Effects of amino acids on bile acid-dependent and independent bile flow in the isolated perfused rat liver.

BACKGROUND/AIMS: Conflicting data on the effects of amino acids on biliary function led us to investigate their interaction with taurocholate in the perfused rat liver model. METHODS: To investigate the influence of amino acids on the bile acid-independent component of bile flow, 12 livers were perfused with (n = 6) and without (n = 6) amino acid addition from t30 min. For the study of bile acid-dependent bile flow, 24 livers were perfused under 8 experimental conditions according to the perfusate taurocholate concentration (12.5, 25, 37.5 or 50 microM) and whether amino acids were or were not added from t30 min. RESULTS: In the absence of taurocholate, amino acids induced a 40% (p<0.01) decrease in bile flow together with an increase in hepatic water content (17.8%, p< 0.05). Thus, amino acids exert an inhibitory effect on bile acid-independent bile flow despite the postulated cell swelling-dependent increase in bile flow. When livers were perfused at various taurocholate concentrations, amino acids induced, in addition to their inhibitory effect on bile acid-independent bile flow, a significant increase in taurocholate apparent choleretic activity (13.2 microl/micromol vs. 10.6 microl/micromol; p = 0.05), while taurocholate intrinsic clearance was significantly decreased (4.5+/-1.2 ml x min(-1) x g(-1) vs. 6.1+/-1.3 ml x min(-1) x g(-1); p<0.01). CONCLUSIONS: These data suggest that at physiological bile acid concentrations amino acids exert an inhibitory effect on both bile acid-dependent and- independent bile flow, whereas at higher taurocholate concentrations this inhibitory effect disappears, probably because of cell swelling-dependent mechanisms.

Direct cytotoxicity of hypoxia-reoxygenation towards sinusoidal endothelial cells in the rat.

AIMS/BACKGROUND: Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury. METHODS: Sinusoidal endothelial cells were isolated from rat liver. After 2 3 days of primary culture, the cells were exposed to hypoxia (N2/CO2 95/5) for 120 min and reoxygenation (O2/CO2 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O2 alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondrial dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively. RESULTS: The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type. CONCLUSIONS: The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.

Is the L-arginine-nitric oxide pathway involved in endotoxemia-induced muscular hypercatabolism in rats?

We investigated the role of the nitric oxide (NO) synthase (NOS) pathway in muscular metabolism during endotoxemia in four groups of male Wistar rats. Two groups were injected with the lipopolysaccharide (LPS) of Escherichia coli (3 mg/kg), with one group treated using N(G)-nitro-L-arginine methylester ([L-NAME] 85 mg/kg/d) and the other not. The two control groups included one treated with L-NAME and the other not. After 24 hours of fasting, the rats were fed by controlled enteral nutrition and killed on day 3. The results showed that (1) NOS inhibition was detrimental during endotoxemia, increasing lethality from 20% to 80.5%, and (2) NOS inhibition did not modify the hypercatabolic state consecutive to endotoxemia, particularly at the muscular level (nitrogen balance, total-body and muscular weight loss, and muscular protein and glutamine concentrations). However, myofibrillar catabolism was delayed in the LPS-NAME group. In conclusion, NO production is of major importance for survival after an endotoxemic challenge, but contributes weakly to the metabolic response of muscle to injury.

Intestinal absorption and permeability in human immunodeficiency virus-infected patients.

BACKGROUND: Impaired intestinal function could account for diarrhoea and weight loss, which are common features of advanced human immunodeficiency virus (HIV) infection. METHODS: We assessed intestinal permeability to lactulose and mannitol and absorption of D-xylose in 96 HIV-infected patients (group I: asymptomatic subjects (CDC-A); group II: symptomatic subjects (CDC-B or C) without body weight loss and/or diarrhoea; group III: 25 acquired immunodeficiency syndrome (AIDS) patients (CDC-C) with severe body weight loss and/or diarrhoea) and 10 healthy subjects as controls. RESULTS: An incremental decrease in urinary D-xylose recoveries was observed, with all groups statistically different from each other. Impaired intestinal permeability was only found in patients of group III (statistically different from all other groups). CONCLUSIONS: These findings suggest a loss of intestinal functional absorptive surface as HIV disease progresses. This process may be present at the early stage of infection. Impaired intestinal permeability is observed later in AIDS patients when digestive signs are present, particularly diarrhoea.

Plasma concentration, kinetic constants, and gene polymorphism of angiotensin I-converting enzyme in centenarians.

We have determined serum activity and kinetic constants of angiotensin I-converting enzyme (ACE), parallel to an insertion/deletion (I/D) polymorphism in its gene, in French centenarians and controls 20-70 years of age because this enzyme could have an impact on cardiovascular risk, and thus on longevity. Both the ACE D allele and ACE D/D genotype were more frequent in centenarians in comparison with controls, without sex-related differences nor significant correlation with a cardiovascular pathology. In centenarians, I/D polymorphism was correlated with circulating ACE activity (D/D genotype, 89.0 +/- 36.8 U/L; I/D genotype, 63.5 +/- 26.0 U/L; and I/I genotype, 55.1 +/- 39.4 U/L). The Michaelis constants for two substrates were identical whatever the genotype and were not different between centenarians and controls, i.e., 0.30 +/- 0.03 mmol/L for furylacryloyl-phenylalanyl-glycyl-glycine and 1.35 +/- 0.05 mmol/L for hippuryl-histidyl-leucine; for the latter, the optimal pH and activating concentration of chloride did not depend on I/D polymorphism. The maximal velocities with both substrates reflected the distribution of serum ACE activity as a function of the genotypes, in centenarians and in controls. In conclusion, plasma ACE activity is subject to a similar genotypic influence in centenarians as in adults 20-70 years of age; however, ACE itself appears to be functionally similar for each genotype. Furthermore, the D allele as well as the higher serum ACE activities associated with the D/D genotype cannot discriminate individuals at high risk for cardiovascular diseases, major causes of mortality before the age of 100 years.

TNF-alpha and IL-6 synergistically inhibit ketogenesis from fatty acids and alpha-ketoisocaproate in isolated rat hepatocytes.

BACKGROUND: During sepsis, lipid metabolism is shunted toward triacylglycerol synthesis and hepatic lipogenesis. A decrease in ketogenesis from free fatty acids also is observed, probably mediated by cytokines involved in host response to infection. Whether such an inhibition of ketogenesis occurs with other ketone body precursors such as ketoacids is not known. The aim of this study was to determine the effects of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) on hepatic ketone body production from octanoic acid, a medium-chain fatty acid, and from alpha-ketoisocaproate (KIC), the ketoanalogue of leucine. METHODS: The experiments were conducted in cultured hepatocytes isolated from 24-hour-fasted Sprague-Dawley rats. Hepatocyte monolayers were incubated for 6 hours, with either KIC or octanoic acid (1 mmol/L), in the presence of glucagon and TNF-alpha (25 micro/L) IL-6 (15 microg/L) and/or IL-6. Acetoacetate, beta-hydroxybutyrate, and free fatty acids were determined in culture medium by enzymatic methods and KIC was measured by high-performance liquid chromatography. RESULTS: KIC and octanoic acid uptake by hepatocytes was 79% and 92%, respectively, over 6 hours, and cytokines had no influence. However, TNF-alpha and IL-6 caused inhibition of ketogenesis from alpha-ketoisocaproate (5.6% +/- 2.3% and 4.4% +/- 3.0%, respectively), and from octanoic acid (7.9% +/- 2.9%, 5.7% +/- 3.2%, respectively). In addition, when the two cytokines were present together in the culture medium, the inhibition was enhanced (inhibition of ketogenesis from KIC: 14.0% +/- 4.8%; from octanoic acid: 11.6% +/- 3.4%). CONCLUSIONS: In our experimental conditions, cytokines mediate an inhibition of ketogenesis; this process could be explained by a direct effect of cytokines on metabolic pathways of octanoic acid and KIC oran indirect effect by modification of the mitochondrial redox state.

A randomized controlled trial of the influence of the mode of enteral ornithine alpha-ketoglutarate administration in burn patients.

To investigate appropriate mode and daily dose of enteral ornithine alpha-ketoglutarate (OKG) administration, 54 burn patients (total burn surface area: 20-50%) were included in a randomized controlled trial and assigned to receive either a supplement of OKG (10, 20 or 30 g/d) as bolus or continuous infusion, or a continuous infusion of an isonitrogenous amount of a soy protein mixture (Protil-1: 10, 20 or 30 g/d) in addition to their enteral diet. The influence of these treatments on clinical outcome and biological indices was evaluated. OKG administration significantly improved nitrogen balance and reduced 3-methylhistidine and hydroxyproline urinary elimination. This was associated with a gradual rise in plasma glutamine over time. Given as a bolus, OKG significantly improved wound healing, assessed both clinically [day of last graft: (mean +/- SEM) OKG bolus 23.7 +/- 2.1 d versus Protil-1, 39.9 +/- 9.9 d; P < 0.05] and by hydroxyproline excretion, and biological markers of nitrogen metabolism, and tended to reduce duration of enteral nutrition (P = 0.12). The higher catabolic status in the patients administered 20 g OKG/d at the onset of the study, despite randomization, precludes any definite conclusion (concerning the dose-effect relationship). However, based on 3-methylhistidine elimination, our data indicate a benefit of 30 g OKG/d administration over 10 g/d. This study further supports OKG supplementation in burn patients. In addition, this is the first trial based on objective data that favors bolus over continuous infusion of OKG in critically ill patients.

Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing.

We developed two capillary isoelectric focusing (CIEF) assays, in narrow pH gradients, with the aim of routinely separating and quantitating normal and abnormal hemoglobins (Hbs): a one-step CIEF assay where residual electroosmotic flow mobilizes the proteins during focalization, and a two-step CIEF assay where focused Hbs are mobilized by low pressure by maintaining high-voltage. The resolution of 0.10 pH unit obtained with the one-step assay allowed the separation of the Hbs A, F, S and C; but Hb A2, which represents about 2-3% of whole Hb, could not be quantitated. The better resolution of 0.02 pH unit obtained with the two-step assay allowed the separation of some Hb variants of very close isoelectric points. The reproducibility of retention times was satisfactory (C.V.<5%). Moreover, in this configuration quantitation of Hb A2, Hb F and Hb S led to a standard deviation of less than 5%, allowing the diagnosis of thalassemias. The one-step assay could be useful only for the detection of abnormal variants, while the two-step assay could be applied to the routine analysis of Hbs, with quantitation of minor fractions and presumptive identification of variants.

Standardization of the lactulose-mannitol test in rats: application to burn injury.

Intestinal permeability can be assessed by measuring urinary mannitol and lactulose excretion after oral administration. This test may be useful as a tool in experimental studies to explore the effects of specific diets intended to promote the repair of the integrity of the gut barrier. In this study we standardized the lactulose-mannitol test in rats and applied it to a burned-rat model. The conditions were: oral administration of an isotonic mixture of 50 mg of mannitol and 66 mg of lactulose, followed by aseptic collection of urine over 4 h. The increase in the lactulose/mannitol ratio in burned rats was due to higher lactulose excretion. These results corroborate those obtained in burn patients and show that our model is suitable for further experiments on nutritional manipulation.

Pharmacokinetic assessment of an oligopeptide-based enteral formula in abdominal surgery patients.

The specific effect of the molecular form of the nitrogen supply (oligopeptides and whole proteins) on amino acid kinetics during enteral feeding after surgery has not been assessed previously. In a prospective, randomized study, patients having undergone esophagectomy or gastrectomy for cancer received jejunal infusions of oligopeptide-based or whole-protein-based complete formulas (OPD and WPD, respectively) during two 9-h periods on 2 consecutive days in a crossover design. The OPD and WPD had identical energy compositions and amino acid profiles. Amino acid peripheral bioavailability (measurements of area under the curve of arterial blood concentrations), amino acid arteriovenous differences, and insulin and glucagon responses were measured. Amino acid peripheral bioavailability was higher (leucine: 54%, P < 0.01; essential amino acids: 48%, P < 0.01; total amino acids: 53%, P < 0.02) and peripheral appearance of amino acids was more homogeneous (variation around the calculated plateau of plasma leucine was 39% for OPD and 78% for WPD, P < 0.001) with the OPD than with the WPD. With the OPD, insulin stimulation was faster and plasma concentrations of leucine and insulin were correlated (r = 0.77, P < 0.01). The OPD led to a higher amino acid peripheral bioavailability than the corresponding WPD. These results could be useful for a better definition of clinical indications of semi-elemental diets.

Capillary isoelectric focusing and high-performance cation-exchange chromatography compared for qualitative and quantitative analysis of hemoglobin variants.

We have developed two assays for complete analysis of hemoglobins (Hbs) in the field of hemoglobinopathies: a high-performance cation-exchange liquid chromatography (HPLC) assay on the weak cation-exchanger Poly Cat A and a two-step capillary isoelectric focusing (CIEF) assay on the neutral-coated capillary from Beckman in a narrow pH gradient. The resolution was satisfactory for both HPLC and CIEF and allowed separation of normal and common abnormal Hbs, i.e., Hb A, Hb F, Hb A2, Hb S, Hb C, and Hb E; slight differences were shown for the resolution of unusual variants such as Hb C-Harlem and Hb D-Punjab. The reproducibility of retention times was satisfactory as well for HPLC (CV 3.3%) and CIEF (CV 4.9%). The imprecision of quantification of Hb A2, evaluated at two concentrations, and of Hb F and Hb S was < 5%, except for low concentrations of Hb A2 quantified by CIEF. Quantitative data obtained for these three Hb forms were highly correlated between the two assays. These results suggest that the new CIEF assay can be competitive with HPLC for complete routine analysis of Hb variants.

Amino acid determination in biological fluids by automated ion-exchange chromatography: performance of Hitachi L-8500A.

The Hitachi L-8500A is a newly available apparatus for amino acid (AA) analysis that allows automatic on-line mixing of the ninhydrin reagent. The within-run precision (human plasma pools at three different concentrations) showed CVs < 3.8% except for the lowest concentration of citrulline (4.4%), Tyr (4.5%), and alpha-aminobutyric acid (7.6%), and for the intermediate concentration of Asp (8.7%). Between-run precision (CV) was < 3.1% for 17 AAs and < 8.0% for 24 of 25 AAs (CV Asp = 12.0%). For retention times, within-run precision was < 0.4% and between-run precision < 1.8%. Excellent relations were found between the results from the Hitachi L-8500A and the widely used Beckman 6300 analyzer (0.929 < or = r < or = 0.999). The detection was still linear at 5 mumol/L except for Pro and hydroxyproline (20 mumol/L). The upper limit was at least 2500 mumol/L for 13 AAs and at least 1000 mumol/L for 27 of 29 AAs (anserine = 500, Val = 600 mumol/L). Values from 100 human plasma samples agreed with previously published data. We conclude that the results obtained with the Hitachi L-8500A are satisfactory when compared with those of other AA analyzers utilizing the same method. Furthermore, the Hitachi L-8500A displays several advantages including programming flexibility, microsample capacity, low noise plotting, ammonia filtering, and manual repacking of the analytical column.

Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme.

We enzymatically deglycosylated pig lung angiotensin I-converting enzyme (ACE) to study the involvement of its glycanic chains in its physicochemical and catalytic properties. The effects of endoglycosidases F2 and H, and of N-glycanase were assessed by ACE mobility in SDS-PAGE. N-Glycanase only was completely effective with or without previous denaturation, leading to a shift in ACE M(r) from 172 to 135 kDa; endoglycosidase F2 produced the same shift but only without previous denaturation. Deglycosylated ACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine, and an identical Stokes radius as measured by size-exclusion high performance liquid chromatography. Neuraminidase had no effect on ACE Stokes radius but slightly decreased its kcat which could be related to variations in ionization of the active site. The isoelectric point of ACE, as, determined by isoelectric focusing, increased from 4.5-4.8 to 5.0-5.3 after either endoglycosidase F2 or neuraminidase digestion, but still with microheterogeneities which thus did not seem to be related to ACE glycans. Deglycosylated ACE did not bind onto agaroselectins in contrast to native ACE which bound strongly to concanavalin A showing interactions involving oligomannosidic or biantennary and sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin, an inhibitor of N-glycosylation, did not modify ACE secretion by endothelial cells. Thus, ACE glycans have no drastic effects on structural and biological properties of the protein, but they may have a functional role on intracellular targeting of both secreted and membrane-bound ACE isoforms, also for the protection of the soluble plasma form against hepatic lectins and the maintenance of its hydrosolubility.

[Fibronectin and hyaluronic acid in seminal fluid: relationship with sperm count and percentage of typical forms]. / Fibronectine et acide hyaluronique dans le liquide séminal: relations avec la numération spermatique et le pourcentage des formes typiques.

Two components of the extra cellular matrix, hyaluronic acid (HA) and fibronectin (Fbn), were assessed in the seminal fluids of 29 patients submitted for diagnosis of infertility. The concentrations of HA and Fbn were elevated in seminal plasma in comparison to their ranges of concentration in normal sera. No correlations were found between seminal Fbn and sperm count per ml and/or ejaculate, normal sperm cytology and multiple anomaly index (MAI). The concentrations of HA were negatively correlated with sperm count per ml (p < 0.01, r = -0.5) and ejaculate (p < 0.005, r = -0.5), and also with sperm cytology (p < 0.001, r = -0.6). A positive correlation was found between seminal concentrations of HA and MAI (p < 0.001, r = +0.7). It seems that high seminal HA concentrations were in relation with the bad results of sperm count and sperm cytology. These data suggest that spermatozoon could be implicated, in part, in the seminal HA metabolism.

Deletion of hydroxyethylstarch from University of Wisconsin solution induces cell shrinkage and proteolysis during and after cold storage of rat liver.

Among the numerous components of the University of Wisconsin (UW) solution used for organ preservation, the usefulness of hydroxyethylstarch (HES), the colloido-osmotic support of this solution, is controversial. The aim of our study was to determine the influence of HES on hepatic metabolism and intracellular hydration state during hypothermic preservation and after reperfusion in a model of isolated perfused rat liver. Three groups of eight livers were perfused either immediately or after 18 hours of cold storage in a UW-based preservation solution with or without HES. Omission of HES results in 1) a stimulation of protein degradation shown by the marked increase in branched-chain amino acid (BCAA) release (211 +/- 55 vs. 87 +/- 28 nmol/min/g; P < .05, modified UW group vs. UW group), 2) an increase in oxygen consumption (81.7 +/- 4.8 vs. 61.5 +/- 5.0 micromol/h/g; P < .05), 3) a decrease in glucose production (2.3 +/- 0.6 vs. 5.0 +/- 0.6 micromol/min/g; P < .05), and 4) a reduction in intracellular volume (414 +/- 36 vs. 557 +/- 41 microL/g; P < .05). We conclude that HES plays an important role in liver preservation by limiting proteolysis, possibly through the observed preservation of cell volume.

Ornithine alpha-ketoglutarate metabolism after enteral administration in burn patients: bolus compared with continuous infusion.

Ornithine alpha-ketoglutarate (OKG) has been successfully used as an enteral supplement in the treatment of catabolic states, including burn injury. However, specific questions remain unanswered concerning burn patients, including OKG metabolism and metabolite production, appropriate mode of administration, and dose. We thus performed a kinetic study and followed plasma ornithine and OKG metabolite concentrations on day 7 postburn in 42 (35 men, 7 women) consecutive burn patients aged 33 +/- 2 y with a mean (+/-SEM) total burn surface area (TBSA) of 31 +/- 1%. Patients were randomly assigned to receive OKG as a single bolus (10 g; n = 13) or in the form of a continuous gastric infusion (10, 20, or 30 g/d over 21 h; n = 13) or an isonitrogenous control (n = 16). Plasma pharmacokinetics of ornithine followed a one-compartment model with first-order input (r = 0.993, P < 0.005). OKG was extensively metabolized in these patients (absorption constant = 0.028 min-1, elimination half-life = 89 min), with the production of glutamine, arginine, and proline; proline was quantitatively the main metabolite [in OKG bolus, area under the curve (AUC)0-7h: proline, 41.4 +/- 5.6 mmol.min/L; glutamine, 20.4 +/- 5.7 mmol.min/L; and arginine, 7.3 +/- 1.9 mmol.min/L]. Proline production was dose-dependent and quantitatively similar between modes of OKG administration. Glutamine and arginine production were not dose-dependent and were higher in the bolus group than in the infusion group. Overall, the bolus mode of OKG administration appeared to be associated with higher metabolite production compared with continuous infusion in burn patients, especially for glutamine and arginine.

Cytotoxicity of amiodarone in cultured human endothelial cells.

As damage to the pulmonary vascular endothelium may be responsible for the lung toxicity of amiodarone, we evaluated the cytolytic toxicity of the drug in cultures of endothelial cells. Cells were cultured from human umbilical cord veins. Amiodarone caused a vacuolization of the cells with liberation of both lactate dehydrogenase (LDH) and angiotensin-converting enzyme (ACE) in the culture medium. These effect were both concentration and time dependent, and were correlated between them. The first toxic effects were shown as soon as 2 hours after contact with the drug and at 0.1 mg/ml, a concentration that can be reached in plasma of amiodarone-treated patients. A decrease of ACE activity in the cells was delayed to 24 hours and only with the 10 mg/ml concentration. This event correlated with cell death and detachment from the extracellular matrix. LDH increases corresponded to its isoenzymes 3 and 4. These data support the hypothesis of a direct toxic effect of amiodarone on the endothelium and show the need for evaluating LDH, total activity and isoenzymic profile, and ACE determinations in the plasma of patients treated with amiodarone for ischemic heart disease or arrhythmia.

Serum levels of endothelial injury markers creatine kinase-BB and soluble thrombomodulin during human liver transplantation.

Endothelial damage within the sinusoids of the liver probably plays a key role in primary liver dysfunction following transplantation. The aim of this work was to study the serum levels of two potential markers of endothelial damage, creatine kinase-BB and soluble thrombomodulin, during human graft revascularization. Thirteen human liver grafts were preserved in UW solution (mean time: 13.8 h). Creatine kinase-BB and transaminase activities and soluble thrombomodulin levels were measured: 1) in effluent and 2) in serum samples sequentially collected before revascularization, then during the first 120 min of revascularization and first post-operative week. No correlation was observed between serum values (peak) and effluent values. In serum, pre-operative creatine kinase-BB activities were correlated with soluble thrombomodulin levels (p = 0.01). Both increased significantly during the first minutes of the revascularization, then decreased markedly. In contrast, AST activity was maximal at day 1. This detectable and early release of creatine kinase-BB and soluble thrombomodulin in blood is in keeping with the early occurence of endothelial damage. Together with previous data, these findings suggest that serum determination of these two markers may be a useful tool in the assessment of endothelial injury in liver transplantation.

Kinetic nephelometric determination of albumin produced by rat hepatocytes in culture.

A kinetic immunonephelometric method for the determination of albumin produced by rat hepatocytes in culture is described. This assay is simple, rapid and sensitive. The methodology allows detection of 0.7 mg/l albumin in 200 microliters of culture medium. Within-run precision CVs for three levels of concentrations were under 1.0% and between-day precision CVs were under 4.1%. The range of measurement obtained using appropriately diluted samples was 1.2 to 74 mg/l. The rabbit IgG fraction to rat albumin used in this method did not cross-react with albumin from cow, allowing the use of fetal calf serum in the medium. The method described can thus be used easily for the assessment of albumin synthesis in cellular studies using isolated hepatocytes.