million deathsworldwide, is the leading cause of global mortalityand morbidity.
. There is a growing interest in the use of probiotics,“live microorganisms which when administered inadequate amounts confer a health benefit on thehost,” for the treatment of CVD, specifically relatedto hypercholesterolemia.
. BSH activity of selected probiotic bacteria is associatedwith probiotic cholesterol-lowering properties inpreclinical and clinical trials, leading to decreased TC,LDL-C, non-HDL-C and apoB-100, with no significantside effects.
. There are a number of potential mechanisms of actionresponsible for BSH-active probiotic cholesterol lowering,including influencing bile acid binding of FXR anddecreased inward cholesterol transport throughABCG5/G8 and/or NPC1L1.
. BSH-active probiotic bacteria show potential, aloneor in combination with other cholesterol-loweringtherapeutics, for the treatment and prevention of CVD.
This box summarizes key points contained in the article.
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agents that affect cholesterol through a different mechanismof action. For example, additive effects in reducing LDL-Cmay be achieved by combining a statin with either a bileacid sequestrant or a cholesterol absorption inhibitor (e.g.,ezetimibe), or with either of these classes plus niacin. Inpatients with elevated triglycerides, in those whom the pri-mary concern is pancreatitis rather than CAD, the combina-tion of a fibrate plus niacin or one of these agents plus fishoil (omega-3 fatty acids), or all three may be optimal [26].The combination of a statin with niacin or fenofibrate canprovide increased benefits in raising high-density lipoproteincholesterol (HDL-C) and reducing triglycerides in patientswith mixed dyslipidemias [26]. Niacin may also be added toan existing regimen to lower lipoprotein(a) levels. In additionto the potential for augmenting beneficial changes in lipidlevels, combination therapy may be based on the consider-ation that the use of lower doses of multiple drugs may resultin fewer or less severe side effects than the use of higher ormaximum doses of a single agent [26]. However, reports haveindicated that new drugs such as ezetimibe and torcetrapib,either have not shown an incremental reduction of secondaryendpoints in atherosclerotic disease, such as carotid intima-media thickness [27,28], or have had unanticipated adverseeffects [29]. Consequently, additional agents, such as pro-biotics, that target the metabolism of lipoproteins to improveoutcomes in patients with CVD could prove to be beneficial,with no or little risk for side effects.
1.4 Bile and bile saltsBile salts are synthesized from cholesterol in the liver, throughthe rate-limiting enzyme cholesterol 7a-hydroxylase(CYP7A1), and are the primary route of cholesterol excretionin the body. After synthesis, more than 99% of primary bileacids are conjugated either with the amino acid glycine ortaurine. The majority of bile salts are stored in the gallbladderand associated into mixed micelles containing bile salts,cholesterol and phosphatidylcholine. Upon gallbladder con-traction, often as a result of a meal, bile is released into theduodenum thus entering the enterohepatic circulation. Bilesalts have a number of functional roles, principally facilitatingthe intestinal digestion and absorption of dietary fats and fatsoluble vitamins [30]. Conjugated bile salts undergo severalimportant bacterial transformations in the gastrointestinaltract, including deconjugation and dehydroxylation by gutmicrobes, resulting in secondary bile salts such as deoxycho-late and lithocholate [31,32]. Bile salts are efficiently conservedin the enterohepatic circulation. After secretion into theintestine, the vast majority of bile salts are re-absorbed, eitherthrough active transport in the terminal ileum and possiblythe jejunum for conjugated bile salts, or passive diffusionin the small and large intestine for unconjugated bile salts [33].Following re-absorption, bile salts are transported through theportal circulation and returned to the liver, completing theenterohepatic circulation. In every bile cycle, approximately4% of bile salts are lost in the feces and de novo bile salt
synthesis from cholesterol is used to maintain the poolsize under steady state conditions. Bile salts are essential inmaintaining cholesterol homeostasis. Approximately 10% ofcholesterol is excreted in an unmetabolized form and evenless is utilized for other pathways, such as the production ofsteroid hormones and vitamin D [34]. Therefore, the conver-sion of cholesterol to bile salts in the liver and their subse-quent secretion and fecal excretion provides the major route(~ 90%) for the elimination of excess cholesterol. Hence,one can speculate that probiotic bacteria, that target themetabolism of bile salts, could prove beneficial to patientswith CVD.
1.5 Probiotic bacteriaOver the past two decades, there has been a growing interestin both the basic and clinical science of probiotics, whichare defined by the WHO as “live microorganisms whichwhen administered in adequate amounts confer a healthbenefit on the host,” and are being examined for their efficacyin preventing or treating a host of diseases [35,36]. This interesthas resulted in more than 6000 publications in the biomedicalliterature, with over 60% published since 2008 [37]. The inter-est in probiotics and GI microbiota has been echoed in themedia [38] and the business [39] worlds. The human GI tracthouses over 1014 bacterial cells, the most numerically pre-dominant of which are from the Firmicutes and Bacteroidetesphyla, with the most abundant bacteria from the Bacteroidesgenus [35,40,41]. Probiotic strains belong mostly to the generaLactobacillus and Bifidobacterium. Evidence from over700 randomized, placebo-controlled, clinical trials has shownthat certain probiotic bacterial strains may aid in: preventingmetabolic syndrome [40,42], reducing the duration of acutediarrhea [43,44], reducing the risk of nosocomial diarrhea androtavirus gastroenteritis [45], preventing sepsis and severe acutepancreatitis [46], reducing post-operative infection rate [46,47],preventing periodontal diseases [48], preventing allergy andreducing colonization by bacterial pathogens [49], improvingsymptomatology of inflammatory bowel disease [50] andreducing lipids in hyperlipidemic adults [51-56].
2. The gut microbiome and BSH-activebacterial cells
The gut microbiome plays a crucial role in human health anddisease [35,57]. Interestingly, the gut microbiome, whichincludes all gut microorganisms, their genetic informationand their environmental interactions, is 150 times largerthan the human genome [58]. The gut microbiota’s relation-ship with the epithelial layers of the GI and the gut-associatedlymphoid tissue highlights the symbiotic relationship betweenthe microbiota and the eukaryotic host [59]. The gut micro-biome has evolved to perform metabolic functions otherwisenot performed by human eukaryotic cells. For example,early research demonstrated that the synthesis of riboflavin,vitamin B, pantothenic acid, vitamin B12, folic acid,
Cholesterol lowering by bile salt hydrolase-active probiotic bacteria: mechanism, clinical evidence and heart health applications
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nicotinic acid, thiamine and biotin is performed by the GImicrobiota [60].It was first observed in the 1960s that germ-free animals
accumulated cholesterol in greater quantities and at a fasterrate than their conventionally raised counterparts [61]. The pro-posed explanation was that germ-free animals catabolize choles-terol at a slower rate, and that the intestinal microbiota wasresponsible for accelerating cholesterol catabolism through anincrease in the elimination of bile acids [62]. This was furthersubstantiated when it was shown that germ-free animals haveelevated levels of conjugated bile acids throughout the intestine,significantly reduced fecal biliary excretion and three times thebile acid concentration in bile [63-67].More recently, comprehensive porcine studies showed that,
by increasing the total BSH activity of the GI microbiota, asignificant increase in the deconjugated bile acid pool wasobserved [68,69]. BSH activity is responsible for the deconjuga-tion of bile acids, by hydrolysis of the amide bond, of the con-jugated bile acid, and liberation of the glycine/taurine moietyfrom the steroid core. In addition, the GI microbiota hasbeen shown as responsible for the elimination of sterols, withthe intestinal flora promoting cholesterol catabolism in conven-tional, as compared to germ-free animals [62]. BSH activity isspecific to the microbiota and is not present in eukaryotic cells,substantiating the importance of the gut microbiota incholesterol metabolism. BSH activity has been characterizedin Lactobacillus [70-73], Bifidobacterium [74-77], Clostridium [78],Enterococcus [79] and Bacteroides [80,81]. Functions of BSH inbacterial cells are still not completely understood, but thereare several hypotheses. Potential roles, discussed in a previousreview, include nutrition, cell surface modifications, biledetoxification and GI persistence [82]. In terms of cholesterolmetabolism, mice treated with a three-day course of oral anti-biotics increased biliary bile acid output threefold while fecaloutput decreased by 70% [83]. These findings support thehypothesis that the intestinal microbiota specifically increaseddeconjugation of intestinal bile acids by BSH, and increasedlevels of deconjugated bile acids entering the enterohepaticcirculation and via the portal vein.
3. Probiotic bacteria as cholesterol-loweringtherapeutics: in vitro, pre-clinical, andclinical evidence
3.1 Cholesterol-lowering with probioticsProbiotic bacteria have been investigated for their clinicalefficacy in lowering LDL-C. A crossover study, in 30 normoli-pidemic male participants, reported a decrease in TC andLDL-C by 4.4% and 5.4%, respectively, following the con-sumption of yogurt enriched with Lactobacillus acidophilusand fructooligosaccharides three times daily [55]. A rando-mized, blinded, clinical study using 32 participants withmild to moderate hypercholesterolemia, reported a decreasein TC and LDL-C by 5.3% and 6.15%, respectively, follo-wing consumption of a fermented milk product containing
Enterococcus faecium [53]. In addition, a randomized, blinded,study in 58 non-obese, normocholesterolemic middle-agedmen, reported a decrease in LDL-C by 10% following theconsumption of a fermented milk product containingE. faecium and two strains of Streptococcus thermophilus [51].On the other hand, several placebo-controlled studies havereported little or no effect following the daily consumptionof various supplements and foods containing probioticbacteria [84-90]. These contradictory results can be largelyattributed to inadequate strain selection, small sample size orpoor clinical design [91]. In an attempt to increase statisticalpower, a recent meta-analysis integrating results from 13 indi-vidual lipid-lowering probiotic clinical studies [92] demon-strated a LDL-C mean net change of -0.12 mmol/L (95%confidence interval: -0.206 to -0.049, p < 0.01) or approxi-mately 3%. A number of research groups have also shownthat BSH-active probiotic bacteria can be used to lowercholesterol in murine models [93-95].
3.2 Cholesterol lowering with BSH-active probioticsRecently, a randomized, placebo-controlled clinical trialshowed a significant reduction in plasma TC and LDL-C asa result of the administration of BSH-active L. acidophilusCHO-220 combined in a capsule with the prebiotic inulin [54].The BSH phenotype of selected probiotic strains can be asso-ciated with the capacity of an organism to reduce cholesterollevels, as demonstrated in pre-clinical and clinical studies, aswell as improved survival of the organism in the GI tract, asBSH is associated with bile tolerance [96]. In addition, ourgroup has previously published an extensive in vitro characte-rization of Lactobacillus reuteri NCIMB 30242 using a combi-nation of molecular and metabolic analysis techniques tosupport its safe use as a lipid-lowering probiotic inhumans [97]. We have performed a double-blind, randomized,placebo-controlled, parallel-arm, multi-center study to evalu-ate the cholesterol-lowering effect of microencapsulatedBSH-active L. reuteri NCIMB 30242 (~ 1 � 1010 CFU/yogurt), in yogurt format, over 6 weeks [56]. Subjects con-suming the probiotic yogurts attained significant reductionsin LDL-C of 8.92%, TC of 4.81% and non-HDL-C of6.01%, as compared to placebo [56]. There was also a signifi-cant absolute change in apolipoprotein B-100 (apoB-100)of -0.19 mmol/L over the 6-week treatment period [56].Importantly, the placebo and treatment groups remainedcomparable for biomarkers of safety at the study endpoint [56].In a second double-blind, randomized, placebo-controlled,parallel-arm, multi-center study, we have evaluated the safetyand efficacy of an improved L. reuteri NCIMB 30242 capsuleformulation (~ 3 � 109 CFU/capsule) over 9 weeks [98].Subjects consuming the probiotic capsules attained significantreductions in LDL-C of 11.64%, TC of 9.14%, non-HDL-Cof 11.30% and apoB-100 of 8.41%, over the 9-week treat-ment period, as compared to placebo. Additionally, signifi-cant reductions in cardiovascular risk factors were observedfor fibrinogen of 14.24% and high-sensitivity-C-reactive
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protein (hs-CRP) of 192.92%. In support of the proposedmechanism of action, we found significant increases in decon-jugated bile acids, indicating increased intraluminal BSHactivity, and significant reductions in the surrogate absorptionof sterols, indicating a reduction in inward cholesteroltransport [98]. A comparison of individual LDL-C responses,at the study endpoint, indicates that, despite a proportion ofsubjects that experienced unchanged LDL-C values, a generalLDL-C lowering effect was observed. In addition, weobserved significant reductions in cholesterol esters, whichindicated a reduction in the storage form of cholesterol seenin foamy cells and atherosclerotic plaques. Finally, no adverseevents or biochemical safety endpoints were determined to besubstantial or associated with the treatment [99,100].
4. Cholesterol-lowering mechanisms ofaction of BSH
There are a number of mechanisms of action potentiallyinvolved in lowering cholesterol by BSH-active probioticbacteria, as shown in Figure 1. BSH-active probiotic bacteria
may act in the lumen of the intestine as well as in the liverand other organs. BSH-active probiotics have been shownto increase intraluminal bile acid deconjugation, resultingin increased levels of circulating deconjugated bile salts inhumans [56] and pigs [69]. The increased deconjugation ofbile salts decreases cholesterol absorption by enterocytes,simply due to a decrease in primary and secondary bileacids and the micellization capacity of conjugated andunconjugated bile salts.
In addition, by modifying the bile acid pool profile,BSH-active bacteria may influence the farnesoid X receptor(FXR), a bile acid nuclear receptor [56,101]. Endogenous bileacids activate FXR with different efficacy (chenodeoxycholicacid > lithocholic acid = deoxycholic acid > cholic acid), withthe conjugated forms being less potent [102]. It has been shownthat decreased FXR activity results in the downregulation ofsmall heterodimer partner (SHP) and increased catabolism ofcholesterol and synthesis of bile acids by CYP7A1 [101]. Thedownregulation of SHP results in the upregulation of the liverX receptor (LXR) [101]. This, in turn, leads to the upregulationof adenosine triphosphate-binding cassette transporters G5 and
LDL-Receptors
LDL-C (Blood)
7α-hydroxylase
BA (Hepatocyte)
Bile acid(CBA + BA)
(Enterohepatic)
FXR SHP
CYP7A1
LXR ABCG5/G8
BA
Cholesterol(Hepatocyte)
LDL-Receptor
ABCA1ABCG1
ABCA1ABCG1
ABCG5/G8
BSH-activeLactobacillus
ApoA-1ABCA1ABCG1
NPC1L1
LXR
BA and cholesterolexcretion (Feces)
CBA BA
BSH
(GI lumen)
SR-BIApoA-1
HDL-CCholesterol
(Macrophage)Cholesterol(Enterocyte)
CholesterolBile
(Biliary)
Cholesterol(GI lumen)
LDL-C(Blood)
Cholesterol(Hepatocyte)
BA
H
C
O
HN H
HDCA
H2O+
OH
HO
DCA
H
C OH + OH
OH
HO
Hydrolysis
Bile salt hydrolase (BSH)
O
H2N
S O
O
O
OHH2N
O
Figure 1. Schematic representation of the potential effects of probiotic BSH activity on cholesterol metabolic pathways. BSH
enzymatic activity hydrolyzes conjugated bile acids (CBA) to deconjugated bile acids (BA).
Cholesterol lowering by bile salt hydrolase-active probiotic bacteria: mechanism, clinical evidence and heart health applications
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G8 (ABCG5/G8) [103]. The upregulation of ABCG5/G8results in a decrease in the intestinal absorption of cholesterol,while promoting their biliary excretion. Support for thishypothesis can be found in the finding that the oral feedingof Lactobacillus plantarum KCTC3928 to mice resulted in sig-nificant LDL-C and triglyceride lowering effects, increasedfecal bile acid excretion, increased hepatic bile acid synthesisand increased expression of CYP7A1, enhancing cholesterolcatabolism and bile synthesis [93]. Also, it was found that theoverexpression of ABCG5/G8 in transgenic mice limits sterolabsorption and promotes neutral sterol excretion [104]. Further-more, the deconjugated bile acids preferentially increaseATPase activities of ABCG5/G8 transporters found on theapical membranes of enterocytes and hepatocytes, limitingthe accumulation of cholesterol by transporting it into theintestinal lumen and bile [105]. In addition, BSH-active Lactoba-cillus, using Caco-2 cells, resulted in the upregulation of LXRconcomitantly with the elevated expression of ABCG5/G8heterodimeric transporters, and the promotion of cholesterol
efflux, with no noticeable effects on Niemann-Pick C1 Like 1(NPC1L1) expression [103].
Another proposed mechanism of action associated withBSH activity involves the inhibition of NPC1L1, similarto plant sterols [106]. It has also been demonstrated thatBSH activity and increased circulating deconjugated bileresult in significant reductions in serum plant sterols,surrogate markers for cholesterol absorption. This is similarto the mechanism of action of the drug ezetimibe, a potentcholesterol absorption inhibitor [107]. NPC1L1 is responsiblefor the bulk movement of cholesterol into the enterocytes,with genetic inactivation resulting in a significant reductionof cholesterol absorption [108]. Indeed, a decrease inNPC1L1 was demonstrated in rats administered a BSH-active L. acidophilus [109,110]. In addition, in recent research,Yoon et al. have demonstrated a downregulation ofNPC1L1 in Caco-2 colon epithelial cells by Lactobacillusrhamnosus and L. plantarum, as well as increased cholesterolefflux in macrophages [111,112].
Table 1. Potential effects of BSH-active bacteria on cholesterol metabolic pathways.
BSH-active probiotic targets BSH-active probiotic
effect on target
Impact of modified expression on
cholesterol metabolic pathways
Refs.
ATP binding cassette A1 (ABCA1) Upregulated Increased cholesterol efflux to lipoproteinsIncreased plasma HDL-C levels
[110,112]
ATP binding cassette G1 (ABCG1) Upregulated Increased cholesterol efflux [110,112]
*Effective dose in grams of dry ingredient.zApproved Food and Drug Administration (FDA) heart health claim.§Study performed with BSH-active Lactobacillus and prebiotic inulin [54].
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In addition, a recent study by Jones et al. demonstrated thatincreased circulating bile was correlated with a reductionin serum LDL-C and apoB-100 [56]. ApoB-100 is found innon-HDL-C cholesterol originating from the liver, and itslevels are well-correlated with CAD [113]. ApoB-100 is aprimary structural protein of the atherogenic lipoproteins,and so its decrease results in reducing LDL-C serum levels.Indeed, Sniderman et al. have demonstrated an inverse rela-tion between bile acid synthesis and apoB-100 secretion [114].This could point to an alternate mechanism of actionof bacterial BSH activity. Of interest is the fact that thecholesterol-reducing drug-candidate Mipomersen, an anti-sense oligonucleotide, acts on a similar mechanism of actioninvolving the inhibition of apoB-100 synthesis in the liverthrough RNA interference technology [115].
Despite a growing understanding of the mechanismsthrough which increased deconjugation of bile may lead toincreased cholesterol catabolism, reduced cholesterol absorp-tion and reduced serum cholesterol, detailed studies arerequired to elucidate the precise mechanisms of action.
5. Conclusion
CVD is the leading cause of global mortality and morbidity,but current treatment modalities are inefficient and presentimportant side effects and toxicity issues. Using germ-free animal studies, the role of the GI microbiota in choles-terol metabolism was demonstrated. A number of probioticbacteria have been successfully used as cholesterol-loweringbiotherapeutics. BSH, an enzyme expressed solely by bacteria,including a number of probiotic bacteria, is responsible forthe deconjugation of bile salts in the intestinal lumen, limitingthe absorption of cholesterol through the enterocytes. Theadministration of BSH-active probiotic bacteria has demon-strated significant cholesterol-lowering effects in vitro and inpreclinical and human clinical studies. There are a numberof hypothesized mechanisms of action by which the BSH-active probiotic bacteria may be exerting their cholesterol-lowering effects. Players of the cholesterol metabolic pathwaysthat may be modulated by probiotic BSH activity include, butare not limited to, FXR, SHP, CYP7A1, LXR, ABCG5/G8,NPC1L1 and apoB-100. Despite the abundance of existingdata on the beneficial effects of BSH-active probioticbacteria and their modulation of important regulatorsinvolved in cholesterol metabolism, detailed mechanisticstudies are nonetheless required for a complete understandingof the cholesterol-lowering effects. This is crucial for thefurther optimization and development of a probioticcholesterol-lowering therapeutic.
6. Expert opinion
Probiotic bacteria have gained much interest in recent yearsfor the prevention and treatment of a number of healthdisorders. BSH-active probiotic bacteria have shown promise
for their cholesterol-lowering properties and as a potentialadjunct therapy for the management of CVD, in several largerandomized placebo-controlled clinical trials. As described inthis review, an important consideration for BSH-active probi-otics is the need for a better understanding of the mechanismsof action responsible for the observed cholesterol-loweringeffects. Table 1 presents a list of hypothesized targets ofBSH-active probiotic bacteria for their cholesterol-loweringeffects. A second consideration is safety, as with the deliveryof all live probiotic microorganisms. On this point, clinicaltoxicological studies have been published confirming thesafety and tolerability of BSH-active probiotic bacteria [99,100].These studies have reported no significant changes inhematological markers of safety, biochemical markers ofsafety or adverse effects [56]. Moreover, an improvement ingastrointestinal symptoms, including GI discomfort, bloating,constipation and diarrhea, was detected by a Rome III GIquestionnaire following BSH-active probiotic administra-tion [116]. Of interest is the comparison of BSH-active probi-otics with available nutritional supplements for cholesterollowering, specifically in terms of a significantly lower effectivedose, as presented in Table 2.
The potential link between widespread antibiotic use and thehigh prevalence of hypercholesterolemia in Western societies isalso of interest. The intestinal microbiota plays an importantrole in the metabolism of cholesterol by interacting with bileacids and cholesterol. In the feces of man and other mammals,the major microbial cholesterol derivative is coprostanol. Itis well-established that germ-free animals lack cholesterol-metabolizing microbes and do not have an intestinal microbialexcretion pathway for cholesterol. As a result, they have reducedde novo synthesis of cholesterol and higher serum cholesterollevels than their conventional counterparts [117]. Antibiotic usehas been shown to permanently change the GI microbiotaresulting in significantly reduced diversity, as well as reducedconversion of cholesterol to coprostanol. Hence, althoughthe Western diet is commonly cited as a causative factor forhypercholesterolemia, reduced BSH activity due to widespreadantibiotic use may also play a causative role and should be inves-tigated. A metagenomic analysis of patients exposed to anti-biotics compared to naive patients should be examined toevaluate the relative quantity and conservation of the BSHphenotype following antibiotic use.
BSH-active probiotic formulations may have potential forcholesterol lowering in combination with plant sterols,omega-3 fatty acids, fish oils, and statins. Plant sterols act bycompetitively inhibiting cholesterol absorption by NPC1L1transporters and have been shown to increase levels of serumplant sterols [118]. Recently, plant sterols have been foundas part of atherosclerotic plaques [119] and in the retina oflong-term plant sterol and stanol users [120]. BSH-activeprobiotic bacteria are postulated to function by inhibitingthe absorption and/or increasing the excretion of neutralsterols, likely through the upregulation and increased activityof ABCG5/G8 transporters. In addition, they have been
Cholesterol lowering by bile salt hydrolase-active probiotic bacteria: mechanism, clinical evidence and heart health applications
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shown to reduce circulating cholesterol and plant sterols [98].Thus, a combination of plant sterols and BSH-active pro-biotics should be evaluated for an improved cholesterol-lowering effect while reducing plant sterol serum levels,as well as potentially improving the safety profile of sterolproducts. On the other hand, fish oils, omega-3 fatty acids,medium chain triglycerides, and alpha-linoleic acids aimat improving the ratio of ‘good’ to ‘bad’ fatty acids in theblood. The result of consuming beneficial fatty acids is thatthe esterified cholesterol profile is improved by increasingthe proportion of monounsaturated and polyunsaturatedcholesterol esters at the expense of saturated fatty acidcholesterol esters, a shift that has been correlated with a lowerrisk of atherosclerosis and cardiovascular events. A com-bination of beneficial fatty acids and BSH-active probioticsmay result in a significant decrease in serum cholesterol whileimproving the relative molar fraction of beneficial fattyacids esterified to cholesterol. Finally, it is postulated thatBSH-active bacteria would work in a complementary fashionwith statins to amplify LDL receptor activity and the clear-ance of serum cholesterol [56], as they increase bile salt decon-jugation and reduce sterol absorption. Therefore, a greaterreduction in serum LDL-C is hypothesized when admini-stering BSH-active probiotic bacteria concomitantly to statin
monotherapy. Well-designed randomized controlled clinicalstudies should be performed to investigate the use of suchcombination therapies. There is also a need for meta-analyses to better examine the variation in study outcomesbetween studies. Factors that may impact on outcomes ofcholesterol-lowering strategies with BSH-active probioticbacteria must also be examined.
Acknowledgments
The authors would like to acknowledge a Canadian Instituteof Health Research (CIHR) Grant (MPO 64308) andgrants from Micropharma Ltd. to S Prakash and a DoctoralAlexander Graham Bell Canada Graduate Scholarship fromthe Natural Sciences and Engineering Research Councilof Canada (NSERC) to C Tomaro-Duchesneau. ML Jonesand C Tomaro-Duchesneau contributed equally to this work.
Declaration of interest
ML Jones and S Prakash are co-founders and shareholders ofMicropharma Ltd and report a conflict of interest. CJ Martoniis employed by and is a shareholder of Micropharma Ltd.C Tomaro-Duchesneau reports no conflict of interest.
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