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Possible Role of Endogenous Retinoid (Vitamin A) Toxicity in the Pathophysiology of Primary Biliary Cirrhosis
J. theor. Biol. (2000) 206, 47}54 doi:10.1006/jtbi.2000.2102, available online at http://www.idealibrary.com on
Possible Role of Endogenous Retinoid (Vitamin A) Toxicity in the Pathophysiology of Primary Biliary Cirrhosis JASON M. ERICKSON*
AND
ANTHONY R. MAWSON-?
*Department of Internal Medicine, Iowa Methodist Medical Center, 1215 Pleasant St., Suite 300, Des Moines, IA 50309, ;.S.A. and -Public Health Program, Des Moines ;niversity, Osteopathic Medical Center, 3200 Grand Avenue, Des Moines, IA 50312, ;.S.A. (Received on 10 June 1999, Accepted in revised form on 10 May 2000)
Primary biliary cirrhosis (PBC) is a chronic, cholestatic disease of unknown etiology commonly a!ecting women. It is characterized by progressive destruction of the small intrahepatic bile ducts and portal in#ammation, leading to "brosis and cirrhosis. The major signs and symptoms of PBC, which include pruritus, lethargy, the sicca syndrome, and osteoporosis, closely resemble the manifestations of hypervitaminosis A. Based on a review of the literature and other observations connecting PBC with retinoid metabolism (vitamin A and its derivatives), the hypothesis is proposed that exposure to excess endogenous retinoids contributes to the pathogenesis of PBC and may be to the cause of some of the signs and symptoms associated with the disease. 2000 Academic Press
Background Primary biliary cirrhosis (PBC) is an uncommon liver disease of unknown etiology and pathogenesis. PBC is characterized by progressive destruction of the small intrahepatic bile ducts and portal in#ammation, leading to impaired secretion and consequently to increased concentrations of toxic bile acids in the hepatic lobule. These bile acids are associated with damage to hepatic membranes and secondary destruction of both bile ducts and hepatic parenchyma. Longstanding in#ammation eventually leads to hepatic "brosis, cirrhosis, end-stage liver failure, and premature death (Sherlock, 1994; Kaplan, 1996; Mackay & Gershwin, 1997; Neuberger, 1997). The disease is relatively rare and predominantly ?Author to whom correspondence should be addressed. E-mail: [email protected] 0022}5193/00/170047#08 $35.00/0
a!ects women between the ages of 35 and 60. Prevalence estimates range from 19 to 151 cases per million per year, and incidence rates range from 4 to 30 cases per million per year. There is also a strong familial predisposition for the disease (Myszor & James, 1990; Metcalf et al., 1996). Some patients with PBC are initially asymptomatic and the disease may simply be detected by elevations of serum alkaline phosphatase. Of those with symptoms, lethargy and pruritus are the most commonly reported, with pruritus a!ecting about 50% of the patients. Swelling of liver and spleen is common in the early stages, with signs of portal hypertension developing before the onset of cirrhosis. Liver tests usually show a cholestatic pattern with raised alkaline phosphatase, 5-nucleotidase, and gamma glutamyl transpeptidase. Bilirubin and aminotransferases are usually within the normal range. The diagnosis is supported by an elevated 2000 Academic Press
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J. M. ERICKSON AND A. R. MAWSON
antimitochondrial antibody (titer'1 : 40). This is relatively sensitive and speci"c. From 90 to 95% of asymptomatic patients are positive. Liver biopsy is performed to con"rm the diagnosis. Retinoids Retinoids are dietary-derived, fat-soluble signaling molecules that are stored principally in the liver and are essential for numerous life processes. These functions include normal cell homeostasis, di!erentiation and growth, embryonic development, vision, and mucus secretion (Ho!mann & Eichele, 1994). The retinoids are present in many forms: an alcohol (retinol), esters (retinyl acetate and retinyl palmitate), an aldehyde (retinal), and an acid (retinoic acid), as well as numerous natural derivatives. Retinoic acid is the most biologically active metabolite of retinol. It has important hormonal functions and participates in the regulation of numerous genes, including those controlling morphology in embryonic development, growth hormone, numerous metabolic enzymes, and cellular di!erentiation. In fact, it has been shown that animals grow normally on retinoic acid alone (Ross, 1991; Hinds et al., 1997) and that all-trans-retinoic acid induces hypervitaminosis A much more e!ectively than retinol (Olson, 1996). Retinoids are predominantly stored as retinyl esters. Over 90% is stored in the liver in the nonparenchymal Ito (stellate) cell (Goodman, 1984). A small amount is also stored in hepatocytes within lipid droplets and in the kidney and adrenal glands. A carefully regulated transport system ensures that target tissues receive the necessary amounts of retinoids despite #uctuations in dietary intake (Blomho! et al., 1990). Retinoids are absorbed from the gastrointestinal tract and converted to retinol. Retinol then forms a mixed micelle and is taken up by the enterocyte. Provitamin A carotenoids in the intestine are directly absorbed and converted into retinaldehyde which is then converted into retinol. Once retinoids are converted to retinol they are esteri"ed, packaged within chylomicrons, released into lymphatic drainage and deposited into the liver, the main storage site. Stored retinyl esters then are converted back to retinol and bound in the serum with retinol-binding protein.
Retinol is in turn converted to retinaldehyde and "nally to retinoic acid, the most active form. Retinoic acid exerts its e!ects through binding to nuclear receptors (Allenby et al., 1993). These nuclear receptors are of two types: retinoic acid receptors (RAR) and retinoid X receptors (RXR). Retinoic acid receptors and RXRs exist as three distinct gene products*a, b, and c. Retinoic acid receptors bind all-trans-retinoic acid (tretinoin) and 9-cis-retinoic acid, whereas RXRs only bind 9-cis-retinoic acid. These transcription factors regulate their expression by forming homodimers (RXR/RXR) or heterodimers (RXR/RZR) and subsequent gene expression occurs as a result of RXR/RAR binding to two directly repeated consensus motifs (AGGTCA) separated by 1, 2, or 5 non-speci"c nucleotides (Manglesdorf & Evans, 1992; Gri$ths, 1999). Hypervitaminosis A is most commonly a result of self-medication. Following excessive intake, the liver becomes saturated with vitamin A so that considerable amounts of the retinyl esters, the storage form, spill over into the circulating blood. In hypervitaminotic humans, the retinyl ester values can be as high as 67% of the total plasma vitamin A (Smith & Goodman, 1976). Retinyl esters react more randomly with the membranes of the cells than retinol bound to RBP and hence are a major form of vitamin A toxicity. Furthermore, endogenous and/or administered retinoic acid (and other acidic retinoids), although not forming similar esters, is considerably more biologically active and more toxic than retinol itself (Olson, 1996). Interestingly, serum retinol levels may be normal or low due to feedback inhibition to the liver. It is important, therefore, to demonstrate increased hepatic stores along with serum retinyl ester and/or retinoic acid concentration in determining retinoid toxicity. Hypothesis The cause of PBC is unknown but it is thought to involve both genetic and environmental factors. Most data point to an inherited abnormality of immunoregulation (Kaplan, 1987, 1996). However, since PBC has been seen in only one of two identical twins (Kaplan et al., 1994), additional factors are likely to play a role. Based on
ROLE OF ENDOGENOUS RETINOID TOXICITY
similarities between the signs and symptoms of PBC and those of hypervitaminosis A, including pruritus, lethargy, osteoporosis, the sicca syndrome and arthralgia, we hypothesize that increased levels of retinoids (vitamin A and its derivatives) in the liver contribute to the pathogenesis of PBC. We also hypothesize that some of the signs and symptoms of PBC may be caused by excess retinoids. In this theory, the cholestasis-induced regurgitation of cytotoxic retinoids contained in bile aid in the progressive destruction of the small intrahepatic bile ducts characteristic of PBC. Furthermore, damaged hepatocytes leak toxic retinyl esters into the circulation, causing some of the symptoms seen in PBC, such as pruritus, arthralgias, and bone changes such as osteoporosis. The systemic picture speculatively includes circulating levels of retinol and retinol-binding protein that are known to be low, along with high concentrations of retinyl esters, retinoic acid and other polar metabolites. Low levels of retinol and retinol-binding protein in PBC may be due to the known inhibitory e!ect of retinoic acid on the secretion of non-toxic retinol and retinol-binding protein from the liver (Keilson et al., 1979; Barua et al., 1997). Evidence in support of this hypothesis is reviewed below under the following headings: 1. Retinoid concentrations increase in liver. 2. Cholestasis in PBC leads to the regurgitation of biliary retinoids causing local and systemic e!ects. 3. Retinoid toxicity and PBC: parallel signs, symptoms and laboratory values. 4. Retinoids toxicity and PBC: histopathology. 5. Conditions that a!ect PBC also in#uence retinoid metabolism. RETINOID CONCENTRATIONS INCREASE IN LIVER
It is postulated that PBC evolves, in part, from the gradual accumulation of retinoids in the liver resulting in retinoid accumulation and toxicity. In PBC, there are high liver levels of retinoids (Moore, 1957). For instance, it has been shown that the number and size of cells, as well as the cellular retinol-binding protein-staining intensity of Ito cells, are higher in PBC patients than in controls (Nyberg et al., 1988).
49
In conjunction with high liver levels of retinoids, plasma retinol-binding protein and serum retinol levels are decreased in PBC (Moore, 1957; Kaplan et al., 1988). These decreased serum levels in the face of high liver levels of retinoids are not due to malabsorption or retinoid de"ciency but due to decreased mobilization of retinoids from the liver (Mezey, 1982). Decreased mobilization may be due in part to the known inhibitory e!ect of increased retinoic acid levels on the hepatic secretion of RBP (Keilson et al., 1979; Barua et al., 1997). The initial accumulation of vitamin A in the liver may be associated with a change in estrogen levels. Since PBC tends to present near or after the menopause, when estrogen levels are no longer at their highest (Moreno-Otero et al., 1989), a state of hypoestrogenemia may be responsible. Estrogen (oral contraceptive use) strongly a!ects the hepatic synthesis and release of vitamin A, increasing plasma retinol-binding protein levels by 35}50%, due to the stimulatory e!ect of estradiol (Moore, 1957; Vahlquist et al., 1979; Underwood, 1984). Declining estrogen levels would therefore be expected to have the e!ect of inhibiting retinolbinding protein secretion from the liver. On the other hand, since the great majority of patients are adult women, and the disease can present at any age, even during pregnancy, hyperestrogenemia with associated increased sensitivity may be a more likely scenario. The estrogen component of the oral contraceptive pill is either ethinyl estradiol or mestranol (which is demethylated to ethinyl estradiol) (Lundberg, 1992), and estradiol is a known cholestatic agent. PBC-prone persons may have an unusual sensitivity to estrogen, which may increase the potential for cholestasis (Myers et al., 1981) and possibly PBC. Women who develop cholestasis during pregnancy or with oral contraceptives, may likewise have an increased sensitivity to estrogen (Reyes, 1982). CHOLESTASIS IN PBC LEADS TO THE REGURGITATION OF BILIARY RETINOIDS CAUSING LOCAL AND SYSTEMIC EFFECTS
Bile acids are hydroxylated, acidic derivatives of cholesterol. They are synthesized in the liver and secreted in high concentration in bile. When intrahepatic ducts are damaged, enterohepatic
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J. M. ERICKSON AND A. R. MAWSON
bile acid circulation is impaired and bile acids accumulate within the liver (Kaplan, 1987). Bile eventually regurgitates through the altered bile ducts, raising the level of all biliary substances in the blood. Retinoid metabolites are also present in the bile and include retinoyl beta-glucuronide, retinoic acid, retinotaurine, and other unidenti"ed metabolites of vitamin A (Frolik, 1984, pp. 189}190; Olson, 1996). Similar to the pathway outlined above, our hypothesis proposes that the increased amounts of retinoids found in the liver in PBC also accumulate in the bile and cause local cytotoxic e!ects and are spilled into the circulation to cause some of the signs and symptoms seen in PBC. RETINOID TOXICITY AND PBC: PARALLEL SIGNS, SYMPTOMS AND LABORATORY VALUES
A review of signs and symptoms found many similarities between PBC and retinoid toxicity
(see Table 1). Based on our hypothesis, elevated retinol esters and/or retinoic acid levels may cause some of these e!ects in PBC. Similarities with respect to the musculoskeletal system include arthritis, osteoporosis, bone pain and spontaneous fractures. E!ects that are not similar include osteomalacia, which is found in only a small number of patients with PBC, and calci"cation of soft tissues, which is found in some patients with retinoid toxicity. Interestingly, two of the most common presenting symptoms of PBC are often quoted as side e!ects of retinoid toxicity: fatigue and pruritus. Other manifestations common to both conditions are keratoconjunctivitis sicca (dry eyes), xerostomia (dry mouth) and xanthomas (likely caused by the hyperlipidemia found in each condition). Retinoid intoxication is also associated with dry skin and alopecia which, however, are not seen typically in PBC. Splenomegaly and
TABLE 1 Symptoms and signs in primary biliary cirrhosis and hypervitaminosis A Symptoms/signs
PBC*
Hypervitaminosis A-
Increased cerebrospinal #uid pressure and associated neurologic changes including headache, confusion, irritability, etc. Fatigue
Yes
Yes Yes
Hepatomegaly Hepatic "brosis and cirrhosis Portal hypertension Palmar erythema Spider angiomas Ascites
Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes
Splenomegaly Right upper quadrant discomfort Gastrointestinal symptoms
Yes Yes
Yes
Joint pains Bone pain Osteoporosis/osteopenia Osteomalacia Calci"cation of soft tissue Muscle pain
Yes Yes Yes Yes
Keratoconjunctivitis (dry eyes) Xerostomia (dry mouth) Pruritus Xanthomas Skin hyperpigmentation Excessive dryness of skin Alopecia (loss of hair)
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
*PBC (Kaplan, 1996; Neuberger, 1997; Bodenheimer, 1995; Moreno-Otero et al., 1989; Nyberg et al., 1988). -Hypervitaminosis A (Moore, 1957; Olson, 1996; Leo & Lieber, 1988; Safran et al., 1991; DiGiovanna et al., 1995; De Luca & Creek, 1986).
51
ROLE OF ENDOGENOUS RETINOID TOXICITY
hepatomegaly are also found in both conditions as well as hepatic "brosis and cirrhosis. The following features are common to end-stage liver disease associated with both conditions: portal hypertension, palmar erythema, spider angiomas, and ascites. Other symptoms that are found in retinoid toxicity but are not typically seen in PBC are muscle pain, increased cerebrospinal #uid pressure, and many neurologic symptoms associated with the latter, including headache, nausea, ataxia, confusion, irritability, weight loss, and anorexia. Similarities between PBC and retinoid toxicity are also seen in the laboratory "ndings (see Table 2). With regard to liver enzymes, one common "nding is an elevation in alkaline phosphatase. This is often the laboratory "nding that initiates the investigation into PBC in patients. Gamma-glutamyltransferase has been found to be variable in retinoid toxicity but is typically elevated in PBC. The aminotransferases are elevated in retinoid toxicity and normal to slightly elevated in PBC. Bilirubin and prothrombin time are found to be elevated in some cases of retinoid toxicity and are initially normal in PBC but increase as the disease progresses. An interesting "nding is the increase in hyperlipidemia/hypercholesterolemia found in both conditions. Retinol and retinol-binding protein are found to be
decreased in PBC and are highly variable in retinoid toxicity, possibly due to the feedback inhibition from elevated retinoic acid. Hypercalcemia is also found in both conditions. Increased levels of IgM are found in both conditions (Feldman et al., 1998; Bodenheimer, 1995). The signi"cance of this is uncertain. Finally, antimitochondrial antibodies are elevated in PBC, but their presence in retinoid toxicity has not been reported. In summary, there are many similarities in clinical signs, symptoms, and laboratory values found between PBC and retinoid toxicity. RETINOID TOXICITY AND PBC: HISTOPATHOLOGY
There are obvious di!erences as well as similarities in the histopathology found in classic hypervitaminosis A and the initial "ndings in PBC. However, with rare exceptions (e.g. Nyberg et al., 1988), we are not aware of studies of patients with PBC that have speci"cally looked for histological signs of retinoid toxicity in the liver. The classic changes seen in hypervitaminosis A are found predominantly in the fat storing (Ito) cells. These changes include increased size and number of Ito cells along with numerous lipid"lled vacuoles. There is also a transition of the Ito cells into "broblastic forms with activated, collagen-synthesizing rough endoplasmic reticulum.
TABLE 2 ¸aboratory ,ndings in primary biliary cirrhosis and hypervitaminosis A Lab. tests
PBC*
Hypervitaminosis A-
Alkaline phosphatase c-Glutamyltransferase Alanine aminotransferase Aspartate aminotransferase Bilirubin
Elevated Elevated Normal to slightly elevated Normal to slightly elevated Normal initially and elevated later in the disease Normal initially and elevated later in the disease
Elevated Variable Elevated Elevated Elevated
Hyperlipidemia/hypercholesterolemia
Present
Present
Retinol Retinyl esters
Normal to Low Unknown
Variable Elevated
AMA IgM
Elevated Elevated
Unknown Elevated
Hypercalcemia
No
Yes
Prothrombin time
Elevated
*PBC (Bodenheimer, 1995; Sherlock, 1994; Mezey, 1982). -Hypervitaminosis A (Ganguly, 1989; Leo & Lieber, 1988; Sarles et al., 1990).
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J. M. ERICKSON AND A. R. MAWSON
These activated cells may cause obliteration of the space of Disse by the deposition of basement membrane-type material (type IV collagen) into the perisinusoidal spaces. There is also an associated damage to the sinusoidal barrier (Leo & Lieber, 1988). In the earliest stages of PBC, histologic changes are largely found in the bile ducts that are about 100 lm in diameter. At this level, there are about 6}12 epithelial cells in cross section. The epithelium is hyperplastic, with nuclei that are distorted and hyperchromatic. The cytoplasm is vacuolated. There is portal in"ltration by lymphocytes along with plasma cells, macrophages, and eosinophils. Also seen are granulomas in the portal spaces adjacent to the damaged ducts. At this stage of the disease cholestasis is not a prominent feature. In these early stages, the hepatocytes, fat-storing (Ito) cells, and endothelial cells in the sinusoids are largely una!ected (Bodenheimer, 1995). However, later in the disease, an increase in the number and size of Ito cells in PBC has been found, which is similar to that seen in hypervitaminosis A (Nyberg et al., 1988). New information has also pointed to the hepatic stellate (Ito) cell as a key component in the pathogenesis of hepatic "brosis that is known to occur in both PBC and hypervitaminosis A. These vitamin-A-rich stellate cells are thought to be the primary source of the extracellular matrix deposition in liver "brosis and also a source for the apoptotic mediators involved in "brosis (Li & Friedman, 1999). During hepatic "brosis, hepatic stellate (vitamin A-containing) cells transform into myo"broblastic cells and lose their intracellular droplets of retinyl esters, the storage form of vitamin A. Okuno et al. (1999) have reported evidence suggesting that the loss of retinyl esters is associated with the increased retinoic acid formation, and this facilitates TGF-beta-mediated liver "brogenesis. In summary, there are both similarities and di!erences in the histopathology of the two conditions but as mentioned, data on this subject are incomplete. CONDITIONS THAT AFFECT PBC ALSO INFLUENCE RETINOID METABOLISM
The hypothesis that retinoid intoxication contributes to the pathogenesis of PBC is supported
by observations indicating that the same factors that a!ect PBC also a!ect retinoid metabolism. Successful liver transplantation is associated with substantial increases in bone density in patients with PBC (Porayko et al., 1991), suggesting that substances retained in the liver and excreted into plasma during cholestasis impair osteoblast function. This hypothesis was tested using a new bioassay for measuring plasma mitogenic activity for normal human osteoblast-like cells. In jaundiced patients, this value was found to be 56% of the value found in normal subjects. Relatively selective removal of bilirubin from the plasma by photobleaching normalized the decreased plasma mitogenic activity in "ve jaundiced patients, but produced no apparent change in the "ve nondiseased controls. These results were interpreted as indicating that hyperbilirubinemia or possibly other photolabile substances impair osteoblast proliferative capacity, and thus contribute to the osteoporosis associated with PBC (Janes et al., 1995). The present hypothesis suggests that the photolabile substance in question may be retinoic acid. Ursodeoxycholic acid is a naturally occurring hydrophilic bile acid that makes up 1% of the total bile acid pool in humans (Parquet et al., 1985). It is one of the few compounds that has been found to improve liver-test pro"les in PBC and other cholestatic disorders (Kaplan, 1996), but its mechanism of action is uncertain. Consistent with the proposed retinoid toxicity hypothesis of PBC, a study of patients with cystic "brosis and liver disease showed that ursodeoxycholic acid signi"cantly lowered the concentration of retinyl esters in serum (which normally account for less than 3% of circulating retinol) from 13.7 to 8.1% (p(0.05) (Lepage et al., 1997). Conclusions Based on this review of observations connecting PBC with retinoid (vitamin A) metabolism, we have proposed that exposure to excess endogenous retinoids contributes to the pathogenesis of PBC and also contributes to some of the classic signs and symptoms seen in PBC. It was shown that hepatic retinoid levels are increased in patients with PBC. These increased levels may cause direct cellular damage to bile ducts.
ROLE OF ENDOGENOUS RETINOID TOXICITY
Spillage of retinoids into the circulation with systemic consequences is also expected. The signs and symptoms of the two conditions were compared and shown to be similar in many respects. The histopathology was also discussed and comparisons made. Finally, two interesting relationships between retinoid metabolism and PBC were noted. If the model is correct, patients with PBC should have higher tissue concentrations of retinoids than controls, and a higher ratio of serum retinyl esters to retinol. Moreover, dietary retinoid restriction should prevent and/or ameliorate the disease. Subject to obtaining experimental support for the model, the e$cacy of retinoid antagonists could also be investigated for the treatment of PBC. A study by the authors comparing serum retinoid levels in PBC patients and controls is currently underway. REFERENCES ALLENBY, G., BOCQUEL, M. T., SAUNDERS, M., KAZMER, S., SPECK, J., ROSENBERGER, M., LOVEY, A., KASTNER, P., GRIPPO, J. F., CHAMBON, P., et al. (1993). Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proc. Nat. Acad. Sci. ;.S.A. 90, 30}34. BARUA, A. B., DUITSMAN, P. K., KOSTIC, D., BARUA, M. & OLSON, J. A. (1997). Reduction of serum retinol levels following a single oral dose of all-trans-retinoic acid in humans. Int. J.
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HINDS, T. S., WEST, W. L. & KNIGHT, E. M. (1997). Carotenoids and retinoids: a review of research, clinical, and public health applications. J. Clin. Pharmacol. 37, 551}558. HOFFMAN, C. & EICHELE, G. (1994). Retinoids in development. In: ¹he Retinoids: Biology, Chemistry, and Medicine (Sporn, M. B., Roberts, A. B. & Goodman, D. S., eds), pp. 387}441. New York: Raven Press. JANES, C. H., DICKSON, E. R., OKAZAKI, R., BONDE, S., MCDONAGH, A. F. & RIGGS, B. L. (1995). Role of hyperbilirubinemia in the impairment of osteoblast proliferation associated with cholestatic jaundice. J. Clin. Invest. 95, 2581}2586. KAPLAN, M. M. (1987). Primary biliary cirrhosis. N. Engl. J. Med. 316, 521}528. KAPLAN, M. M. (1996). Primary biliary cirrhosis. N. Engl. J. Med. 335, 1570}1580. KAPLAN, M. M., ELTA, G. H., FURIE, B., SADOWSKI, J. A. & RUSSELL, R. M. (1988). Fat-soluble vitamin nutriture in primary biliary cirrhosis. Gastroenterology 95, 787}792. KAPLAN, M. M., RABSON, A. R., LEE, Y. M., WILLIAMS, D. L. & MONTAPERTO, P. A. (1994). Discordant occurrence of primary biliary cirrhosis in monozygotic twins. N. Engl. J. Med. 331, 951. KEILSON, B., UNDERWOOD, B. A. & LOERCH, J. D. (1979). E!ects of retinoic acid on the mobilization of vitamin A from the liver in rats. J. Nutr. 109, 787}795. LEO, M. A. & LIEBER, C. S. (1988). Hypervitaminosis A: a liver lover's lament. Hepatology 8, 412}417. LEPAGE, G., PARADIS, K., LACAILLE, F., SENECHAL, L., RONCO, N., CHAMPAGNE, J., LENAERTS, C., ROY, C. C. & RASQUIN-WEBER, A. (1997). Ursodeoxycholic acid improves the hepatic metabolism of essential fatty acids and retinol in children with cystic "brosis. J. Pediatr. 130, 52}58. LI, D. & FRIEDMAN, S. L. (1999). Liver "brogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. J. Gastroenterol. Hepatol. 14, 618}633. LUNDBERG, M. C. (1992). Hepatobiliary complications of oral contraceptives. J. Gen. Intern. Med. 7, 199}209. MACKAY, I. R. & GERSHWIN, M. E. (1997). The nature of autoimmune disease. Semin. ¸iver Dis. 17, 3}11. MANGLESDORF, D. J. & EVANS, R. M. (1992). Vitamin A receptors: new insights on retinoid control of transcription. In: Retinoids in Normal Development and ¹eratogenesis (Morris-Kay, G., ed.), pp. 27}50. New York: Oxford University Press. METCALF, J. V., MITCHISON, H. C., PALMER, J. M., JONES, D. E., BASSENDINE, M. F. & JAMES, O. F. (1996). Natural history of early primary biliary cirrhosis. ¸ancet 348, 1399}1402. MEZEY, E. (1982). Liver disease and protein needs. Annu. Rev. Nutr. 2, 21}50. MOORE, T. (1957).
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NESHER, G. & ZUCKNER, J. (1995). Rheumatologic complications of vitamin A and retinoids. Semin. Arth. Rheum. 24, 291}296. NEUBERGER, J. (1997). Primary biliary cirrhosis. ¸ancet 350, 875}879. NYBERG, A., BERNE, B., NORDLINDER, H., BUSCH, C., ERIKSSON, U., LOOF, L. & VAHLQUIST, A. (1988). Impaired release of vitamin A from liver in primary biliary cirrhosis. Hepatology 8, 136}141. OKUNO, M., SATO, T., KITAMOTO, T., IMAI, S. et al. (1999). Increased 9,13-di-cis-retinoic acid in rat hepatic "brosis: implication for a potential link between retinoid loss and TGF-beta-mediated "brogenesis in vivo. J. Hepatol. 30, 1073}1080. OLSON, J. A. (1996). Vitamin A. In: Present Knowledge in Nutrition, 7th Edn., pp. 109}119. (Ziegler, E. E. & Filer, Jr. L. J., eds), Washington, DC: International Life Sciences Institute Press. PARQUET, M., METMAN, E. H., RAIZMAN, A., RAMBAUD, J. C., BERTHAUX, N. & INFANTE, R. (1985). Bioavailability, gastrointestinal transit, solubilization and faecal excretion of ursodeoxycholic acid in man. Eur. J. Clin. Invest. 15, 171}178. PORAYKO, M. K., WIESNER, R. H., HAY, J. E., KROM, R. A., DICKSON, E. R., BEAVER, S. & SCHWERMAN, L. (1991).
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Possible Role of Endogenous Retinoid (Vitamin A) Toxicity in the Pathophysiology of Primary Biliary Cirrhosis JASON M. ERICKSON*
AND
ANTHONY R. MAWSON-?
*Department of Internal Medicine, Iowa Methodist Medical Center, 1215 Pleasant St., Suite 300, Des Moines, IA 50309, ;.S.A. and -Public Health Program, Des Moines ;niversity, Osteopathic Medical Center, 3200 Grand Avenue, Des Moines, IA 50312, ;.S.A. (Received on 10 June 1999, Accepted in revised form on 10 May 2000)
Primary biliary cirrhosis (PBC) is a chronic, cholestatic disease of unknown etiology commonly a!ecting women. It is characterized by progressive destruction of the small intrahepatic bile ducts and portal in#ammation, leading to "brosis and cirrhosis. The major signs and symptoms of PBC, which include pruritus, lethargy, the sicca syndrome, and osteoporosis, closely resemble the manifestations of hypervitaminosis A. Based on a review of the literature and other observations connecting PBC with retinoid metabolism (vitamin A and its derivatives), the hypothesis is proposed that exposure to excess endogenous retinoids contributes to the pathogenesis of PBC and may be to the cause of some of the signs and symptoms associated with the disease. 2000 Academic Press
Background Primary biliary cirrhosis (PBC) is an uncommon liver disease of unknown etiology and pathogenesis. PBC is characterized by progressive destruction of the small intrahepatic bile ducts and portal in#ammation, leading to impaired secretion and consequently to increased concentrations of toxic bile acids in the hepatic lobule. These bile acids are associated with damage to hepatic membranes and secondary destruction of both bile ducts and hepatic parenchyma. Longstanding in#ammation eventually leads to hepatic "brosis, cirrhosis, end-stage liver failure, and premature death (Sherlock, 1994; Kaplan, 1996; Mackay & Gershwin, 1997; Neuberger, 1997). The disease is relatively rare and predominantly ?Author to whom correspondence should be addressed. E-mail: [email protected] 0022}5193/00/170047#08 $35.00/0
a!ects women between the ages of 35 and 60. Prevalence estimates range from 19 to 151 cases per million per year, and incidence rates range from 4 to 30 cases per million per year. There is also a strong familial predisposition for the disease (Myszor & James, 1990; Metcalf et al., 1996). Some patients with PBC are initially asymptomatic and the disease may simply be detected by elevations of serum alkaline phosphatase. Of those with symptoms, lethargy and pruritus are the most commonly reported, with pruritus a!ecting about 50% of the patients. Swelling of liver and spleen is common in the early stages, with signs of portal hypertension developing before the onset of cirrhosis. Liver tests usually show a cholestatic pattern with raised alkaline phosphatase, 5-nucleotidase, and gamma glutamyl transpeptidase. Bilirubin and aminotransferases are usually within the normal range. The diagnosis is supported by an elevated 2000 Academic Press
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J. M. ERICKSON AND A. R. MAWSON
antimitochondrial antibody (titer'1 : 40). This is relatively sensitive and speci"c. From 90 to 95% of asymptomatic patients are positive. Liver biopsy is performed to con"rm the diagnosis. Retinoids Retinoids are dietary-derived, fat-soluble signaling molecules that are stored principally in the liver and are essential for numerous life processes. These functions include normal cell homeostasis, di!erentiation and growth, embryonic development, vision, and mucus secretion (Ho!mann & Eichele, 1994). The retinoids are present in many forms: an alcohol (retinol), esters (retinyl acetate and retinyl palmitate), an aldehyde (retinal), and an acid (retinoic acid), as well as numerous natural derivatives. Retinoic acid is the most biologically active metabolite of retinol. It has important hormonal functions and participates in the regulation of numerous genes, including those controlling morphology in embryonic development, growth hormone, numerous metabolic enzymes, and cellular di!erentiation. In fact, it has been shown that animals grow normally on retinoic acid alone (Ross, 1991; Hinds et al., 1997) and that all-trans-retinoic acid induces hypervitaminosis A much more e!ectively than retinol (Olson, 1996). Retinoids are predominantly stored as retinyl esters. Over 90% is stored in the liver in the nonparenchymal Ito (stellate) cell (Goodman, 1984). A small amount is also stored in hepatocytes within lipid droplets and in the kidney and adrenal glands. A carefully regulated transport system ensures that target tissues receive the necessary amounts of retinoids despite #uctuations in dietary intake (Blomho! et al., 1990). Retinoids are absorbed from the gastrointestinal tract and converted to retinol. Retinol then forms a mixed micelle and is taken up by the enterocyte. Provitamin A carotenoids in the intestine are directly absorbed and converted into retinaldehyde which is then converted into retinol. Once retinoids are converted to retinol they are esteri"ed, packaged within chylomicrons, released into lymphatic drainage and deposited into the liver, the main storage site. Stored retinyl esters then are converted back to retinol and bound in the serum with retinol-binding protein.
Retinol is in turn converted to retinaldehyde and "nally to retinoic acid, the most active form. Retinoic acid exerts its e!ects through binding to nuclear receptors (Allenby et al., 1993). These nuclear receptors are of two types: retinoic acid receptors (RAR) and retinoid X receptors (RXR). Retinoic acid receptors and RXRs exist as three distinct gene products*a, b, and c. Retinoic acid receptors bind all-trans-retinoic acid (tretinoin) and 9-cis-retinoic acid, whereas RXRs only bind 9-cis-retinoic acid. These transcription factors regulate their expression by forming homodimers (RXR/RXR) or heterodimers (RXR/RZR) and subsequent gene expression occurs as a result of RXR/RAR binding to two directly repeated consensus motifs (AGGTCA) separated by 1, 2, or 5 non-speci"c nucleotides (Manglesdorf & Evans, 1992; Gri$ths, 1999). Hypervitaminosis A is most commonly a result of self-medication. Following excessive intake, the liver becomes saturated with vitamin A so that considerable amounts of the retinyl esters, the storage form, spill over into the circulating blood. In hypervitaminotic humans, the retinyl ester values can be as high as 67% of the total plasma vitamin A (Smith & Goodman, 1976). Retinyl esters react more randomly with the membranes of the cells than retinol bound to RBP and hence are a major form of vitamin A toxicity. Furthermore, endogenous and/or administered retinoic acid (and other acidic retinoids), although not forming similar esters, is considerably more biologically active and more toxic than retinol itself (Olson, 1996). Interestingly, serum retinol levels may be normal or low due to feedback inhibition to the liver. It is important, therefore, to demonstrate increased hepatic stores along with serum retinyl ester and/or retinoic acid concentration in determining retinoid toxicity. Hypothesis The cause of PBC is unknown but it is thought to involve both genetic and environmental factors. Most data point to an inherited abnormality of immunoregulation (Kaplan, 1987, 1996). However, since PBC has been seen in only one of two identical twins (Kaplan et al., 1994), additional factors are likely to play a role. Based on
ROLE OF ENDOGENOUS RETINOID TOXICITY
similarities between the signs and symptoms of PBC and those of hypervitaminosis A, including pruritus, lethargy, osteoporosis, the sicca syndrome and arthralgia, we hypothesize that increased levels of retinoids (vitamin A and its derivatives) in the liver contribute to the pathogenesis of PBC. We also hypothesize that some of the signs and symptoms of PBC may be caused by excess retinoids. In this theory, the cholestasis-induced regurgitation of cytotoxic retinoids contained in bile aid in the progressive destruction of the small intrahepatic bile ducts characteristic of PBC. Furthermore, damaged hepatocytes leak toxic retinyl esters into the circulation, causing some of the symptoms seen in PBC, such as pruritus, arthralgias, and bone changes such as osteoporosis. The systemic picture speculatively includes circulating levels of retinol and retinol-binding protein that are known to be low, along with high concentrations of retinyl esters, retinoic acid and other polar metabolites. Low levels of retinol and retinol-binding protein in PBC may be due to the known inhibitory e!ect of retinoic acid on the secretion of non-toxic retinol and retinol-binding protein from the liver (Keilson et al., 1979; Barua et al., 1997). Evidence in support of this hypothesis is reviewed below under the following headings: 1. Retinoid concentrations increase in liver. 2. Cholestasis in PBC leads to the regurgitation of biliary retinoids causing local and systemic e!ects. 3. Retinoid toxicity and PBC: parallel signs, symptoms and laboratory values. 4. Retinoids toxicity and PBC: histopathology. 5. Conditions that a!ect PBC also in#uence retinoid metabolism. RETINOID CONCENTRATIONS INCREASE IN LIVER
It is postulated that PBC evolves, in part, from the gradual accumulation of retinoids in the liver resulting in retinoid accumulation and toxicity. In PBC, there are high liver levels of retinoids (Moore, 1957). For instance, it has been shown that the number and size of cells, as well as the cellular retinol-binding protein-staining intensity of Ito cells, are higher in PBC patients than in controls (Nyberg et al., 1988).
49
In conjunction with high liver levels of retinoids, plasma retinol-binding protein and serum retinol levels are decreased in PBC (Moore, 1957; Kaplan et al., 1988). These decreased serum levels in the face of high liver levels of retinoids are not due to malabsorption or retinoid de"ciency but due to decreased mobilization of retinoids from the liver (Mezey, 1982). Decreased mobilization may be due in part to the known inhibitory e!ect of increased retinoic acid levels on the hepatic secretion of RBP (Keilson et al., 1979; Barua et al., 1997). The initial accumulation of vitamin A in the liver may be associated with a change in estrogen levels. Since PBC tends to present near or after the menopause, when estrogen levels are no longer at their highest (Moreno-Otero et al., 1989), a state of hypoestrogenemia may be responsible. Estrogen (oral contraceptive use) strongly a!ects the hepatic synthesis and release of vitamin A, increasing plasma retinol-binding protein levels by 35}50%, due to the stimulatory e!ect of estradiol (Moore, 1957; Vahlquist et al., 1979; Underwood, 1984). Declining estrogen levels would therefore be expected to have the e!ect of inhibiting retinolbinding protein secretion from the liver. On the other hand, since the great majority of patients are adult women, and the disease can present at any age, even during pregnancy, hyperestrogenemia with associated increased sensitivity may be a more likely scenario. The estrogen component of the oral contraceptive pill is either ethinyl estradiol or mestranol (which is demethylated to ethinyl estradiol) (Lundberg, 1992), and estradiol is a known cholestatic agent. PBC-prone persons may have an unusual sensitivity to estrogen, which may increase the potential for cholestasis (Myers et al., 1981) and possibly PBC. Women who develop cholestasis during pregnancy or with oral contraceptives, may likewise have an increased sensitivity to estrogen (Reyes, 1982). CHOLESTASIS IN PBC LEADS TO THE REGURGITATION OF BILIARY RETINOIDS CAUSING LOCAL AND SYSTEMIC EFFECTS
Bile acids are hydroxylated, acidic derivatives of cholesterol. They are synthesized in the liver and secreted in high concentration in bile. When intrahepatic ducts are damaged, enterohepatic
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J. M. ERICKSON AND A. R. MAWSON
bile acid circulation is impaired and bile acids accumulate within the liver (Kaplan, 1987). Bile eventually regurgitates through the altered bile ducts, raising the level of all biliary substances in the blood. Retinoid metabolites are also present in the bile and include retinoyl beta-glucuronide, retinoic acid, retinotaurine, and other unidenti"ed metabolites of vitamin A (Frolik, 1984, pp. 189}190; Olson, 1996). Similar to the pathway outlined above, our hypothesis proposes that the increased amounts of retinoids found in the liver in PBC also accumulate in the bile and cause local cytotoxic e!ects and are spilled into the circulation to cause some of the signs and symptoms seen in PBC. RETINOID TOXICITY AND PBC: PARALLEL SIGNS, SYMPTOMS AND LABORATORY VALUES
A review of signs and symptoms found many similarities between PBC and retinoid toxicity
(see Table 1). Based on our hypothesis, elevated retinol esters and/or retinoic acid levels may cause some of these e!ects in PBC. Similarities with respect to the musculoskeletal system include arthritis, osteoporosis, bone pain and spontaneous fractures. E!ects that are not similar include osteomalacia, which is found in only a small number of patients with PBC, and calci"cation of soft tissues, which is found in some patients with retinoid toxicity. Interestingly, two of the most common presenting symptoms of PBC are often quoted as side e!ects of retinoid toxicity: fatigue and pruritus. Other manifestations common to both conditions are keratoconjunctivitis sicca (dry eyes), xerostomia (dry mouth) and xanthomas (likely caused by the hyperlipidemia found in each condition). Retinoid intoxication is also associated with dry skin and alopecia which, however, are not seen typically in PBC. Splenomegaly and
TABLE 1 Symptoms and signs in primary biliary cirrhosis and hypervitaminosis A Symptoms/signs
PBC*
Hypervitaminosis A-
Increased cerebrospinal #uid pressure and associated neurologic changes including headache, confusion, irritability, etc. Fatigue
Yes
Yes Yes
Hepatomegaly Hepatic "brosis and cirrhosis Portal hypertension Palmar erythema Spider angiomas Ascites
Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes
Splenomegaly Right upper quadrant discomfort Gastrointestinal symptoms
Yes Yes
Yes
Joint pains Bone pain Osteoporosis/osteopenia Osteomalacia Calci"cation of soft tissue Muscle pain
Yes Yes Yes Yes
Keratoconjunctivitis (dry eyes) Xerostomia (dry mouth) Pruritus Xanthomas Skin hyperpigmentation Excessive dryness of skin Alopecia (loss of hair)
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
*PBC (Kaplan, 1996; Neuberger, 1997; Bodenheimer, 1995; Moreno-Otero et al., 1989; Nyberg et al., 1988). -Hypervitaminosis A (Moore, 1957; Olson, 1996; Leo & Lieber, 1988; Safran et al., 1991; DiGiovanna et al., 1995; De Luca & Creek, 1986).
51
ROLE OF ENDOGENOUS RETINOID TOXICITY
hepatomegaly are also found in both conditions as well as hepatic "brosis and cirrhosis. The following features are common to end-stage liver disease associated with both conditions: portal hypertension, palmar erythema, spider angiomas, and ascites. Other symptoms that are found in retinoid toxicity but are not typically seen in PBC are muscle pain, increased cerebrospinal #uid pressure, and many neurologic symptoms associated with the latter, including headache, nausea, ataxia, confusion, irritability, weight loss, and anorexia. Similarities between PBC and retinoid toxicity are also seen in the laboratory "ndings (see Table 2). With regard to liver enzymes, one common "nding is an elevation in alkaline phosphatase. This is often the laboratory "nding that initiates the investigation into PBC in patients. Gamma-glutamyltransferase has been found to be variable in retinoid toxicity but is typically elevated in PBC. The aminotransferases are elevated in retinoid toxicity and normal to slightly elevated in PBC. Bilirubin and prothrombin time are found to be elevated in some cases of retinoid toxicity and are initially normal in PBC but increase as the disease progresses. An interesting "nding is the increase in hyperlipidemia/hypercholesterolemia found in both conditions. Retinol and retinol-binding protein are found to be
decreased in PBC and are highly variable in retinoid toxicity, possibly due to the feedback inhibition from elevated retinoic acid. Hypercalcemia is also found in both conditions. Increased levels of IgM are found in both conditions (Feldman et al., 1998; Bodenheimer, 1995). The signi"cance of this is uncertain. Finally, antimitochondrial antibodies are elevated in PBC, but their presence in retinoid toxicity has not been reported. In summary, there are many similarities in clinical signs, symptoms, and laboratory values found between PBC and retinoid toxicity. RETINOID TOXICITY AND PBC: HISTOPATHOLOGY
There are obvious di!erences as well as similarities in the histopathology found in classic hypervitaminosis A and the initial "ndings in PBC. However, with rare exceptions (e.g. Nyberg et al., 1988), we are not aware of studies of patients with PBC that have speci"cally looked for histological signs of retinoid toxicity in the liver. The classic changes seen in hypervitaminosis A are found predominantly in the fat storing (Ito) cells. These changes include increased size and number of Ito cells along with numerous lipid"lled vacuoles. There is also a transition of the Ito cells into "broblastic forms with activated, collagen-synthesizing rough endoplasmic reticulum.
TABLE 2 ¸aboratory ,ndings in primary biliary cirrhosis and hypervitaminosis A Lab. tests
PBC*
Hypervitaminosis A-
Alkaline phosphatase c-Glutamyltransferase Alanine aminotransferase Aspartate aminotransferase Bilirubin
Elevated Elevated Normal to slightly elevated Normal to slightly elevated Normal initially and elevated later in the disease Normal initially and elevated later in the disease
Elevated Variable Elevated Elevated Elevated
Hyperlipidemia/hypercholesterolemia
Present
Present
Retinol Retinyl esters
Normal to Low Unknown
Variable Elevated
AMA IgM
Elevated Elevated
Unknown Elevated
Hypercalcemia
No
Yes
Prothrombin time
Elevated
*PBC (Bodenheimer, 1995; Sherlock, 1994; Mezey, 1982). -Hypervitaminosis A (Ganguly, 1989; Leo & Lieber, 1988; Sarles et al., 1990).
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J. M. ERICKSON AND A. R. MAWSON
These activated cells may cause obliteration of the space of Disse by the deposition of basement membrane-type material (type IV collagen) into the perisinusoidal spaces. There is also an associated damage to the sinusoidal barrier (Leo & Lieber, 1988). In the earliest stages of PBC, histologic changes are largely found in the bile ducts that are about 100 lm in diameter. At this level, there are about 6}12 epithelial cells in cross section. The epithelium is hyperplastic, with nuclei that are distorted and hyperchromatic. The cytoplasm is vacuolated. There is portal in"ltration by lymphocytes along with plasma cells, macrophages, and eosinophils. Also seen are granulomas in the portal spaces adjacent to the damaged ducts. At this stage of the disease cholestasis is not a prominent feature. In these early stages, the hepatocytes, fat-storing (Ito) cells, and endothelial cells in the sinusoids are largely una!ected (Bodenheimer, 1995). However, later in the disease, an increase in the number and size of Ito cells in PBC has been found, which is similar to that seen in hypervitaminosis A (Nyberg et al., 1988). New information has also pointed to the hepatic stellate (Ito) cell as a key component in the pathogenesis of hepatic "brosis that is known to occur in both PBC and hypervitaminosis A. These vitamin-A-rich stellate cells are thought to be the primary source of the extracellular matrix deposition in liver "brosis and also a source for the apoptotic mediators involved in "brosis (Li & Friedman, 1999). During hepatic "brosis, hepatic stellate (vitamin A-containing) cells transform into myo"broblastic cells and lose their intracellular droplets of retinyl esters, the storage form of vitamin A. Okuno et al. (1999) have reported evidence suggesting that the loss of retinyl esters is associated with the increased retinoic acid formation, and this facilitates TGF-beta-mediated liver "brogenesis. In summary, there are both similarities and di!erences in the histopathology of the two conditions but as mentioned, data on this subject are incomplete. CONDITIONS THAT AFFECT PBC ALSO INFLUENCE RETINOID METABOLISM
The hypothesis that retinoid intoxication contributes to the pathogenesis of PBC is supported
by observations indicating that the same factors that a!ect PBC also a!ect retinoid metabolism. Successful liver transplantation is associated with substantial increases in bone density in patients with PBC (Porayko et al., 1991), suggesting that substances retained in the liver and excreted into plasma during cholestasis impair osteoblast function. This hypothesis was tested using a new bioassay for measuring plasma mitogenic activity for normal human osteoblast-like cells. In jaundiced patients, this value was found to be 56% of the value found in normal subjects. Relatively selective removal of bilirubin from the plasma by photobleaching normalized the decreased plasma mitogenic activity in "ve jaundiced patients, but produced no apparent change in the "ve nondiseased controls. These results were interpreted as indicating that hyperbilirubinemia or possibly other photolabile substances impair osteoblast proliferative capacity, and thus contribute to the osteoporosis associated with PBC (Janes et al., 1995). The present hypothesis suggests that the photolabile substance in question may be retinoic acid. Ursodeoxycholic acid is a naturally occurring hydrophilic bile acid that makes up 1% of the total bile acid pool in humans (Parquet et al., 1985). It is one of the few compounds that has been found to improve liver-test pro"les in PBC and other cholestatic disorders (Kaplan, 1996), but its mechanism of action is uncertain. Consistent with the proposed retinoid toxicity hypothesis of PBC, a study of patients with cystic "brosis and liver disease showed that ursodeoxycholic acid signi"cantly lowered the concentration of retinyl esters in serum (which normally account for less than 3% of circulating retinol) from 13.7 to 8.1% (p(0.05) (Lepage et al., 1997). Conclusions Based on this review of observations connecting PBC with retinoid (vitamin A) metabolism, we have proposed that exposure to excess endogenous retinoids contributes to the pathogenesis of PBC and also contributes to some of the classic signs and symptoms seen in PBC. It was shown that hepatic retinoid levels are increased in patients with PBC. These increased levels may cause direct cellular damage to bile ducts.
ROLE OF ENDOGENOUS RETINOID TOXICITY
Spillage of retinoids into the circulation with systemic consequences is also expected. The signs and symptoms of the two conditions were compared and shown to be similar in many respects. The histopathology was also discussed and comparisons made. Finally, two interesting relationships between retinoid metabolism and PBC were noted. If the model is correct, patients with PBC should have higher tissue concentrations of retinoids than controls, and a higher ratio of serum retinyl esters to retinol. Moreover, dietary retinoid restriction should prevent and/or ameliorate the disease. Subject to obtaining experimental support for the model, the e$cacy of retinoid antagonists could also be investigated for the treatment of PBC. A study by the authors comparing serum retinoid levels in PBC patients and controls is currently underway. REFERENCES ALLENBY, G., BOCQUEL, M. T., SAUNDERS, M., KAZMER, S., SPECK, J., ROSENBERGER, M., LOVEY, A., KASTNER, P., GRIPPO, J. F., CHAMBON, P., et al. (1993). Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proc. Nat. Acad. Sci. ;.S.A. 90, 30}34. BARUA, A. B., DUITSMAN, P. K., KOSTIC, D., BARUA, M. & OLSON, J. A. (1997). Reduction of serum retinol levels following a single oral dose of all-trans-retinoic acid in humans. Int. J.
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NESHER, G. & ZUCKNER, J. (1995). Rheumatologic complications of vitamin A and retinoids. Semin. Arth. Rheum. 24, 291}296. NEUBERGER, J. (1997). Primary biliary cirrhosis. ¸ancet 350, 875}879. NYBERG, A., BERNE, B., NORDLINDER, H., BUSCH, C., ERIKSSON, U., LOOF, L. & VAHLQUIST, A. (1988). Impaired release of vitamin A from liver in primary biliary cirrhosis. Hepatology 8, 136}141. OKUNO, M., SATO, T., KITAMOTO, T., IMAI, S. et al. (1999). Increased 9,13-di-cis-retinoic acid in rat hepatic "brosis: implication for a potential link between retinoid loss and TGF-beta-mediated "brogenesis in vivo. J. Hepatol. 30, 1073}1080. OLSON, J. A. (1996). Vitamin A. In: Present Knowledge in Nutrition, 7th Edn., pp. 109}119. (Ziegler, E. E. & Filer, Jr. L. J., eds), Washington, DC: International Life Sciences Institute Press. PARQUET, M., METMAN, E. H., RAIZMAN, A., RAMBAUD, J. C., BERTHAUX, N. & INFANTE, R. (1985). Bioavailability, gastrointestinal transit, solubilization and faecal excretion of ursodeoxycholic acid in man. Eur. J. Clin. Invest. 15, 171}178. PORAYKO, M. K., WIESNER, R. H., HAY, J. E., KROM, R. A., DICKSON, E. R., BEAVER, S. & SCHWERMAN, L. (1991).
Bone disease in liver transplant recipients: incidence, timing and risk factors. ¹ransplant Proc., Part 1, 23, 1462}1465. REYES, H. (1982). The enigma of cholestasis of pregnancy: lessons from Chile. Hepatology 2, 87}96. ROSS, A. C. (1991). Vitamin A: current understanding of the mechanisms of action. Nutr. ¹oday 26, 6}12. SAFRAN, A. B., HALIOUA, B., ROTH, A. & SAURAT, J.-A. (1991). Ocular side e!ects of oral treatment with retinoids. In: Retinoids: 10 >ears On (Saurat, J.-A. ed.), pp. 315}326. Basel: Karger. SARLES, J., SCHEINER, C., SARRAN, M. & GIRAUD, F. (1990). Hepatic hypervitaminosis A: a familial observation. J. Pediatr. Gastroenterol. Nutr. 10, 71}76. SHERLOCK, S. (1994). Primary biliary cirrhosis: clarifying the issues. Am. J. Med. 96, 27S}33S. SMITH, F. R. & GOODMAN, D. S. (1976). Vitamin A transport in human vitamin A toxicity. N. Engl. J. Med. 294, 805}808. UNDERWOOD, B. A. (1984). Vitamin A in animal and human nutrition. In: ¹he Retinoids (Sporn, M. B., Roberts, A. B. & Goodman, D. S., eds), Vol. 1, pp. 281}392. New York: Academic Press. VAHLQUIST, A., JOHNSON, A. & NYGREN, K. G. (1979). Vitamin A transporting plasma proteins and female sex hormones. Am. J. Clin. Nutr. 32, 1433}1438.