Biliary Excretion of Iron

Introduction In these experiments, we assessed the role of hepatocyte lysosomes in biliary excretion of iron. We loaded rats with iron by feeding 2% carbonyl iron and collected bile for 24 h via bile fistulae from iron-loaded and control rats. In additional rats, bile was collected before and after the administration of colchicine. Rats were then killed and their livers were homogenized and fractionated for biochemical analyses or processed for electron microscopy and x-ray microanalysis. Inclusion of 2% carbonyl iron in the diet caused a 45-fold increase (P < 0.001) in hepatic iron concentration compared with controls (1,826±159 vs. 38±6.7 ,gg/g liver, mean±SE). Electron microscopy with quantitative morphometry and x-ray microanalysis showed that the excess iron was sequestered in an increased number of lysosomes concentrated in the pericanalicular region of the hepatocyte. Iron loading was also associated with a twofold increase in biliary iron excretion (4.06±0.3 vs. 1.75±0.1 pg/g liver/24 h; P < 0.001). In contrast, the biliary outputs of three lysosomal enzymes were significantly lower (P < 0.0005) in iron-loaded rats compared with controls (mean±SE) expressed as mU/24 h/g liver: N-acetyl-B-glucosaminidase, 26.7±4.6 vs. 66.2±13A; 0glucuronidase, 10.1±13 vs. 53.2±17.9; 9-galactosidase, 8.9±1.0 vs. 15.4±23. In iron-loaded rats but not in controls, biliary iron excretion was coupled to the release into bile of each of the three lysosomal hydrolases as assessed by linear regression analysis (P < 0.001). In contrast, no relationships were found between biliary iron excretion and the biliary outputs of a plasma membrane marker enzyme (alkaline phosphodiesterase I) or total protein. After administration of colchicine, there was a parallel increase in biliary excretion of iron and lysosomal enzymes in iron-loaded rats, but not controls. We interpret these data to indicate that, in the rat, biliary iron excretion from hepatocyte lysosomes is an important excretory route for excess hepatic iron. Portions of this work were presented at the meeting of the American Gastroenterological Association, 1983; and at the meeting of the American Association for the Study ofLiver Diseases, 1984; and were published in abstract form, 1983, Gastroenterology. 84:1381; and 1984. Hepatology. 4:1077. Dr. LeSage was a Research Fellow in the Gastroenterology Unit. His present address is Scott and White Clinic, Temple, TX 76508. Address correspondence to Dr. LaRusso, Professor of Medicine and Associate Professor ofCell Biology, Gastroenterology Unit, Mayo Medical School, Clinic, and Foundation, Rochester, MN 55905. Receivedfor publication 3 May 1985 and in revisedform 6 August 1985. Lysosomes are intracellular, membrane-bound organelles that contain over 40 hydrolytic enzymes with an acid pH optimum. The major functions oflysosomes include intracellular digestion, transcellular transport, extracellular release or secretion of their contents, and the sequestration and storage of endogenous or exogenous cell components (1). Several years ago, we proposed that exocytosis of lysosomal contents into biliary canaliculi was an important excretory pathway in the liver (2), a hypothesis originally suggested by de Duve and Wattiaux (3). Subsequent studies have produced results consistent with this hypothesis and have included: (a) the demonstration of coordinate release of several lysosomal hydrolases into bile under basal (2) and cholestatic (4) conditions; (b) the observation that an exogenous lysosomotropic agent is excreted into bile in parallel with endogenous lysosomal constituents under basal (5) or stimulated conditions; and (c) the finding that the biliary excretion of lysosomal enzymes can be altered by pharmacologic agents (6) and is a microtubule-dependent process (7). Thus, our studies have been consistent with the possibility that this lysosome-tobile hepatic excretory pathway is involved in the metabolism and subsequent biliary excretion of a variety of circulating molecules (e.g., proteins [8], lipoproteins [9], and trace metals) destined for sequestration in hepatocyte lysosomes. It is well known that iron may accumulate in the liver in genetic hemochromatosis (10), after multiple blood transfusions ( 1) or excessive oral intake of iron (12). In all these disorders, the excess hepatic iron conspicuously accumulates in hepatocyte lysosomes, as shown by both biochemical (13) and morphologic (14) studies. The mechanism whereby iron is sequestered in hepatocyte lysosomes in conditions ofiron overload is not known. Nevertheless, the consistent pericanalicular location ofiron-laden lysosomes within hepatocytes and the demonstration that there are significant amounts of iron in bile (15) has led investigators to suggest that iron may be released into bile directly from hepatocyte lysosomes by exocytosis after fusion of lysosomes with the canalicular membrane. There is, however, no direct morphologic or biochemical evidence for the existence of this excretory pathway. Therefore, we examined in detail the role of hepatocyte lysosomes in the biliary excretion of iron. To this end, we applied an animal model of iron overload to examine the subcellular distribution of iron in the livers of normal and iron-loaded rats, assessed the relationship between biliary iron excretion and the release into bile of lysosomal and plasma membrane enzymes, and examined the effect of a microtubule binding agent, colchicine, on biliary iron excretion. Our results show that, in the condition of hepatic iron overload, excess iron is sequestered in hepatocyte lysosomes, biliary iron excretion is increased, the accelerated release of excess iron into bile is closely coupled to biliary excretion of lysosomal but not plasma membrane en90 G. D. LeSage, L. J. Kost, S. S. Barham, and N. F. LaRusso J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 002 1-9738/86/01/0090/08 $1.00 Volume 77, January 1986, 90-97 zymes, and colchicine augments biliary excretion of both iron and lysosomal enzymes in a coordinate fashion.