The biliary cycle of Moritz Schiff

Cycles and circles, loops and revolutions seem to be fundamental to our universe in which they are, simply put, universal. The trajectories of the planets around their stars and the moons around their planets are circles (or, more strictly, ellipses) on a grand scale, as are the motions of electrons around their nuclei at the subatomic level. The rotation of our globe provides the cycles of night and day, and its orbit around the sun the familiar cycle of the seasons—captured vividly in the art of Pieter Bruegel the Elder (or was it Pieter Brueghel the Elder?) and melodically by Antonio Vivaldi in Le Quattro Staggioni (The Four Seasons) from his twelve-violin concerti collection entitled Il Cimento dell'armonia e dell'inventione (The Contest of Harmony and Invention). The seasons in turn drive the rhythm of terrestrial life and the labors and cyclical rituals of human existence, all of which are aided by the replenishing cycles of water and carbon. Cycles of biochemical reactions, even occasionally futile ones, underpin the success of living organisms, while innumerable engines and machines devised with human ingenuity have at their core that primitive circular invention, the wheel. In animal physiology, the best known and scientifically perhaps the most sacred cycle is William Harvey's circulation of the blood1 that supplanted the far-fetched and fanciful flux and reflux schemes imagined by Aristotle, Hippocrates, and Galen, which were later promulgated by their disciples in the East and the West for fifteen hundred years or more. Embedded in Harvey's discovery was also the concept of the feedback loop, which in the case of the circulation of the blood is a positive one. According to Ernest Henry Starling,2 the greater the venous return to the heart the greater the cardiac output, until the ventricles fail. In the domains of hepatobiliary physiology and pathophysiology there are cycles and feedback loops too, both positive and negative in nature. The notion that there exists a biliary cycle in liver physiology dates back to at least the 17th Century, when the composition and functions of bile began to be appreciated3 and shortly after Harvey unveiled his paradigm for the circulation of the blood. Until then, according to anatomist Thomas Gibson, bile was dismissed as "…a mere excrement, and of no other use than by its acrimony to promote the excretion of the Guts."4 In the 16th Century, Andreas Vesalius thought of bile as "…the thinner refuse of the liver,"5 and Realdo Colombo of Cremona wrote that "bile is discharged…into the intestines to excite their activity, and…compel them to excrete the faeces."6 Even Francis Glisson in the mid-17th Century concluded that bile was "…solely an excrementitious fluid."7 Gibson, undaunted by these luminaries of anatomy, nonetheless realized that bile was necessary for life, for he reasoned of the liver that "…so bulky a Bowel was never made for the separation of a mere excrement."4 But it was Flemish alchemist turned chemist, physiologist, and physician Johann Baptista van Helmont, the man who coined the term "gas," who vindicated bile as viscus nobile et vitale.8 Mauritius van Reverhorst, inspired by Harvey's reasoning that the large volume of daily blood flow can only occur if blood re-circulates, applied a similar argument to bile flow. In other words, he proposed that there must be a motus bilis circularis; i.e., "an enterohepatic circulation of bile." On December 17, 1691, Reverhorst cannulated the bile duct of a living dog and calculated from its output in just a few hours that the amount of bile that would enter the duodenum each day would surely exceed the amount that could be excreted from the gut.9 Reverhorst reasoned that the "earthy" sediment fraction of bile is excreted and the more "volatile parts" enter the mesenteric veins via "pores" in the gut, whence they are returned to the liver.9 Beguiled by the concept of re-cycling, Reverhorst argued that there should be a circulatory movement not only of blood and bile, but also of saliva, gastric juice, pancreatic juice, lymph, and, believe it or not, even semen. The task of assessing the kinetics of the enterohepatic circulation of the bile was assumed by Giovanni Alfonso Borelli,10 mathematician, physicist, astronomer, physiologist, and early biophysicist. Borelli had been a pupil of Galileo Galilei and sometime teacher of Marcello Malpighi, the renowned anatomist who not only described the microscopic "bile capillaries" of the liver11 but who also proved conclusively that it is the liver and not the gallbladder that makes bile.12 Borelli's arithmetic and geometry may not have been as sharp as the rest of his science, since he appears to have made errors in calculating the cycling frequency of the bile pool that he thought moved around 16 or 17 times daily, and in his mensuration of the vessels of the liver, at least according to his translator Paul Maquet.10 Yet, Borelli's propositions concerning the exclusive formation of bile from the blood that flows through the liver, the anatomic sites of the enterohepatic circulation, and the roles played by bile in the digestion of fat and the promotion of muscular activity in the intestine10 were made with uncanny prescience some four hundred years ago. Armed with the observations of his former pupil, Malpighi, of the microscopic anatomy of the liver lobule and the hepatic origin of bile, Borelli reasoned that bile flows from the liver into the duodenum, where it mixes with digested food and then passes through pores in the intestinal wall to enter the mesenteric veins for carriage in portal venous blood back to the liver. Borelli even inferred that only a small amount of bile enters the stools. The propositions of van Reverhorst and Borelli, revived early in the 18th Century,13 were developed further by Edward Barry,14 Regius Professor of Physic at Trinity College in the University of Dublin, Ireland. Barry realized that the enterohepatic circulation was a mechanism for the body to conserve what he viewed as "…the most concocted Humour"; i.e., a most complex fluid. Upwards of 40 years before the earliest work on bile acids was done at the beginning of the 19th Century, and some 80-odd years before the Great German chemist Justus von Liebig suggested "bile acids" as a generic term,15 Barry predicted the function of this group of compounds that was yet to be discovered and described their key role in the enterohepatic circulation. Barry wrote, "it is likewise more than probable, that the more acrid and active Parts of the Bile, which remain dissolved in the Intestines, after the Chyle has passed the Lacteals, are likewise received into the Meseraic Veins, which…soon return again, by this short Circulation, to the Liver; and by these Means supply it with active, genuine Materials, fit for the more easy Preparation and Secretion of new bile."14 Support for Barry's prediction that the acrid part of the bile is absorbed in the intestine came indirectly as several investigators showed the paucity of bile products in the stools,15-18 whereas others using isolated bowel segments and loops, beginning with Hermann von Tappeiner, Director of the Institute of Pharmacology at the University of Munich,19 showed directly that bile acids could be absorbed in the small intestine.20, 21 The significance of the differential absorption of different bile salts by different regions of the small intestine was not appreciated until the presence of an active transport system for bile salts was found in the ileum,22 and John Dietschy and colleagues23, 24 found passive absorption for unconjugated bile acids in the jejunum and active transport for bile acids in the ileum. By the latter third of the 19th Century, the whim that the intestine could and would salvage for the body precious bile acids that the liver had squandered in the bile25 had become axiomatic.26 But the proof of this plausible hypothesis was lacking until Moritz Schiff, an eminent, and some would say the most pre-eminent, physiologist in Europe, conducted a simple ingenious and utterly convincing experiment that closed the loop, so to speak, of an enterohepatic circulation27 and possibly helped to seal his fate in Florence too. What Schiff, his contemporaries, and his followers for 100 years did not envisage, however, was that the intestine also exerts considerable influence on the liver with respect to the latter's transport and metabolism of bile acids. It may come as a surprise and disappointment to some hepatologists that the liver may take instructions from the gut in matters biliary. But more of that later. With the shock of his shoulder-length white hair, splendid beard and eyebrows to match, and piercing gray eyes (Fig. 1), Moritz Schiff (1823-1896), one of the most distinguished biologists of the latter half of the 19th Century, was an engaging personality and a striking figure despite his small stature.28 Of towering intellect and indefatigable industry, as recalled by fellow student Adolf Kussmaul29 Schiff was a kindly man with a fondness of teasing who gave short shrift to societal conventions.28 He was a ceaseless and untiring worker in nearly every field of physiology,30 focusing not only on his favorite topic of neuroscience,31 but also delving into the digestive tract32 and endocrinology,33 including early attempts at thyroid replacement,34 and even into open cardiac resuscitation.35 Born in Frankfurt am Main, Schiff was educated at the gymnasium there and then at the Senckenbergische Institut. He studied anatomy in Heidelberg under Friedrich Tindemann and physiology under Johann Müller in Berlin; subsequently, he obtained his medical degree at the University of Göttingen in 1844. In Paris, he studied neurophysiology under François Magendie and François Achille Longet at the Collège de France, and at the Museum des Jardins des Plantes he incidentally became an expert on the flora of South America. Eschewing medical practice for zoology he returned to Frankfurt in 1845 and was given charge of the ornithological section of the zoological museum there and became an authority here too, later collaborating with the renowned ornithologist Prince Charles-Luçien-Jules-Laurent Bonaparte, nephew of the first emperor Napoléon. His active support and participation as a military surgeon for the liberal revolutionaries in Baden during the turmoil of the 1848 uprisings in Germany and the rest of Europe later precluded his appointment as a privatdozent (i.e., a professorial aspirant) in experimental physiology at the University of Göttingen in the mid-1850s. Although his academic credentials were more than acceptable to the faculty, the government in Hanover refused to confirm Schiff's appointment on the grounds that his teaching would allegedly be dangerous to the students because of his liberal views. Instead he moved to Berne, Switzerland, as Professor of Anatomy and Pathological Physiology, and after a 7-year stint here he became Professor of Physiology in Florence, Italy, at the Museo di Storia Naturale (known colloquially at La Specola). In Florence, in the Istituto di Studi Superiori, his scientific studies flourished, including those of the enterohepatic circulation, and so did his political and legal struggles in bizarre and sinister circumstances that led to his flight to Geneva, Switzerland, where he ended his days 20 years later. Although Schiff nutured throughout his life in Italy and Switzerland a love of Germany, this certainly did not include the Prussians,28 who had brutally put down the Baden uprising, condemned him and his friend Carl Vogt (later to become the Rector who invited him to the University of Geneva) to death, and court-martialed and executed one of the leaders of the Baden revolt, the son of his old professor in Heidelberg Friedrich Tiedemann. Moritz Schiff: biliary cyclist and humane vivisectionist. In common with previous investigators, Schiff studied the enterohepatic circulation in the bile-fistula dog model. But instead of simply comparing the output of bile products via the fistula and in the stools, as others had done before him,17, 26, 27 Schiff with inventive simplicity returned to the dog's duodenum and by feeding, the fistula bile he had collected and observed a two-fold choleresis within 15 minutes, which peaked three-fold at 45 minutes.27 This suggested that the administered endogenous bile salts were absorbed by the dog's gastrointestinal tract and returned to the liver, to be secreted again to promote bile flow in a positive feedback loop. Skepticism of this interpretation was ultimately assuaged by the results of further innovative experiments, in which Weiss in Moscow fed glycocholic acid to dogs36 and Schiff fed ox bile to guinea pigs.37 In each case, the exogenous "foreign" bile acids appeared in substantial quantities in the respective animals' bile.36, 37 Whipple and Smith confirmed and extended Schiff's original observations by showing that the daily bile salt secretion rate in a bile-fistula dog is eight times greater if the drained bile is returned to the animal rather than being discarded.38 To complete the description of Schiff's biliary cycle, it remained only to show that portal blood contains bile salts, and this was demonstrated in the latter part of the 1930s.39, 40 The animal experiments that brought Moritz Schiff international recognition and acclaim as a physiologist—his published works, collected after his death by two former students, numbered more than 200 and filled 4 volumes41—led to his harassment and trial by the loosely organized anti-vivisectionist movement in Florence of well-to-do and intellectual locals and foreigners, as well as to his ill-advised late countersuit against his accusers and detractors and to his eventual escape to Switzerland.28, 42-44 Whereas opposition to vivisection had its roots in the 18th Century, supported and publicized by philosophers, literary figures, and artists,45 it made little headway then against the tide of scientific endeavor and especially not against the swell of physiological discovery by animal experimentation. Nowhere in Europe was concern over cruelty to animals more vocal and successful than in Great Britain, where its prevention became a watchword of 19th Century Victorian morality that promoted and was in turn spurred on by the passage of Richard Martin's Act in 1822 concerning the humane treatment of domestic farm animals. Other European countries had active anti-vivisectionist movements too, but these were often marginalized by the scientific community, and Great Britain was the only state to have enacted such animal rights laws by the end of the 19th Century. For Schiff, his troubles began shortly after his arrival in Florence, probably by chance because of the vacation there, with the English colony, in 1863 of Anglo-Irish social reformer and feminist theorist Frances Power Cobbe, who was arguably the most experienced and determined of British anti-vivisectionists.46 Following the December 1863 publication in the English newspaper The Daily News of false claims of Schiff's cruelty to animals, a heated debate ensued in the local Italian press, La Nazione, Lo Zenzero, the Gazetta Del Popalo and other media, to which Schiff and his supporters responded with lively defense. Ironically, Schiff had always openly advocated humane care of animals; where feasible he proposed the use of organs for research rather than whole animals, and he insisted on the universal administration of adequate analgesia and anesthesia.43 After a somewhat fallow period, the anti-vivisectionist debate was rejoined in Britain and abroad, aggravated by a well-publicized absinthe experiment on dogs that was perpetrated by French psychiatrist Valentin Jaques Joseph Magnan at the 1874 meeting of the British Medical Association. Among other events, this led to the appointment of a Royal Commission of inquiry and the passage a couple of years later of the 1876 Cruelty to Animals Act. There had been vitriolic articles published against Schiff in the Times, the Morning Post, the Spectator, and other British newspapers as well. By 1873, Schiff had already been forced to move his laboratory because of popular local complaints about animal noises,43 and in 1874, on questionable legal advice, he brought suit against his former antagonists, which embroiled him and his supporters in distasteful public hearings. These were reported in detail by his assistant and former student Alexandre Herzen,42 son of the famous Russian democratic socialist, pro-Western writer, and thinker Aleksandr Ivanovich Herzen. In 1876, exhausted by the 12-year–long wearisome fight with the anti-vivisectionists in Florence, Schiff fled and took up his last position at the University of Geneva, which was offered to him by his longtime friend Carl Vogt. It must be added as a footnote to this unfortunate history that Schiff's relations with the society for the prevention of cruelty to animals in Geneva were cordial and collaborative,28, 43 and he pursued his work there unmolested. Once the reality of the enterohepatic circulation of bile acids was accepted, it only remained for modern cholephiles to derive its kinetic parameters, find out the consequences when the process goes awry, define the mechanisms and forces that propel bile acids around the circuit in their perpetuum mobile, and elucidate how bile acids are synthesized and how this synthesis is regulated. It took the best part of the 20th Century, however, to accomplish these tasks, which early on encompassed the elegance of isotope-derived kinetic and metabolic analyses47-51 and marathon tour de force multi-lumen intestinal marker perfusion studies52-54 that, in the author's personal experience of both ends of the perfusion tube,55 try the patience of experimental subject and investigator alike.55 For the dynamics of the enterohepatic circulation and the metabolism of bile acids in health and disease, authoritative reviews abound.56-62 With respect to the forces which drive bile acids around their circuit, it soon became clear that the mechanical contraction of gallbladder and intestinal peristalsis63 act in series with the energy-requiring sodium-dependent uptake of bile acids, like taurocholate, by the basolateral membranes of hepatocytes and the apical membranes of terminal ileal enterocytes.64, 65 Of course, many transporters for bile acids and other ligands have now been identified on hepatocytes, bile duct cells, and intestinal cells that all participate in the enterohepatic circulation, but the pioneer landmark discoveries in the modern era in this field were the identification of the so-called Na+-taurocholate cotransporting polypeptide (NTSCP) that is expressed normally on the basolateral membrane of hepatocytes,66 and the identification of the so-called apical sodium-dependent bile salt transporter (ASBT) that is expressed on the apical membrane of terminal ileal enterocytes.67 These transporters that belong to a common sodium bile salt cotransport family (SLC10)68 were discovered by similar expression cloning strategies in the rat66 and the hamster,67 respectively, but have since been found in other species, including humans. And yet, the saga of the enterohepatic circulation of bile acids has not ended. For as Edmund, bastard son to Gloucester, says in King Lear,69 "The wheel is come full circle." What once was seen as a passive passage of bile products through "pores" in the intestine may now turn out to play a significant regulatory role in hepatic bile acid synthesis and possibly transport too. The discovery just a few years ago that bile acids are ligands for nuclear receptors, such as the farnesoid X receptor (FXR) and the pregnane X receptor (PXR), which were subsequently shown to downregulate the expression of genes of bile acid synthesis and upregulate those of cholesterol metabolism now provides a plausible mechanism for the well-known negative feedback effect of bile acids on bile acid synthesis and positive feedback by cholesterol.70, 71 What was puzzling, heretofore, was the observation that bile acid administration in vivo enterally is more potent in repressing cholesterol 7α-hydroxylase expression (the rate-determining enzyme in the bile acid biosynthetic pathway) than bile acids administered parenterally.72 In fact, even administration of bile acids via the portal vein (as well as intravenous administration) cannot suppress bile acid synthesis as effectively as intraduodenal administration,73 which suggests that a regulatory factor produced by the intestine may be involved in bile acid synthesis in vivo, possibly by interacting with the aforementioned nuclear receptors. The recent demonstration that FGF-19, a secreted member of the fibroblast growth factor family, signals through the fibroblast growth factor receptor 4 (FGFR4) cell surface receptor tyrosine kinase to suppress cholesterol 7α-hydroxylase expression in isolated hepatocytes (and, incidentally, FGF-19 is itself induced by FXR74), strongly supports the notion that a similar gut-derived growth factor, induced by intestinal bile acid flux, could mediate the feedback control exerted by the "enteric part" of the enterohepatic circulation. How much better could the wheel turn full circle? If only Moritz Schiff, a revolutionary in so many ways, had transitioned from whole animal physiology to cellular genetics in time, he might yet be considered the center of Florentine scientific circles to this day. Schiff's obituary in the Lancet in 1896 was nothing short of a eulogy.75 Doggedly Schiff would insist that nothing should be taken for granted or accepted on authority but must be verified, checked, and re-verified until doubt regarding it was at an end. Perhaps his greatest gift was to inspire young investigators in physiology whose attention he arrested, whose interest he engaged, whose intelligence he enlightened, and whose reasoning power he exercised.75 Once again, the author expresses his gratitude to Alan F. Hofmann, M.D., the supreme pedallar and peddler of Schiff's Biliary Cycle, for his friendship, mentoring, enlightenment, gentle corrections, and many helpful reprints and suggestions in the preparation of this and other Landmarks in Hepatology. The author thanks Chiara Luberto, Ph.D., Muriel Labonté, R.N., B.S.N., C.C.T.C. and Radu Tutuian, M.D. for their help, respectively, with Italian, French, and German translation. The author continues to enjoy and benefit from Margie Myers's manuscript mastery.