Loss Of Liver Mass Research Articles

Polyploidy and liver proliferation

Organisms containing an increase in DNA content by whole number multiples of the entire set of chromosomes are defined as polyploid. Cells that contain more than two sets of chromosomes were first observed in plants about a century ago, and it is now recognized that polyploid cells form in many eukaryotes under a wide variety of circumstances. Although it is less common in mammals, some tissues, including the liver, show a high percentage of polyploid cells. Thus, during post-natal growth, the liver parenchyma undergoes dramatic changes characterized by gradual polyploidization during which hepatocytes of several ploidy classes emerge as a result of modified cell-division cycles. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence and to both lead to a progressive loss of cell pluripotency and to a markedly decreased replication capacity. In adults, liver polyploidization is differentially regulated upon loss of liver mass and liver damage. Here we review the current state of understanding about how polyploidization is regulated during normal and pathological liver growth, and detail by which mechanisms hepatocytes become polyploid.

Glycogen synthase kinase 3β-dependent Snail degradation directs hepatocyte proliferation in normal liver regeneration

Liver regeneration proceeds under the well-orchestrated control of multiple transcription factors that lead hepatocytes to reenter the cell cycle, proliferate, and renew quiescence. Here, we found an important role of the zinc-finger transcription factor Snail in liver regeneration. Snail was typically expressed in quiescent adult hepatocytes, but was rapidly degraded when the liver needed to regenerate itself. Decreased levels of Snail induced DNA synthesis in hepatocytes through up-regulation of cell cycle-related proteins. Snail degradation was dependent on phosphorylation by glycogen synthase kinase (GSK)-3β, whose quantity and activity were immediately increased after loss of liver mass or hepatic injury. Inactivation of GSK-3β resulted in suppression of Snail degradation and DNA synthesis in hepatocytes, leading to impaired liver growth during regeneration. This GSK-3β-dependent Snail degradation occurred as a result of cytokine, growth factor, and bile acid signals that are known to drive liver regeneration. Thus, GSK-3β-dependent Snail degradation acts as a fundamental cue for the initiation of hepatocyte proliferation in liver regeneration.

Regeneration and DNA demethylation do not trigger PDX-1 expression in rat hepatocytes

To explore the possibility that PDX-1 gene is reactivated as a consequence of molecular events that occur during liver regeneration. Rat hepatocytes were maintained in DMEM-F12, 10% fetal bovine serum (FBS), penicillin/streptomycin and geneticin when applicable. Rat insulinoma RIN 1046-38 cells were maintained in M-199-10% FBS and penicillin/streptomycin. The final concentration of glucose was 11.1 mmol/L. During regeneration, lateral and medial liver lobes of adult male Wistar rats were surgically removed, with up 70% loss of liver mass. In methylation experiments, 5-aza-deoxycytidine (5-aza-dC) was used. Primer3 software was used for polymerase chain reaction (PCR). Quantitative real time PCR (qRT-PCR) was performed using SYBR Green technology; primers were designed by Beacon Designer 6 software. Western blotting and SDS-PAGE were performed according to standard procedures. Antibodies were purchased from commercial suppliers. We explored the possibility that liver regeneration could trigger PDX-1 expression, and hence insulin production. Twenty-four hours after surgical liver removal, regeneration was active as demonstrated by the increased proliferating cell nuclear antigen; however, all the other checked genes (involved in insulin gene expression): PC-1, Ngn3, NeuroD1, Btc, PDX-1 and Ins-1, were not related to the molecular events caused by this process. The only marker detected in regenerating liver was E47: a transcription factor of the the basic helix-loop-helix family known to be expressed ubiquitously in mammalian cells. In the rat pancreas, almost all of the tested genes were expressed as shown by RT-PCR, except for Ngn3, which was silenced 2 d after birth. Therefore, the molecular events in liver regeneration are not sufficient to promote PDX-1 expression. DNA methylation is a known mechanism to achieve stable repression of gene expression in mammals: Hxk 2 gene is silenced through this mechanism in normal hepatocytes. The administration of 5-aza-dC to cultured cells is in fact able to upregulate Hxk 2 mRNA. We investigated whether PDX-1 silencing in liver cells could be exerted through methylation of CpG islands in both the promoter and the gene coding regions. The results show that the drug increased the expression level of the Hxk 2 control gene but failed to rescue the expression of PDX-1, thus DNA demethylation is not sufficient to override repression of the PDX-1 gene. During liver regeneration, PDX-1 gene is not reactivated. Demethylation does not de-repress PDX-1 gene expression. Therefore gene silencing is not achieved through this epigenetic mechanism.

TLRs, NF-κB, JNK, and Liver Regeneration

While hepatocytes rarely undergo proliferation in normal livers, they quickly induce proliferation in response to loss of liver mass by toxin or inflammation-induced hepatocyte injury, trauma, or surgical resection, leading to a restoration of liver mass to its original size. Recent studies suggest that Toll-like receptor (TLR) signaling participates in this regenerative response. Myeloid differentiation factor (MyD88), a common adaptor molecule in the TLR, IL-1 and IL-18 receptor signaling, plays a key role, at least, in the early phase of liver regeneration. Currently, definite ligands which bind to TLRs and initiate this process are still unclear. TLRs stimulated by their corresponding ligands, as well as tumor necrosis factor (TNF) receptors (TNFRs), can activate downstream signal molecules, including transcription factor nuclear factor (NF)-κB and c-Jun N-terminal kinase (JNK). Previous studies have revealed the important role of TNF receptor signaling, NF-κB, and JNK in liver regeneration by using hepatocyte-specific gene-modified animals. This review will summarize the current knowledge of TLR signaling and their related molecules in liver regeneration. We will also discuss whether modulating these factors may become new therapeutic strategies to promote liver regeneration in various clinical situations.

A Model of Liver Regeneration

The network of interactions underlying liver regeneration is robust and precise with liver resections resulting in controlled hyperplasia (cell proliferation) that terminates when the liver regains its lost mass. The interplay of cytokines and growth factors responsible for the inception and termination of this hyperplasia is not well understood. A model is developed for this network of interactions based on the known data of liver resections. This model reproduces the relevant published data on liver regeneration and provides geometric insights into the experimental observations. The predictions of this model are used to suggest two novel strategies for speeding up liver mass recovery and a strategy for enabling liver mass recovery in cases where a resection leaves <20% of the liver that would otherwise result in complete loss of liver mass.

Cerebral Blood Flow Autoregulation in Experimental Liver Failure

Patients with acute liver failure (ALF) display impairment of cerebral blood flow (CBF) autoregulation, which may contribute to the development of fatal intracranial hypertension, but the pathophysiological mechanism remains unclear. In this study, we examined whether loss of liver mass causes impairment of CBF autoregulation. Four rat models were chosen, each representing different aspects of ALF: galactosamine (GlN) intoxication represented liver necrosis, 90% hepatectomy (PHx90) represented reduction in liver mass, portacaval anastomosis (PCA) represented shunting of blood/toxins into the systemic circulation thus mimicking intrahepatic shunting in ALF, PCA+NH(3) provided information about the additional effects of hyperammonemia Rats were intubated and sedated with pentobarbital. We measured CBF with laser Doppler, intracranial pressure (ICP) was measured in the fossa posterior and registered with a pressure transducer, brain water was measured using the wet-to-dry method, and cerebral glutamine/glutamate was measured enzymatically. The CBF autoregulatory index in both the GlN and PHx90 groups differed significantly from the control group. Conversely, CBF autoregulation was intact in the PCA and PCA+NH(3) groups despite high arterial ammonia, high cerebral glutamine concentration, and increased CBF and ICP. Increased water content of the brainstem or cerebellum was not associated with defective CBF autoregulation. In conclusion, impairment of CBF autoregulation is not caused by brain edema/high ICP. Nor does portacaval shunting or hyperammonemia impair autoregulation. Rather, massive liver necrosis and reduced liver mass are associated with loss of CBF autoregulation.

Therapeutic applications of bone marrow-derived stem cells in liver transplantation for end-stage liver diseases

Today, liver transplantation (LT) is the only established treatment for end-stage liver diseases. The development of LT, including OLT, cadaveric LT, split LT, living donor LT (LDLT), brings hopes to patients with these diseases. However, increasing donor shortage, rejection and life-long immunosuppression with its side effects are the major limitations of this therapy strategy. Bone marrow-derived stem cells (BMDSCs) are capable of differentiating into hepatocyte-like cells and contribute to liver injury repair. The microenvironment of liver injury caused by rejection, ischemia/reperfusion, loss of liver mass, recurrence of HCV and “small-for-size syndrome” after LT can attract a variety of bone marrow-derived stem cell population to the peripheral circulation and then migration to the injury liver to promote the hepatic function restoration. Additionally, BMDSCs can also take part in the functional regeneration of living donor liver after LDLT. This participation in liver regeneration may be associated to the interaction between SDF-1and its receptor CXCR4, involving HGF, IL-8, MMP9, and VEGF/VEGFR-2. BMDSC with its bio-characteristics could maintain the allograft tolerance from different angles and in different ways. In conclusion, BMDSCs transplantation, as a new assistant therapeutic method for LT, will expand the space of LT, and provide more survival opportunities for the patients suffering liver diseases in the future

During Thioacetamide-Induced Acute Liver Failure, the Proliferative Response of Hepatocytes to Thyroid Hormone Is Maintained, Indicating a Potential Therapeutic Approach to Toxin-Induced Liver Disease

In toxic liver injury, proliferation of preexisting hepatocytes helps restore liver mass and function. While loss of liver mass per se stimulates hepatocyte proliferation, exogenous mitogens have a potential role in enhancing liver regeneration. The aim of this study was to characterize the effects of the mitogen, tri-iodothyonine, on the regenerative capacity of hepatocytes during thioacetamide-induced liver failure. Rats received (two) thioacetamide injections and, 12 hr later, either tri-iodothyonine or vehicle-only control. Liver cell proliferation was assessed and comparison made with other control groups receiving tri-iodothyonine or vehicle only. In rats with thioacetamide-induced hepatitis the proportion of hepatocytes in S-phase was greater in the tri-iodothyonine group (27+/-3.5%) compared to the vehicle-only group (20+/-2.5%; P < 0.05), with, notably, a greater number of midzonal (BrdU) positive hepatocytes in the tri-iodothyonine group. We conclude that the ability of hepatocytes in the midzonal areas of rat liver to proliferate in response to tri-iodothyonine is maintained during severe acute toxic injury.

Restoration of hepatic mast cells and expression of a different mast cell protease phenotype in regenerating rat liver after 70%‐hepatectomy

Mast cells (MC) can undergo significant changes in number and phenotype; these alterations result in the differential expression of growth factors and cytokines. Kit ligand (KL; stem cell factor) is produced by mesenchymal cells, and in the liver by biliary epithelial cells. Recent studies suggest that KL, and its receptor c-kit, may be involved in liver regeneration after loss of liver mass. However, KL is also the major growth, differentiating, chemotactic, and activating factor for MC. The aim of our study was to elucidate the dynamics and phenotype of hepatic MC and KL/c-kit expression during liver regeneration after partial (70%) hepatectomy in the rat. Regenerating livers were harvested after 1, 3, 7, and 14 days, respectively (n = 6 each day). MC were stained for naphthol-AS/D-chloroacetate esterase and counted as MC per bile ductule. MC phenotype was assessed by rat MC protease (RMCP)-1 and -2 immunofluorescence staining, in order to distinguish RMCP-1 positive connective tissue MC (CTMC) from RMCP-2 positive mucosa MC (MMC). mRNA expression of RMCP, c-kit, and the differentially spliced variants of KL was quantified by RT-PCR. MC counts per bile ductule decreased in regenerating rat liver tissue at day 3, compared with native livers, and became normal thereafter. Hepatic MC were predominantly of a CTMC phenotype expressing RMCP-1, as previously published; after hepatectomy, between 76 and 99% of all MC double-expressed RMCP-1 and -2, compatible with an MMC phenotype. The ratio of the two alternatively spliced mRNAs for KL (KL-1 : KL-2), and c-kit mRNA expression did not differ significantly between regenerating livers and the livers of sham operated animals. These results suggest that hepatic mast cells are restored during liver regeneration after partial hepatectomy in the rat. Restored MC express an MMC phenotype, suggesting migration from outside into the regenerating liver. Alternative splicing of KL is affected by the surgical procedure in general, and, together with its receptor c-kit, doesn't seem to be involved in liver regeneration after partial hepatectomy in the rat. Further functional studies, and studies in regenerating human livers might offer the possibility of elucidating the role of the hepatic mast cell, and its different protease phenotypes during liver regeneration after surgical loss of liver mass.

Molecular mechanisms of apoptosis in the liver of rats after portal branch ligation with and without retrorsine

The mechanisms accounting for the atrophy of the portal blood-deprived liver lobes after portal branch ligation (PBL) are still unclear. The first aim of this study was to confirm the role of apoptosis in this process and to determine which apoptotic pathways are involved. The second aim of the study was to evaluate the effect of blocking compensatory hyperplasia of the nonligated lobes with retrorsine on the mechanisms of apoptosis in the ligated lobes. Mitochondrial Bax, Bcl-2 and Bcl-XL, cytosolic cytochrome c, caspase-3, -8 and -9 activities and TNF-α levels were assessed in the liver of rats before and at various time points, ranging from 30 min to 7 days, after PBL. Caspase activities were also measured in rats pretreated with retrorsine. Both the mitochondrial and the death receptor-mediated pathways are activated in the ligated liver lobes after portal branch ligation. Caspase activation is inhibited by retrorsine pretreatment, resulting in fewer apoptotic bodies. Apoptosis accounts for the atrophy of the ligated lobes after PBL. It is inhibited by retrorsine, suggesting an attempt to reduce the loss of liver mass when hyperplasia of the nonligated lobes is impaired.

Food restriction affects energy metabolism in rat liver mitochondria.

To examine the effect of 50% food restriction over a period of 3 days on mitochondrial energy metabolism, liver mitochondria were isolated from ad libitum and food-restricted rats. Mitochondrial enzyme activities and oxygen consumption were assessed spectrophotometrically and polarographically. With regard to body weight loss (-5%), food restriction decreased the liver to body mass ratio by 7%. Moreover, in food-restricted rats, liver mitochondria displayed diminished state 3 (-30%), state 4-oligomycin (-26%) and uncoupled state (-24%) respiration rates in the presence of succinate. Furthermore, "top-down" elasticity showed that these decreases were due to an inactivation of reactions involved in substrate oxidation. Therefore, it appears that rats not only adapt to food restriction through simple passive mechanisms, such as liver mass loss, but also through decreased mitochondrial energetic metabolism.

Liver death and regeneration in paracetamol toxicity.

Paracetamol overdose (POD) is a major clinical problem as the commonest cause of fulminant hepatic failure (FHF) in the UK and the USA. While the main loss of liver mass occurs following hepatocyte necrosis, hepatocyte apoptosis has also been reported to occur during paracetamol toxicity in murine liver. Hepatocyte apoptosis has not previously been identified in human liver and the significance of apoptosis in paracetamol toxicity is not known. In this study of paracetamol toxicity in human liver after POD, hepatocyte apoptosis was identified at time of liver transplantation or death and was associated with striking regenerative activity. The biological significance of apoptosis is unclear but the rates of apoptosis found (0.6%) could account for a significant loss of hepatic parenchyma. The stimulus for apoptosis is not known but it is unlikely to be induced directly by paracetamol since it is absent from serum at this time. The possibility that apoptosis may be induced by Kupffer cell activation with cytokine production is raised. Patients who develop FHF after POD have a poor prognosis, with few therapeutic options apart from liver transplantation; an understanding of the dynamics of liver regeneration and ongoing cell loss by apoptosis may allow the development of new therapies in these patients.

Acid-labile subunit regulation during the early stages of liver regeneration: implications for glucoregulation.

The initiation of liver regeneration is regulated by endogenously produced growth factors and cytokines and is accompanied by suppression of growth hormone (GH) binding to hepatocytes. We have demonstrated some of these factors, particularly GH, which modulate acid-labile subunit (ALS) expression in vitro. Consequently, we investigated ALS hepatic mRNA and serum levels in rats for 24 h after partial hepatectomy (PHx). There was a significant suppression of ALS gene expression (approximately 50%, P < 0.005) and serum levels (approximately 30%, P < 0.02) by 12 h in PHx rats relative to controls. Relative to intact animals, hepatic mRNA and serum levels of ALS were suppressed by approximately 60% at 24 h. Similarly, hepatic GH receptor mRNA levels were significantly reduced in PHx animals. Moreover, hepatocytes isolated from PHx animals were less responsive to GH than those from controls. Overall, our results demonstrate that suppression of ALS gene expression and serum levels during liver regeneration relates to lowered hepatic GH sensitivity. Suppressed circulating ALS may alter insulin-like growth factor bioavailability and constitute a mechanism to maintain relatively normal glucoregulation after loss of liver mass.

Nonregenerative stimulation of hepatocyte proliferation in the rat: variable effects in relation to spontaneous liver growth; a possible link with metabolic induction.

Three procedures were used to stimulate hepatocyte proliferation in the rat without reducing liver mass, resulting in a supplementary growth which differs from the regenerative growth observed after loss of liver mass by hepatectomy or toxic necrosis. They were: (a) the ingestion of cyproterone, a cytochrome P450 inducing drug (b) the injection of an irritant which provokes glycogenesis and synthesis of acute-phase proteins (c) the injection of albumin-bound bilirubin leading to elimination of glucuronated bilirubin in bile. This ensuing supplementary growth was studied in the rat under several conditions of hepatic proliferation: 1. In normal adult rats, in which hepatocyte proliferation is very low, the effect on proliferation was either weak or undetectable. 2. In suckling rats, with a rapid body and liver growth, all the stimulants provoked a synchronized wave of proliferation with a steep increase of the percentage of S-phase hepatocytes from 4.5% in controls to 15-30% in treated rats. This increase was followed by a compensatory period of low proliferation during which a treatment with a second stimulant was much less effective. 3. In 2/3 hepatectomized adult rats, the proliferation induced by cyproterone was higher than the spontaneous regenerative proliferation alone and additional to it during all of the regenerative process. The proliferation induced by acute inflammation was competitive with the synchronous spontaneous proliferation during the early period of synchronized proliferation following surgery, suggesting that both are similar acute responses. Differently, during the late period of lower and unsynchronized regenerative proliferation, the proliferation provoked by acute inflammation was additional to the spontaneous one. A stimulation of proliferation by injection of the albumin-bilirubin complex was observed during the late period after 2/3 hepatectomy. The highest level of stimulation occurred when the liver growth and the hepatocyte proliferation were already high. This suggests that these stimulants are not complete mitogenic stimuli and need cofactors which are present during the spontaneous growth or, alternatively, that the effect of stimulants is opposed by an inhibitory mechanism present in the adult rat.

Early down-regulation of cytochrome P450 3A and 2E1 in the regenerating rat liver is not related to the loss of liver mass or the process of cellular proliferation.

Conflicting data have been reported concerning the modification of cytochrome P450 expression in the regenerating liver. Ligation of branches of the portal vein (PBL) perfusing 70% of the liver parenchyma, which produces regeneration and atrophy within the same liver, constitutes an ideal model to study the relative specificity of the early events in the regenerating liver and their relationship to the loss of liver mass. In this PBL model and in sham models, we studied the expression and the metabolic activities of two major cytochromes, CYP3A and CYP2E1, and the expression of inducible nitric oxide synthase protein (iNOS). They were simultaneously measured in the atrophying and regenerating liver lobes following PBL using Western Blot and HPLC methods. The metabolic activities of both cytochromes were transiently and simultaneously down-regulated in the regenerating and atrophying lobes during the first 2-5 h after PBL. No significant modification was observed at the protein level. In contrast, iNOS protein was significantly induced in both lobes. Similar results were observed after sham operation. The reduction of these CYP activities in both lobes after PBL and in sham livers suggests that other mechanisms than the regenerating process itself or the reduction of the liver mass might account for such down-regulation during the early phase of liver regeneration. The activation of nitric oxide (NO) and/or pro-inflammatory cytokine production provides clues to pathways liable to affect the CYP activities in the regenerating liver.

The Development and Compensation of Biliary Cirrhosis in Interleukin-6-Deficient Mice

In an effort to understand the role of IL-6/gp130 signaling in chronic liver injury, IL-6 deficient (IL-6(-/-)) and wild-type control (IL-6(+/+)) mice were subjected to bile duct ligation (BDL) for 12 weeks. This maneuver causes chronic biomechanical stress and liver injury, fueling sustained biliary epithelial and hepatocyte proliferation. By 12 weeks after BDL, IL-6(-/-) mice develop significantly higher total serum bilirubin levels (23.2 +/- 2.3 versus 14.9 +/- 2.1 mg/dl, P < 0.0001; delta bilirubin subfraction 16.7 +/- 4.0% versus 9.2 +/- 1.8%; P < 0.002), and the majority (15/18) show "black" gallbladder bile, compared to IL-6(+/+) mice (5/16; P < 0.003). The IL-6(-/-) mice also cannot sustain the compensatory liver mass increase commonly seen with chronic obstructive cholangiopathy, because of less hepatocyte proliferation, despite a rate of hepatocyte apoptosis similar to that of IL-6(+/+) mice. Moreover, IL-6(-/-) mice show a more advanced stage of biliary fibrosis and a higher mortality rate than the IL-6(+/+) controls (51% versus 23%; P < 0.02). These phenotypic changes in the IL-6(-/-) mice are associated with decreased expression and phosphorylation of gp130 and the transcription factor STAT3, compared to IL-6(+/+) mice. Daily treatment with exogenous recombinant IL-6 for 3-6 weeks starting at 6 weeks after BDL significantly lowers the serum total bilirubin in both groups. In the IL-6(-/-) mice, exogenous IL-6 treatment also increases the level of gp130 protein expression and completely reverses the loss of liver mass by increasing the hepatocyte proliferation. In conclusion, IL-6 appears to contribute to biliary tree integrity and maintenance of hepatocyte mass during chronic injury.

Early down-regulation of cytochrome P 450 3A and 2E1 in the regenerating rat liver is not related to the loss of liver mass

Early down-regulation of cytochrome P 450 3A and 2E1 in the regenerating rat liver is not related to the loss of liver mass

Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice.

Liver regeneration stimulated by a loss of liver mass leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Mice with targeted disruption of the interleukin-6 (IL-6) gene had impaired liver regeneration characterized by liver necrosis and failure. There was a blunted DNA synthetic response in hepatocytes of these mice but not in nonparenchymal liver cells. Furthermore, there were discrete G1 phase (prereplicative stage in the cell cycle) abnormalities including absence of STAT3 (signal transducer and activator of transcription protein 3) activation and depressed AP-1, Myc, and cyclin D1 expression. Treatment of IL-6-deficient mice with a single preoperative dose of IL-6 returned STAT3 binding, gene expression, and hepatocyte proliferation to near normal and prevented liver damage, establishing that IL-6 is a critical component of the regenerative response.

Hepatic stem cells in liver regeneration

The concept that the liver contains epithelial cells that share some of the major properties of stem cells of the well-characterized, stem cell-fed lineages found in bone marrow, intestinal epithelium, and epidermis is now well supported. Nevertheless, the population dynamics of the major types of liver epithelial cells, hepatocytes, and bile epithelia display a striking difference from the population dynamics of the classic stem cell systems. The focus of this review is on recent studies of the activation and expansion of liver stem cells in vivo and the role these cells may play in regeneration of the liver. The requirement for a selective and sustained expression of growth factors during the early stages of stem cell activation is highlighted. In addition, results are presented supporting the hypothesis that after loss of liver mass, both the quiescent stem cells as well as the residual differentiated hepatocytes and bile duct epithelial cells are activated to proliferate. However, significant contribution of the stem cells to the regeneration process only occurs under circumstances in which the residual differentiated cells are functionally compromised and/or cannot proliferate.

Caffeine demethylation measured by breath analysis in experimental liver injury in the rat

To assess the effects of experimental liver injury on caffeine metabolism, 1 μCi/kg b.w. of [3-methyl 14C]-caffeine (together with 5 mg/kg b.w. of the cold compound) was injected i.p. to four different experimental groups and respective controls of unanesthetized male Sprague-Dawley rats. Exhaled 14CO 2 was completely collected during 4 h and peak exhalation rate and fraction of dose recovered were calculated. 1 3 hepatectomy affected 14CO 2 exhalation to a limited extent, decreasing solely peak exhalation rate ( p<0.05 compared to sham-operated controls). 2 3 hepatectomy, on the other hand, resulted in significant reduction ( p<0.01) in both peak exhalation rate (by 59%) and fraction of dose recovered (by 47%), that were proportionate to the loss of liver mass (59%). End-to-side portocaval shunt led to the well-documented hepatic “atrophy”, liver weight being diminished on average to 50% within 2 weeks of surgery; however, reductions in peak exhalation rate (by 75%) and fraction of dose recovered (by 64%) were even more pronounced. Finally, 48 h bile duct ligation was equivalent to “functional 2 3 hepatectomy”, peak exhalation rate (by 65%) and fraction of dose recovered (by 56%) being markedly diminished despite increased liver weight. These results indicate that 14CO 2 exhalation curves following administration of specifically labelled caffeine are quantitative indicators of acute or chronic loss of functioning liver mass. In addition, the 3-demethylation pathway appears to be particularly sensitive to the inhibitory effects of cholestasis on microsomal function.