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Obesity and its major comorbidities, including type 2 diabetes mellitus, nonalcoholic fatty liver disease (NAFLD), obesity cardiomyopathy, and certain cancers, have caused life expectancy in the United States to decline in recent years. Obesity is the increased accumulation of triglycerides (TG), which are synthesized from glycerol and long-chain fatty acids (LCFA) throughout the body. LCFA enter adipocytes, hepatocytes, and cardiomyocytes via specific, facilitated transport processes. Metabolism of increased cellular TG content in obesity may lead to comorbidities such as NAFLD and cardiomyopathy. Better understanding of LCFA transport processes may lead to successful treatment of obesity and NAFLD.

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36−/−mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36−/−mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36−/−mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36−/−mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.

During differentiation of mouse 3T3-L1 fibroblasts to an adipocyte phenotype, the mitochondrial isoform of aspartate aminotransferase accumulates on the plasma membrane. The determination of whether this reflects translation of an alternatively spliced message lacking the mitochondrial leader sequence required cloning of the enzyme's uncommon a allele, for which these cells are homozygous. The 1.4-kb cDNA sequence of the a allele was obtained from oligo-dT-primed reverse-transcriptase PCR products amplified from FVB mouse RNA. It differed from the b allele at only 2 bp and one amino acid. By contrast, gene-specific primers generated an additional 1.4-kb fragment that differed from the b allele by ≈ 1% of nucleotides, encoding four amino acid substitutions. This sequence proved to represent a recently diverged processed pseudogene. The presence of such pseudogenes can complicate interpretation of expressed-sequence-tag data and single-nucleotide-polymorphism genotyping studies. Using probes derived from the a allele, RNase protection analyses indicated that only a single message for the enzyme was present in 3T3-L1 fibroblasts and adipocytes, despite differences in subcellular protein distribution.

A plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABPpm 's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABPpm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABPpm constitutes approximately 2% of LPM protein, or about 0.7 × 107 sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABPpm is described; it yields an estimate of 3.4 x 107 h-FABPpm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.

The hepatic clearance of unconjugated bilirubin-14C from the plasma of 5 patients with cholestatic liver disease was normal when compared with values obtained in healthy young adults. In 1 patient with benign recurrent cholestasis, clearance was the same during an attack of cholestasis (serum bilirubin concentration 25.7 mg/100 ml) as when the patient was in remission (serum bilirubin concentration 0.65 mg/100 ml). We conclude that the hepatic clearance of unconjugated bilirubin may not be altered in hepatic disorders where cholestasis is the predominant feature and that other explanations should be considered when patients with these disorders manifest an increased plasma concentration of unconjugated bilirubin.

An increase in bile flow after phenobarbital administration occurs in the rat and other species; however, the mechanism(s) of the choleretic effect is incompletely understood and the role of the increase in liver weight is controversial. We therefore measured bile flow, bile acid secretion and pool size in male Sprague-Dawley rats pretreated with phenobarbital (75 mg/kg/day) for 6 days; liver weight, liver cell volume and DNA content were also evaluated. Phenobarbital treatment increased liver weight and mean hepatocyte volume by 39 and 26%, respectively, while total DNA content did not change, thus indicating that the hepatomegaly results principally from hypertrophy rather than hyperplasia. Bile flow was significantly higher in treated rats when expressed per unit of body weight (64.6 ± 2.4 (S.E.) vs 53.3 ± 1.6 μl/min/kg; P< 0.05) but was unchanged when expressed per gram of liver (1.40 ± 0.04 vs 1.37 ± 0.06 μl/min/g; P> 0.5). The initial bile acid secretion rate and pool size were both significantly reduced in the phenobarbital group compared to controls (1224.2 ± 110.4 vs 1656.6 ± 163.2 nmol/kg/min and 562.8 ± 41.5 vs 814.3 ± 78.3 μmol/kg; both P< 0.05), whereas the basal synthetic rate was unchanged. These findings suggest that the enlarged, phenobarbital-treated hepatocyte produces more bile than the normal cell, despite the decreased secretion of bile acids. Therefore, the drug-induced choleresis involves a selective increase in the bile acid-independent fraction of bile flow.

Physicochemical studies have suggested that the 43-kDa plasma membrane fatty acid binding protein (FABPpm) is closely related to the mitochondrial isoform of aspartate aminotransferase (mAspAT). In the present studies, mAspAT was not detected immunohistochemically or by immunoblotting in plasma membranes of proliferating 3T3-L1 fibroblasts. During controlled differentiation to an adipocyte pheno-type, mAspAT became detectable by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated 3T3-L1 adipocytes. The pattern of expression paralleled the previously reported expression both of FABPpmand of the Vmaxfor saturable uptake of long chain free fatty acids. As with anti-FABPpm, antibodies to mAspAT selectively inhibited the uptake of [3H]-oleate in 3T3-L1 adipocytes but not in fibroblasts, while having no effect on uptake of either 2-deoxyglucose or the medium chain fatty acid octanoate. Preabsorption of anti-FABPpmwith mAspAT, or of anti-mAspAT with FABPpm, abolished immunopositivity in immunohistochemical and immunoblotting studies, as well as the ability of either antibody to inhibit [3H]-oleate uptake. These studies provide strong biologic evidence for the identity of FABPpmand mAspAT, and for the hypothesis that FABPpm/mAspAT mediates the uptake of long chain free fatty acids.

To study the relationship between the dose of phenobarbital (PB) and the magnitude of its effects on microsomal enzymes, cytochrome P-450, UDP-glucuronyl transferase (UDPGT), and glucose-6-phosphatase (G6P) activities were determined in liver homogenate and microsome preparations from control rats and rats treated for 6 days with PB at doses ranging from 1 to 125 mg/kg/day. Both P-450 and UDPGT activities were enhanced by PB in a dose-related fashion. However, while the lowest dose of the drug to produce significant induction of both enzymes was the same (3 mg/kg), maximal induction of P-450 (214%) and UDPGT (285%) was obtained with different doses of PB, namely 75 and 125 mg/kg, respectively. UDPGT induction could equally be demonstrated regardless of whether “native” enzyme or enzyme activated by UDP-N-acetyl glucosamine, digitonin or deoxycholate was employed. In contrast to these inducing effects of the drug on P-450 and UDPGT, PB treatment resulted in a dose-related inhibition of G6P activity. The inhibitory effect was observed with both “native” and deoxycholate-activated enzymes, and could be demonstrated whether the data were expressed as enzyme specific activity (nanomoles per minute per milligram microsomal protein) or as total G6P activity (micromoles per minute per 100 g body weight). These results indicate that: (i) enzyme induction by PB is dose-related; (ii) induction of both P-450 and UDPGT is obtained in the rat with doses of the drug similar to those given to man; and (iii) observed inhibition of G6P activity by PB does not solely reflect an enzymatic dilution secondary to the proliferated endoplasmic reticulum.

Long-chain, non-esterified free fatty acids (FFA), the building blocks from which triglycerides are synthesized, are important energy substrates in various tissues, essential building blocks for the lipid components of cell membranes, and precursors for the synthesis of important biological mediators such as the prostaglandins. They are also increasingly being recognized as important intracellular mediators of gene expression. These multiple roles suggest that careful regulation of all aspects of FFA disposition, including cellular uptake, would be advantageous. However, although the intracellular metabolism of FFA and their transport in plasma as complexes with albumin have been extensively studied, little attention was paid until recently to the mechanisms by which FFA enter and leave cells. This neglect occurred both for methodologic reasons and because the cellular uptake of FFA was long considered to be an entirely unregulated process in which FFA partitioned passively into the lipid bilayer of the plasma membrane.Since 1981, a series of studies from several groups clarified the influence of albumin binding on the uptake kinetics of FFA and other bound ligands, making more detailed studies of the uptake process feasible (1-6). Once it became clear that FFA uptake occurred principally from the very small unbound ligand pool in plasma and not from the albumin-bound compartment, it was quickly reported that a major component of FFA uptake in certain cell types, including hepato-cytes, adipocytes, and cardiac and skeletal myocytes, in fact exhibited all the kinetic properties of a facilitated transport process: saturation, trans-stimulation, cis-inhibition, and counter transport (7-18). These observations, which—if valid—cannot be explained by purely passive diffusion, have suggested to many investigators the existence of a facilitated uptake process for FFA, which appears to exist in parallel with a nonsaturable, presumable diffusive pathway.

Hepatic histologic materials (biopsy or autopsy) and associated clinical data from 155 hemophiliacs were collected by an ad hoc hemophilia study group and analyzed retrospectively in an effort to determine the spectrum of liver disease in this population and to examine the relationship between the severity of liver disease and treatment history. Clinical information on the frequency of complications from 126 biopsies in 115 hemophilic patients provided a unique opportunity to assess the safety of liver biopsy in such patients. The incidence of cirrhosis (15%) and chronic active hepatitis (7%) was lower than previously reported. The frequency of severe liver disease (chronic active hepatitis or cirrhosis) in patients receiving large pooled concentrates was no greater than in patients treated principally with cryoprecipitate or plasma. The risks of liver biopsy in this setting are relatively high: clinically significant hemorrhage followed 12.5% of the procedures.

A previously described technique has been modified to permit the continuously recirculating perfusion of the separate maternal and fetal circulations of an isolated cotyledon of human placenta. Viability of the perfused cotyledons was established by measurements of oxygen consumption (average, 0.18 ml/gm/hr), glucose utilization (average, 1.0 mg/gm/hr), and lactate production (<0.01 μmol/gm/hr), and integrity of the placental barrier by the failure of India ink,125I-albumin, or35S-sulfobromophthalein to cross from fetal to maternal circulation. Clearance of3H-inulin from the fetal circuit, 0.0059 ± 0.0005 (SE) ml/min/gm, corresponded to 2.5% of its clearance by the adult human kidney. Clearance of14C-antipyrine was 0.013 ± 0.003 ml/min/gm. After introduction into the fetal circuit, the observed appearance of both inulin and antipyrine in the maternal circuit closely paralleled curves predicted by a dimple mathematical model. The use of a continuously recirculating perfusion system is technically feasible, and has advantages over the single-pass technique for studying transplacental transfer of metabolites with a low efficiency of extraction.

To examine whether fatty acid transport is abnormal in obesity, the kinetics of [3H]oleate uptake by hepatocytes, cardiac myocytes, and adipocytes from adult male Wistar (+/+), Zucker lean (fa/+) and fatty (fa/fa), and Zucker diabetic fatty (ZDF) rats were studied. A tissue-specific increase in oleate uptake was found in fa/faand ZDFadipocytes, in which the Vmaxwas increased 9-fold (p< 0.005) and 13-fold (p< 0.001), respectively. This increase greatly exceeded the 2-fold increase in the surface area of adipocytes from obese animals, and did not result from trans-stimulation secondary to increased lipolysis. Adipocyte tumor necrosis factor-α mRNA levels, assayed by Northern hybridization, increased in the order +/+

Indocyanine green (ICG), a water-soluble tricarbocyanine dye, is rapidly removed from the circulation by the liver and secreted into the bile without conjugation (1–3). Currently, its major medical use is for indicator dilution studies during cardiac catheterization. However, its rapid excretion via the liver without significant extrahepatic removal (1–4) or enterohepatic circulation (1, 3) and freedom from the problems of local tissue necrosis or anaphyllaxis, which may occur with sulfobromophthalein (5), have prompted the increasing use of plasma ICG disappearance curves as a test of liver function. The most commonly used format for this purpose involves determination of the plasma fractional disappearance rate (k) or half-life (T1/2) of ICG following the rapid intravenous injection of dye at a dose of 0.5 mg/kg body weight.We present the results of plasma ICG disappearance studies in a large group of young, healthy normal volunteer subjects. The data unexpectedly indicate a significant difference in the plasma fractional ICG disappearance rates between normal men and women, which must be taken into account in interpreting similar studies in patients with either suspected or established liver disease.Methods. Subjects studied.Plasma ICG disappearance studies were performed in 51 healthy normal individuals, ages 18–29, who had been admitted to the Clinical Center of the National Institutes of Health for the specific purpose of serving as control subjects for a variety of studies. Eighteen were men and 33 were women. All met strict, previously defined criteria for “normality” (6), and none had taken medications of any type within 4 weeks of the studies. Each of the 51 volunteers underwent plasma ICG disappearance studies employing a dose of 0.5 mg/kg body weight. In addition, 19 of the volunteers (9 men and 10 women) underwent three additional studies, employing indocyanine green doses of 2.0, 3.5, and 5.0 mg/kg.

A patient with type Icongenital nonhemolytic jaundice (type ICrigler-Najjar syndrome (CNJ)), who first developed overt neurologic signs at age 18, is reported. Studies with isotopic bilirubin, performed both when she was well and after the onset of brain damage, demonstrated normal hepatic bilirubin uptake and storage capacity, and normal bilirubin turnover (3.0 mg/kg/24hr). As a result of a virtual absence of conjugation, net bilirubin clearance was reduced to 0.0075 ml/min/kg, which is approximately 1 of normal (0.65 ± 0.18 ml/min/kg). Therapy with glutethimide, phenobarbital, blue light, and oral agar were all ineffective in reducing the concentration of unconjugated bilirubin in plasma or accelerating radiobilirubin disappearance.

These studies were carried out to determine whether bovine serum albumin (BSA), which is usually included in the incubation mixture for the in vitrodetermination of bilirubin-UDP-glucuronyl transferase (GT) activity, affects GT activity. Using bilirubin as substrate, addition of BSA to the enzyme reaction mixture at concentrations varying from 2 to 30 mg/ml resulted in a dose-related inhibition of “native” GT activity of rat liver microsomes. When detergent-activated enzyme was employed, increasing concentrations of BSA also required higher concentrations of deoxycholate, digitonin, or Triton X-100 to produce maximal bilirubin conjugation. Low BSA concentrations (2 mg/ml) prevented enzyme activation by both detergents and UDP-N-acetyl glucosamine. When BSA was omitted and bilirubin dissolved in dimethyl sulfoxide, UDP-N-acetyl glucosamine failed to enhance GT activity, and activation by detergents was only 15-25% of that observed in the presence of optimal concentrations of BSA. When rat albumin was substituted for BSA, a similar dose-related inhibition of in vitrobilirubin conjugation by untreated microsomes was observed, although at any given albumin concentration, GT activity was lower with rat than with bovine albumin. Additionally, both detergents and UDP-N-acetyl glucosamine produced similar GT activation regardless of the rat albumin concentration. Finally, these effects of BSA and rat albumin could not be reproduced when β-lactoglobulin was employed and/or when p-nitrophenol was the acceptor substrate of GT. These findings indicate that albumin, in particular BSA, profoundly and selectively influences the in vitroactivity of microsomal GT toward bilirubin as the acceptor substrate.

Background/Aims:The liver loses protein during fasting. This study sought to determine if hepatic protein loss during fasting selectively preserves functions important to survival such as uptake of fatty acids, which are major energy substrates in that condition. Methods:Initial [3H]oleate uptake and efflux rates in hepatocytes from starved (for 48 hours) and fed male rats were measured in media containing 250 μmol/L albumin at oleate/albumin ratios of 0.2:1–2:1. Uptake rates of sulfobromophthalein, taurocholate, and glucose were also determined. Results:Initial oleate uptake rate was saturable with respect to unbound oleate concentration. Maximum initial velocity expressed per cell number did not differ between fasted and fed animals, but measured cell volume and estimated surface area were decreased in starved vs. fed hepatocytes (921 ± 21 vs. 1623 ± 58 μm2, respectively; P< 0.001). Consequently, when expressed per surface area, maximum initial velocity was greater in starved cells (17 ± 3 vs. 10 ± 2 [pmol · min−1· μm2] × 10−7; P< 0.02). Expressed similarly, oleate efflux was also greater from starved hepatocytes and was inhibited by an antibody to plasma membrane fatty acid binding protein (FABPpm). FABPpmconcentration per unit area of plasma membrane also increased in starved hepatocytes (P< 0.05). By contrast, uptake rates of sulfobromophthalein, taurocholate, and glucose by starved hepatocytes were decreased when expressed per cell number and unchanged per unit area. Conclusions:During fasting, the hepatocellular uptake mechanism for oleate is selectively preserved compared with those for sulfobromophthalein, taurocholate, or glucose.

We measured uptake of a representative free fatty acid, oleate, by the single-pass perfused rat liver at oleate : albumin molar ratios of 0.01 to 2:1. For each ratio, uptake was studied at albumin concentrations from 50 to 600 μM. When uptake velocity was plotted as a function of the albumin concentration, the data at each ratio exhibited a pseudosaturation pattern as previously observed in isolated cells (J Clin Invest84: 1325). At a physiologic albumin concentration of 600 μM, a plot of uptake vs. unbound oleate concentrations was best fitted by the Michaelis-Menten equation (Vmax = 235±8.8 nmol · min−1· g · liver−1; Km=130±12 nM). As the albumin concentration was increased from 50 to 250 μM, the unbound oleate clearance, calculated by either the undistributed sinusoidal or venous equilibrium models, increased progressively, in violation of conventional pharmacokinetic theory, indicating an enhancing effect of albumin on ligand uptake at low albumin concentrations. In contrast, there was no significant difference between measures of unbound clearance at albumin concentrations of 350 and 600 μM. To explain this phenomenon, the clearance data were examined for evidence of facilitation (accelerated dissociation of ligand : albumin complexes) by the clearance ratio test (“square root rule”). All deviations from the predictions of conventional theory were entirely attributable to pseudofacilitation. No data required explanation by a true facilitation model.

To clarify mechanisms of hepatic free fatty acid uptake, [3H]oleate uptake by isolated rat hepatocytes was studied. using solutions of 150 μM bovine serum albumin at oleate:albumin molar ratios of 0.033–6.7:1. Oleate partitioning between liver plasma membranes and albumin was also studied, and used to ascertain the membrane binding function for oleate. The experimental uptake curve was complex, but could be resolved by computer fitting into a sum of two components, one a saturable and the second a linear function of the unbound oleate concentration. The saturable component comprises >90% of total oleate uptake when the oleate:albumin molar ratio is <2.5, but <50% when this ratio is >5. Membrane binding also consisted of a sum of a saturable and a linear component. By comparison of the computer-fitted uptake and binding functions, separate rate constants for the transfer into the cell of the saturably and non-saturably bound oleate were estimated to be 0.7 s−1and 0.05 s−1, respectively. The former is compatible with a specific, protein-mediated process. It is 15-times greater than the corresponding rate constant for transfer of non-saturably bound oleate into the cell, which in turn is similar to reported rates of non-specific ‘flip-flop’ of fatty acids across lipid bilayers. The observed kinetics are not consistent with models in which uptake occurs principally from the albumin-bound pool of oleate, or solely from the oleate which has partitioned passively into the lipid bilayer of the plasma membrane.

To determine whether bile formation in the dog is an immature process at birth, several determinants of bile secretion were studied in anesthetized, bile duct-cannulated puppies of 0–42 days of age and adult dogs. Basal canalicular bile flow rate, estimated by 14C-erythritol biliary clearance, averaged 0.182 µl/min/g liver in 0–3 day-old puppies and increased to 0.324 and 0.461 µl/min/g in puppies 7–21 and 28–42 days of age, respectively. The latter two values were not significantly different from that observed in adult dogs (0.348 µl/min/g). This age-related increase in canalicular bile flow could be accounted for primarily by an increase in bile acid independent bile flow (0.103, 0.277, 0.375, and 0.252 µl/min/g in 0–3, 7–21, and 28–42 day-old puppies and adult dogs, respectively). Calculated ductular bile water reabsorption (14C-erythritol biliary clearance-bile flow) was virtually absent in 0–3 day-old puppies (0.005 µl/min/g), and averaged 0.017 and 0.092 µl/min/g in puppies of 7–21 and 28–42 days of age, respectively. In adult dogs, ductular bile water reabsorption was 0.132 µl/min/g. These functional deficiencies of the newborn dog were associated with an increased biliary permeability to 3H-inulin which could not be accounted for solely by an increased solute diffusion due to the lower rate of canalicular bile flow. Administration of taurocholate up to 2000 nmol/min/kg produced in all animals a similar increase in canalicular bile flow and bile acid excretion, and was not associated with changes in ductular bile water reabsorption rate. These findings are interpreted to indicate that, in the dog, bile secretory function is immature at birth and develops during postnatal life.

To determine whether bile formation in the dog is an immature process at birth, several determinants of bile secretion were studied in anesthetized, bile duct-cannulated puppies of 0–42 days of age and adult dogs. Basal canalicular bile flow rate, estimated by 14C-erythritol biliary clearance, averaged 0.182 μl/min/g liver in 0–3 day-old puppies and increased to 0.324 and 0.461 μl/min/g in puppies 7–21 and 28–42 days of age, respectively. The latter two values were not significantly different from that observed in adult dogs (0.348 μl/min/g). This age-related increase in canalicular bile flow could be accounted for primarily by an increase in bile acid independent bile flow (0.103, 0.277, 0.375, and 0.252 μl/min/g in 0–3, 7–21, and 28–42 day-old puppies and adult dogs, respectively). Calculated ductular bile water reabsorption (14C-erythritol biliary clearance-bile flow) was virtually absent in 0–3 day-old puppies (0.005 μl/min/g), and averaged 0.017 and 0.092 μl/min/g in puppies of 7–21 and 28–42 days of age, respectively. In adult dogs, ductular bile water reabsorption was 0.132 μl/min/g. These functional deficiencies of the newborn dog were associated with an increased biliary permeability to 3H-inulin which could not be accounted for solely by an increased solute diffusion due to the lower rate of canalicular bile flow. Administration of taurocholate up to 2000 nmol/min/kg produced in all animals a similar increase in canalicular bile flow and bile acid excretion, and was not associated with changes in ductular bile water reabsorption rate. These findings are interpreted to indicate that, in the dog, bile secretory function is immature at birth and develops during postnatal life.

Extract: A patient with type I congenital nonhemolytic jaundice (type I Crigler-Najjar syndrome (CNJ)), who first developed overt neurologic signs at age 18, is reported. Studies with isotopic bilirubin, performed both when she was well and after the onset of brain damage, demonstrated normal hepatic bilirubin uptake and storage capacity, and normal bilirubin turnover (3.0 mg/kg/24hr). As a result of a virtual absence of conjugation, net bilirubin clearance was reduced to 0.0075 ml/min/kg, which is approximately 1% of normal (0.65 ± 0.18 ml/min/kg). Therapy with glutethimide, phenobarbital, blue light, and oral agar were all ineffective in reducing the concentration of unconjugated bilirubin in plasma or accelerating radiobilirubin disappearance.The acute onset of neurologic disease occurred at age 18, and may possibly have related to a transient increase in the molar bilirubin to albumin ratio to a value greater than 1.0. Exchange plasmapheresis, performed with a continuous flow centrifuge, rapidly reduced the bilirubin concentration in plasma from 41 to 6 mg/100 ml, and was associated with apparent clinical improvement. A total of 1,900 mg bilirubin was removed by this procedure.Although in vivo phototherapy was ineffective, in vitro illumination of the patient's plasma with special blue lamps produced a rapid fall in the bilirubin concentration in plasma. Bilirubin was converted to polar, diazo-negative derivatives by a process in which several steps could be distinguished. The initial photodegradation products were colored, tightly albumin bound and nondialyzable, which explained their tendency to be excreted in the bile in preference to the urine. Continued illumination for up to 24 hr resulted in the production of colorless, nonalbumin-bound compounds which, when injected into rats, were excreted principally in the urine.Speculation: Pending the perfection of techniques for auxiliary hepatic transplantation in man, extracorporeal phototherapy or hemoperfusion over albumin-conjugated agarose columns may provide a means of prevention of the otherwise inevitable development of neurologic disease in patients with type I CNJ.

Human placentas were perfused in vitro through fetal vessels of a single cotyledon with a solution of tritiated androstenedione (A) or tritiated testosterone in oxygenated Krebs-Ringer bicarbonate buffer containing glucose. The same buffer, without labeled steroids, was used to perfuse the maternal side of the placenta. Fractions of unrecycled “fetal” and “maternal” perfusates were collected to estimate the steady-state rates of output into each perfusate of labeled estrone (E1) and estradiol (E2), formed by aromatization of the perfused androgens. An unequal distribution of the two estrogens was observed, i.e., a significantly larger proportion of E2than of E1was released to the maternal perfusate. Addition of unlabeled A or ethinyl estradiol (10−5M) to the fetal perfusion buffer resulted in a redistribution of the E2output toward the fetal perfusate. These in vitro results are similar to the findings of Walsh and McCarthy, who detected by tracer experiments an unequality in the in vivo fetomaternal distribution of the output of placentally produced E1and E2in rhesus monkeys. These results also support the possibility of the existence of saturable specific E2carriers in the syncytiotrophoblast, postulated by the same authors to explain their findings. However, an extrasyncytial conversion of E2to E1could also account for the disproportion in the relative output of the estrogens toward mother and fetus. The perfusion system used in these studies may be useful for the elucidation of factors determining the relative rates of placental secretion of steroids toward mother and fetus

Bilirubin, an organic anion and breakdown product of heme, is excreted chiefly by the liver by a process involving uptake by the hepatocyte and subsequent conjugation to form a more polar compound (1). The availability of purified preparations of bilirubin, especially of radiolabeled bilirubin, has greatly facilitated studies of its metabolism (1-5). Study of the transport of bilirubin from blood to bile, however, must also include consideration of the mechanisms for excretion of bilirubin conjugates. Such study has been limited because of difficulty in obtaining conjugated bilirubin free of significant quantities of contaminants (6-19).Recent studies of albumin-agarose gel affinity chromatography have revealed that the gels have a high affinity for bilirubin and a relatively low affinity and capacity for tri-and dihydroxy bile acids (20, 21), suggesting that this method might effectively separate bilirubin conjugates from other components in bile. We describe here, a rapid and relatively simple method for purification of conjugated bilirubin from bile.Materials and methods.Albumin-agarose gel was prepared by a modification of the cyanogen bromide method (20, 22), and packed in small columns of 0.9-cm i.d. Bile acids were quantitated in bile and column eluates by gas-liquid chromatography (23), cholesterol by the method of Zak et al.(24), and phospholipids by a modification of the method of Chen et al.(25, 26).Absorption spectra were obtained with a Beckman Acta III spectrophotometer, and fluorimetric analyses with a Farrand Mark I spectrofluorimeter.Tritium was quantitated by adding up to 0.5 ml of sample to 15 ml of Aquasol (New England Nuclear) and by counting in a liquid scintillation counter (Nuclear Chicago).All samples were recounted after addition of [3H]toluene (New England Nuclear) and results were corrected for background and quenching.

A lag period of 12-24 hr has frequently been observed to elapse between charcoal hemoperfusion therapy and arousal of patients in stage IV coma due to fulminant hepatic failure. This delay suggests that recovery of consciousness depends on depletion of an extravascular pool of a hypothetical toxin which requires 12-24 hr to equilibrate with plasma. No further definitive information about the nature of the toxin is available. Unconjugated biliru-bin is known to have an extrahepatic, extra-vascular pool which also equilibrates with plasma over a 12- to 24-hr period. Accordingly, a model of unconjugated bilirubin kinetics has been used to investigate the rate of depletion of slowly equilibrating extra-vascular metabolic compartments by hemoperfusion, employing computer simulation techniques. The results indicate that the rate of depletion of slowly equilibrating extravascular pools is critically dependent not merely on the intrinsic efficiency of the hemoperfusion device, but also on the schedule with which it is employed. The conventionally employed regimen of 4-hr of treatment per day is relatively inefficient in achieving depletion of such compartments. These data imply, by analogy, that alternative hemoperfusion regimens, employing longer initial perfusion or perfusions at intervals of approximately 12 hr, would be more effective than current schedules in awakening patients with acute hepatic coma.The author is indebted to Ms. P. Cromwell for assistance with this study and for preparation of the manuscript.

Extract: A Negro female with congenital nonhemolytic jaundice (Crigler-Najjar syndrome) was studied over a period of 15 months. During this period the plasma bilirubin concentration averaged 20.6 mg/100 ml. Administration of phenobarbital did not lower the bilirubin concentration. Studies with isotopically labeled bilirubin showed that phenobarbital had not increased the fractional rate of elimination of bilirubin, and the patient's bilirubin production rate while receiving phenobarbital therapy (3.5 mg/kg/24 hr) was not increased over that seen in normal individuals (3.8 \pm 0.6 mg/kg/24 hr, mean \pm sd).Multicompartmental analysis of the patient's labeled bilirubin clearance data, as well as that from two other reported cases of congenital nonhemolytic jaundice, did not furnish new information on pathways of bilirubin catabolism but did demonstrate that the fractional transfer rate of bilirubin from plasma to liver for all three subjects (0.42–0.87 hr-1) was reduced compared with the range seen in normal individuals (0.9–2.0 hr-1). This finding may reflect saturation of either the hepatic uptake process or intracellular binding sites, in the face of a serum bilirubin concentration 20–40 times normal.Menthol conjugation in the patient's parents was normal, unlike reported findings in parents of children with congenital nonhemolytic jaundice unresponsive to phenobarbital therapy. Labeled bilirubin clearances in the parents demonstrated an abnormality in the mother's pattern of clearance, whereas the father's clearance was normal.The patient was found to excrete normal amounts of testosterone conjugated with glucuronic acid, demonstrating that the defect in glucuronide conjugation does not include all physiological compounds.Speculation: Plasma clearance studies with labeled bilirubin can aid in the investigation of modes of inheritance of unconjugated hyperbilirubinemia.

A Negro female with congenital nonhemolytic jaundice (Crigler-Najjar syndrome) was studied over a period of 15 months. During this period the plasma bilirubin concentration averaged 20.6 mg/100 ml. Administration of phenobarbital did not lower the bilirubin concentration. Studies with isotopically labeled bilirubin showed that phenobarbital had not increased the fractional rate of elimination of bilirubin, and the patient's bilirubin production rate while receiving phenobarbital therapy (3.5 mg/kg/24 hr) was not increased over that seen in normal individuals (3.8 \pm 0.6 mg/kg/24 hr, mean \pm sd).

The plasma unconjugated bilirubin concentration (Br) is related to the bilirubin production rate (BRP) and hepatic bilirubin clearance (CBr) in the following manner: Br = BRP ÷ CBr. The distribution for Br in the normal population was calculated from this relationship, using normal values of BRP and CBr measured with labeled bilirubin. The results indicate that any individual with a Br exceeding 1.0 mg/100 ml (99% level) should be suspected of having abnormal hepatic function or bilirubin overproduction or both. A Br greater than 3.5 mg/100 ml usually indicates abnormal hepatic function, regardless of the BRP. A change in Br means that either bilirubin production or hepatic function has been altered, even if Br remains within the normal range.

A device has been described which facilitates rapid, timed sampling of arterial blood in the rat. This device is especially useful for definition of plasma disappearance curves of various injected compounds.