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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.

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.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.In the absence of taurocholate, amino acids induced a 40% (p0.01) decrease in bile flow together with an increase in hepatic water content (17.8%, p0.05). 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); p0.01).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.

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.

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.

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.

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.

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.

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.

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.

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.

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 μl of culture medium. Within-run precision CVs for three levels of concentrations were under l.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

Diagnostic accuracy of two serum markers of liver fibrosis, hyaluronan (HA) and amino-terminal peptide of type III procollagen (P-III-P), was studied in a cohort of 326 untreated patients with chronic viral hepatitis C. Both P-III-P (RIA-gnost P-III-P, Behring Diagnostic) and HA (HA-test, Pharmacia) serum concentrations correlated with the histological grades of liver fibrosis (P < 0.001). Receiver-operating characteristic (ROC) curves showed that serum HA had greater diagnostic performance than P-III-P, both for discriminating patients with extensive liver fibrosis from those with no or mild fibrosis (area under the ROC curves: 0.864 vs 0.691, P

alpha-Glutathione S-transferase (alpha-GST; EC 2.5.1.18) has been advocated as a better marker of hepatocellular damage than the transaminases in toxic and autoimmune hepatitis. We have assessed the potential interest of plasma alpha-GST determination in 94 anti-hepatitis C virus-positive patients with histologically proven chronic hepatitis C (34 women, 60 men, ages 40.0 +/- 11.9 years). Blood samples were assayed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase, alkaline phosphatase, and alpha-GST on the same day a liver biopsy was performed. alpha-GST concentrations were significantly above reference values in 64% of patients (compared with 58% for AST, 68% for ALT), and this increase was seen in 52% of patients with normal values for transaminases and a Knodell score > 3. Furthermore, there was a significant correlation between alpha-GST and lobular necrosis score (r = 0.31; P < 0.01). Our findings suggest that association of plasma alpha-GST with ALT may improve the biochemical assessment of liver damage in patients with chronic hepatitis C.

Angiotensin I-converting enzyme (ACE) was purified from lungs of pig, rat, monkey and human for comparison of its physicochemical, enzymatic and immunological properties. The protocol involved three chromatographic steps after detergent extraction, i.e. DEAE-Sphérodex ion exchange, lisinopril-Sepharose affinity and Superose 12 HPLC, plus Mono-Q HPLC for monkey ACE. Purified ACE's presented numerous homologies: in particular, closely similar specific activities, catalytic efficiencies, Km's, optimal pH and chloride activations; the molecular weights were about 170 kDa by SDS-PAGE and 320 kDa by gel-filtration on Superose 12; the isoelectric points were about 4.5-4.7. Specific polyclonal antibodies recognized the antigen (porcine ACE) as well as rat, monkey and human ACEs. In contrast, three monoclonal antibodies (F02.4.1, F01.1.3 and F03) produced against porcine ACE showed some differences: they only reacted with pig enzyme and only one (F0.2.4.1) was anticatalytic. Moreover, the cross-reactivity judged on ELISA with porcine ACE characterized different epitopes specific for the porcine enzyme. In particular, the binding of F02.4.1 was not diminished by previous treatment with saturating concentrations of synthetic competitive ACE inhibitors. Thus, the extrapolation to human of data obtained on animal models should be possible at least for pharmacological and medical trials.

Plasma fibronectin (FBN) and angiotensin I-converting enzyme (ACE) were prospectively measured in 50 burn patients from the day of admission to day 28 after the trauma with the aim of finding biochemical markers of pulmonary injury by smoke inhalation. Patients were divided into three groups on the basis of fiberoptic bronchoscopy results (group I: healthy lungs; group II: burned lungs; group III: infected lungs). A decrease in FBN concentrations was observed in the three groups but was larger in group II than in the other groups, especially at day 2 (P < 0.05). A similar profile was observed for plasma ACE activity. Factors other than lung injury may influence plasma FBN and ACE levels, in particular the burned body surface area, an acute event such as septicemia, or outcome. However, repeated measurements of both markers could help in the assessment of lung injury in the follow-up of burn patients.

Although chronic obstructive pulmonary disease (COPD) is associated with weight loss and malnutrition, there is a paucity of relevant data on COPD patients with acute respiratory failure (ARF). We studied 30 consecutive patients on the day of admission to our intensive care unit for ARF. In addition to a clinical work-up, the following biochemical parameters were determined: markers of nutritional status (albumin - ALB, transferrin - TRF, transthyretin - TTR, retinol binding protein - RBP, fibronectin), inflammation (C-reactive protein - CRP, alpha(1) glycoprotein acid - alpha(1)GPA) and catabolism (plasma phenylalanine - PHE, urinary 3-methylhistidine - 3-MH). Values were expressed as mean +/- SD and compared to those of 10 healthy subjects matched for age. COPD-ARF patients had a poor protein status (ALB = 30 +/- 5 vs 42 +/- 3 g.l(-1); TTR = 118 +/- 75 vs 251 +/- 43 mg.l(-1); RBP = 23 +/- 12 vs 46 +/- 8 mg.l(-1); p < 0.001), were hypercatabolic (3-MH Cr = 31 +/- 12 vs 22 +/- 7 mumol.mmol Cr (-1); PHE = 62 +/- 27 vs 46 +/- 10 mumol.l(-1); p < 0.001) and inflamed (CRP = 68 +/- 50 vs 12 +/- 5 mg.l(-1); alpha(1)GPA = 1.2 +/- 0.4 vs 0.5 +/- 0.1 g.l(-1); p < 0.001). Severity of the disease correlated with short half-life proteins and protein catabolism markers but not with inflammation markers. Considering ALB, TTR, RBP, the 3- MH Cr ratio and PHE values, the 30 COPD patients fell into 3 groups: chronic malnutrition (n = 7), acute malnutrition (n = 2), and acute + chronic malnutrition (n = 18). 3 patients had normal nutritional status. We conclude that an assessment of nutritional status at admission to intensive care units could contribute towards a rapid formulation of specific nutritional therapy.

The diagnostic values of aminoterminal propeptide of type III procollagen and hyaluronan serum levels were compared as markers of liver fibrosis in two chronic liver diseases of different etiologies and pathophysiologies, namely primary biliary cirrhosis and chronic viral hepatitis C. The results were analysed in terms of the histological extent of fibrosis. Both serum procollagen-III peptide and hyaluronan were elevated in patients with primary biliary cirrhosis and chronic viral hepatitis C (p0.0001) relative to control values. A positive correlation was found between serum procollagen-III peptide levels and the histological grade of fibrosis in primary biliary cirrhosis (p0.001) but not in chronic viral hepatitis C, while a strong correlation was found between serum hyaluronan levels and histological fibrosis in both primary biliary cirrhosis and chronic viral hepatitis C (p0.001), independent of age. These results suggest that, in chronic liver diseases, serum hyaluronan levels could be an important indicator of the extent of fibrosis and should be assayed to monitor the response to treatment in controlled clinical trials.

Resume Certains acides amines possedent des proprietes immunomodulatrices relevant de mecanismes differents. L'arginine et l'ornithine sont des regulateurs de la fonctionnalite des cellules immunitaires. L'action de l'arginine est liee a son catabolisme en monoxyde d'azote radicalaire (NO°); celle de l'ornithine depend de la synthese de polyamines. La glutamine est un substrat energetique majeur des cellules immunitaires activees, a un effet trophique sur l'epithelium intestinal et participe ainsi aux fonctions de l'intestin de lutte contre l'invasion de l'organisme par les bacteries presentes dans le tube digestif. Des etudes recentes, pour la plupart experimentales, soulignent l'interet potentiel de l'emploi en nutrition artificielle de regimes enrichis en arginine, ornithine et glutamine. L'utilisation d'arginine dans certaines circonstances, telles que l'infection severe, merite reflexion du fait de l'action deletere du NO° produit en exces.

We studied the effect of recombinant human IL-1β (rhIL-1β) on hepatic amino acid (AA) flux in the isolated perfused rat liver model. Two experimental groups were used — a control group (n = 5) and a rhIL-1β-treated group (n = 5). IL-1 was added to the perfusate in two successive boluses of 0.1µg and 0.9µg, respectively 35 min (final concentration 0.67 ng/ml) and 60 min (6 ng/ml) after beginning the perfusion. In the IL-1 treated group, a reduction in flux was observed for only three AA, alanine, phenylalanine and serine. Glucose and urea production in the IL-1-treated group was slightly but not-significantly lower than in the controls. rhIL-1β thus has only minor direct effects on AA flux and gluconeogenesis in the liver and cannot therefore be held responsible for the increase in hepatic amino acid uptake during stress.

Resume Il existe peu de methodes permettant l'estimation de l'etat protidique global de l'organisme ; le bilan d'azote fait partie de celles-ci. Lors de la realisation du bilan d'azote (BA), il se pose toujours la question de sa determination. En effet, si l'on souhaite une bonne precision, il est necessaire de mesurer les pertes azotees par un dosage d'azote total urinaire et fecal. Toutefois, en pratique courante seule la determination de l'azote ureique urinaire est realisee et l'estimation des pertes azotees est faite par l'utilisation de formules adequates. Malgre ses limites, le BA est une mesure incontournable en therapeutique nutritionnelle pour suivre l'efficacite d'un support nutritionnel et pour l'adaptation des apports.

Experimental studies have shown that liver ischemia-reperfusion induces Kupffer cell activation and tumor necrosis factor- α (TNF α ) release. The aim of this work was to determine whether severe hepatic ischemia and subsequent reperfusion triggers TNF α release in man. Serum TNF α was measured before and 3, 10, 30, 60, 120 min after revascularization and postoperatively at day 1 and 2 in 11 patients with orthotopic liver transplantation (group 1 and 4 patients with liver resection with vascular occlusion (group 2). In group 1, TNF α levels decreased during the first few minutes of reperfusion, then increased slightly to peak at 120 min (40 ± 13 pg/ml). Primary non-function occurred in 1 patient in whom low peroperative levels of TNF α levels were measured. In group 2, no significant changes in TNF α levels were observed. These data, in a small number of patients: (a) show that hepatic ischemia reperfusion does not result in major TNF α production; (b) do not support a primary pathogenic role for TNF α in damage after ischemia-reperfusion in humans.

Resume Dans le contexte de la nutrition clinique, l'aminoacidogramme est d'interpretation difficile. Le plus souvent, les hyperaminoacidemies d'origine nutritionnelle sont dues a un exces d'apport (notamment lors d'une nutrition parenterale) ou a un defaut d'utilisation (defaillances polyviscerales). Quant aux hypoaminoacidemies, elles resultent en general d'une insuffisance d'apport (denutrition chronique) ou d'un exces d'utilisation, notamment dans les etats hypercataboliques. Avant tout outil de recherche, la realisation de l'aminoacidogramme est rarement indispensable en routine clinique. Cependant, la phenylalanine merite d'etre integree dans le bilan nutritionnel car sa valeur est un bon reflet du turn-over proteique lorsque la fonction hepatique est preservee, et sa mesure ne necessite pas un recueil urinaire, toujours aleatoire.

Impaired intestinal function could account for diarrhoea and weight loss, which are common features of advanced human immunodeficiency virus (HIV) infection.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.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).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.

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.

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 3 days of primary culture, the cells were exposed to hypoxia ( N 2 /CO 2 95/5) for 120 min and reoxygenation (O 2 /CO 2 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O 2 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.

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

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.

Laudat A, Blum L, Guechot J, Picard O, Cabane J, Imbert JC, Giboudeau J. Changes in systemic gonadal and adrenal steroids in asymptomatic human immunodeficiency virus-infected men: relationship with the CD4 cell counts. Eur J Endocrinol 1995;133:418–24. ISSN 0804–4643 Serum sex hormone-binding globulin (SHBG), testosterone, non-SHBG-bound testosterone, androstenedione, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and cortisol were measured in 58 homosexual men seropositive for human immunodeficiency virus (HIV), all clinically asymptomatic (Centers for Disease Control 1993 classification stage A). The HIV patients were divided into four groups according to the CD4 lymphocyte count—group 1 (more than 500/μl, N= 14), group 2 (between 350 and 500/μl, N= 16), group 3 (between 200 and 349/μl, N = 22) and group 4 (less than 200/μl, N = 6)—and compared with 11 antibody-negative men as controls. The SHBG levels were significantly increased in groups 1, 2, 3 (p < 0.01) and 4 (p < 0.05) compared with controls, with no differences between groups of patients. Compared with controls, testosterone concentrations were significantly lower in group 4 (p < 0.05) and non-SHBG-bound testosterone levels were significantly lower in groups l(p < 0.05), 2 (p < 0.01), 3 (p < 0.001) and group 4 (p < 0.001); DHT and androstenedione levels were significantly lower in group 4 (p < 0.05) and DHEA levels were significantly lower in group 2, group 3 (p < 0.01) and group 4 (p< 0.05) than in controls. Cortisol levels were significantly increased in groups 1 and 4 (p < 0.05) and FSH and LH concentrations were not significantly higher in HIV-infected men than in controls. Also, the DHEA, androstenedione, non-SHBG-bound testosterone and DHT levels were correlated with CD4 cell counts, showing that hypogonadism occurs as the CD4 lymphocytes decrease. A strong reverse correlation between CD4 cell counts and cortisol/DHEA ratios (p < 0.001) confirms the alterations in adrenal steroid secretion, with a shift from androgen to glucocorticoid production as the disease progresses. These data show that in asymptomatic HIV-infected patients serum SHBG levels increase independently of CD4 cells counts, even in patients with undiminished CD4 cell counts; an alteration in serum androgen occurs as the CD4 cell counts decrease; and the cortisol/DHEA ratios increase as the CD4 cell counts decrease, resulting from alterations in adrenal secretion with both a decrease in serum DHEA levels correlated with CD4 cell counts and a slight increase in cortisol levels. Jérôme Guéchot, Laboratoire de Biochimie-Hormonologie, Hôpital Saint-Antoine, 184 rue du Faubourg, Saint-Antoine, 75571 Paris Cedex 12, France

Background/Aims Proteolysis may persist in the liver aliograft during cold storage. The aim of this study was to determine the significance of proteolysis within liver allografts stored at 4°C in University of Wisconsin preservation fluid. Methods Thirty recipients of 32 liver allografts were studied prospectively. Amino acid content of the preservation fluid was analyzed at the end of cold storage and was correlated to graft and patient outcome after transplantation. Results Analysis of the preservation fluid showed the presence of free amino acids, the profile of which was different from that of stored liver parenchyma. Concentrations of amino acids (alanine, cysteine, leucine, isoleucine, methionine, lysine, ornithine, and threonine) and transaminases (alanine aminotransferase and aspartate aminotransferase) in the preservation fluid correlated with the duration of cold ischemia. Indexes of graft dysfunction (serum alanine aminotransferase and aspartate aminotransferase peaks and prothrombin rate) correlated with concentrations of cysteine, alanine, isoleucine, leucine, methionine, lysine, ornithine, and threonine, whereas enzyme concentrations in the fluid were not predictive of graft dysfunction. Conclusions These data suggest that liver proteolysis occurs during cold storage and may have a detrimental effect on the outcome after transplantation. The measurement of the amino acids in the preservation fluid at the end of the cold storage period could help to identify the most severely damaged organs.

This study investigates the short-term effects of glucagon and human recombinant tumor necrosis factor alpha (TNF alpha) singly and in association on 2-methylaminoisobutyric acid (MeAIB) transport in hepatocyte monolayers. As expected, glucagon induced a time-dependent stimulation of MeAIB transport. In our experimental conditions, TNF alpha did not induce cytolysis. A 2 hour exposure to TNF alpha (0.05-500 ng/l) with or without glucagon (10(-9) to 10(-6) M) did not modify the basal or glucagon-stimulated MeAIB transport. Varying the duration of exposure to TNF alpha 5 ng/l up to 6 h was equally ineffective. The presence of hydrocortisone potentiated the glucagon-stimulated transport, but TNF alpha remained ineffective. Finally, the association of interferon (IFN gamma) with TNF alpha and/or glucagon was unable to modify the transport activity. These data demonstrate that TNF alpha does not exert a direct effect on MeAIB transport in hepatocytes, at least on a short-term basis.

To determine the hepatic fate of alpha-ketoisocaproate (KIC) in cirrhosis, six groups of isolated rat livers were perfused with 0, 0.5, 1 (with or without alpha-[1-14C]KIC), 2, and 5 mM KIC; control livers from healthy rats were studied in parallel under similar conditions. KIC was rapidly removed by the normal livers, whereas uptake was lower in the cirrhotic livers at all concentrations tested (at 2 mM, 4.04 +/- 0.33 vs. 6.32 +/- 0.58 mumol/min; P < or = 0.05). The transamination pathway, evaluated by leucine exchanges, was more important in the cirrhotic livers (25.4 vs. 6.8% in controls at 2 mM). The incorporation of alpha-[1-14C]KIC in proteins of cirrhotic liver was increased compared with controls (0.25 +/- 0.04% of alpha-[1-14C]KIC was incorporated in proteins excreted in perfusate vs. 0.20 +/- 0.04 in controls; P < or = 0.05). In addition, a line of evidence suggests that glutamine rather than glutamate is the N donor for leucine synthesis from KIC. The decarboxylation pathway evaluated by beta-hydroxybutyrate production and by 14CO2 release from alpha-[1-14C]KIC was reduced, respectively, by 40-85% (according to KIC dose) and by 24% at 90 min in cirrhotic livers compared with healthy livers. These results indicate a dramatic modification of KIC metabolism in the cirrhotic liver; its uptake by the liver is decreased and its incorporation into proteins is increased via an enhancement of transamination to leucine, probably as a consequence of an inhibition of branched-chain keto acid dehydrogenase.

Ornithine (Orn; α-ketoglutarate (αKG) salt) and arginine (Arg) supplementation of enteral diets has been advocated in the treatment of hypercatabolism of trauma patients, but both compounds are subject to extensive hepatic metabolism. To compare the metabolism of these two compounds and to evaluate the possible influence of the αKG moiety, livers were perfused with αKG, Orn, ornithine α-ketoglutarate (OKG) or Arg (n6 in each group) for 1 h. Arg uptake was nearly fourfold higher than Orn uptake (690 (SD 162) ν. 178 (SD 30) nmol/min per g liver), and Orn uptake was not modified by αKG. Orn was totally metabolized by the liver, whereas Arg led to Orn release (408 (SD 159) nmol/min per g liver) and a threefold stimulation of urea production (Arg 1·44 (SD 0·22) ν. Orn 0·45 (SD 0.09) μol/min per g liver). αKG alone only increased hepatic aspartate uptake but, when associated with Orn as OKG, it led to an increase in giutamate release and in proiine content in the liver and to a decrease in proiine uptake. From these findings we conclude that (1) Arg load is extensively metabolized by the liver, inducing urea production, (2) in enteral use, Orn supplementation appears preferable to Arg as it is less ureogenic (as also recently demonstratedin vivoin stressed rats receiving isomolar amounts of Arg and Orn), (3) the liver participates in the Orn-αKG metabolic interaction, mostly in proiine metabolism, which occurs in the splanchnic area.

It has been suggested that increases in serum hyaluronan levels might be a marker of fibrosis in chronic viral hepatitis C. Patients receiving alpha-interferon therapy are an excellent model to determine the relationship between serum hyaluronan and liver fibrosis, since results suggest that alpha-interferon could reduce liver fibrosis.We studied the relationship between serum hyaluronan and histopathological indices of liver fibrosis, inflammation and necrosis, before and after alpha-interferon therapy (3 MU, three times weekly for 6 months), and the effect of treatment on serum hyaluronan and on histological liver fibrosis, in 52 patients. Hyaluronan levels were measured using a radiometric assay and the liver histopathological indices were scored according to the Knodell system.The serum hyaluronan level correlated with the extent of liver fibrosis both before and after alpha-interferon therapy (p0.0001), but not with the histopathological indices of liver inflammation or necrosis. Parallel changes in serum hyaluronan and liver fibrosis occurred: serum hyaluronan levels fell significantly in patients in whom fibrosis improved (p0.01, n = 11), increased significantly in patients in whom fibrosis worsened (p0.05, n = 10), and did not change significantly in patients in whom fibrosis was unmodified (n = 31). Furthermore, fibrosis improved only when the antiviral effect of alpha-interferon was reflected by persistent normalization of serum alanine aminotransferase, although there was no correlation between serum hyaluronan levels and alanine aminotransferase activities.Serum hyaluronan thus appears to be a non-invasive index of liver fibrosis.

Conflicting reports concerning the hepatic effects of interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) in the metabolic response to injury led us to investigate the influence of physiological concentrations of these cytokines on amino acid metabolism in the isolated perfused rat liver. IL-1 beta was ineffective at a concentration of 1 ng/mL, whereas TNF alpha (0.7 ng/mL) reduced the uptake of some of the main gluconeogenic amino acids (alanine, -55.3 +/- 4.9 v -72.9 +/- 13.7 nmol.min-1.g-1 in controls, P < .05) without affecting urea synthesis. TNF alpha increased glucose uptake by 237% and inhibited that of free fatty acids (-1.6 +/- 1.4 v -9.9 +/- 6.7 nmol.min-1.g-1 in controls, P < .05). IL-1 beta and TNF alpha potentiated glucagon-induced total amino acid uptake by 56% and 87%, respectively. They also affected glucagon-activated gluconeogenesis, leading to an initial potentiation of glucose release. Thereafter, IL-1 beta inhibited glucagon action, leading to an hepatic uptake of glucose. These results indicate that (1) in the conditions of the study, IL-1 beta has no direct effect on hepatic amino acid exchanges and utilization; (2) TNF alpha which exerted an inhibitory effect on these parameters, could be involved in the reduced amino acid exchanges during the end stage of sepsis; (3) the TNF alpha-induced increase in glucose uptake could be related to an inhibition of gluconeogenesis and/or to the activation of glucose utilization by Kupffer cells; (4) IL-1 beta and TNF alpha both potentiate the action of glucagon on hepatic amino acid uptake and utilization; and (5) complex interactions between Kupffer cells and hepatocytes on the one hand and between cytokines and hormones on the other hand could account for the differences in hepatic metabolism according to the stage of the response to injury.

Plasma levels of interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL-6), and markers of protein metabolism were determined in 12 burn patients throughout the healing period (day 2 to 21 post-injury) to determine the pattern of variations in plasma cytokine concentration. To establish the relationship between cytokine production and the nutritional status a wide range of severity standpoints (burn surface area ranging from 9% to 82%) was chosen. Interleukin 6 levels were increased in all patients throughout the study period; maximum concentrations (615 +/- 198 pg/mL) were reached on day 4 and correlated (p < 0.01) with the extent of burn injury. Tumor necrosis factor alpha levels were also elevated; they were significantly higher on day 7 in the patients who developed sepsis than in the other patients (67 +/- 21 pg/mL vs. 20 +/- 7 pg/mL; p < 0.05) but did not correlate with the extent of burn injury. Interleukin 1 beta was rarely detected. Cortisolemia on day 7 was inversely correlated with levels of TNF alpha but not with those of IL-6. Interleukin 6 levels correlated positively with protein turnover (phenylalaninemia) and catabolism (3-methylhistidine/creatinine ratio) and negatively with levels of fibronectin and transthyretin. Our data indicate that the systemic cytokine response to burn injury is mainly represented by IL-6. These data also support the hypothesis that IL-6 is a key mediator of the variations in protein metabolism following burn injury.

The effects of liver transplantation on the pituitary-gonadal axis and sex-hormone metabolism were evaluated by studying hormonal status (androgens, oestrogens, and gonadotropins) and sex-hormone-binding globulin levels in men with advanced liver disease of both alcoholic and viral origins. Comparison of the results prior to and 6 months after liver transplantation showed that successful liver transplantation in men induced significant differences in sex-hormone levels and in pituitary-gonadal function in both alcoholic and post-hepatitis patients. Plasma testosterone and dihydrotestosterone levels increased, oestrogen (oestrone and oestradiol) and androstenedione levels fell while gonadotropin (FSH and LH) levels increased. There was also a fall in plasma prolactin levels. Sex-hormone binding globulin plasma levels were elevated prior to transplantation and decreased thereafter. These data show that male patients with advanced liver disease have biological hypogonadism and feminization, irrespective of the aetiology, and that these abnormalities rapidly improve after successful liver transplantation. Therefore in men with advanced liver disease the biochemical signs of sex hormone disturbance are reversible and may be largely related to the liver disease.

We used near and far UV spectrophotometry for the re-evaluation of the molar extinction coefficient at 280 nm (epsilon 280) of pulmonary angiotensin-converting enzyme (ACE), for the determination of its tryptophan and tyrosine contents and to follow-up guanidine denaturation. ACE purity was assessed by both SDS-PAGE and capillary electrophoresis performed in denaturing conditions. The maxima of the near UV spectrum of purified ACE, dissolved in phosphate buffer pH 6.5, was at 279 nm; with an estimated M(r) of 160 kD, epsilon 280 of native ACE was 1.5 +/- 0.05 x 10(5) (mol/l)-1 x cm-1. Denaturation of ACE by 6 mol/l guanidine hydrochloride produced a hypochromic effect of 23% at 280 nm and led to a blue shift of 3.5 nm. In guanidine solution, absorbance measurements at 288 and 280 nm predicted a ratio of 1 between tyrosine and tryptophan, whereas it was 1.8 with the measure of the amplitude of the spectral bands at 283 and 292 nm of the second derivative of the near UV spectrum. Unfolding of the peptide chain in 6 mol/l guanidine was also well characterized by the second derivative of the far UV spectrum, in parallel with the complete loss of enzymatic activity although the protein remained whole as judged on SDS-PAGE. We also re-evaluated ACE zinc content by atomic absorption spectroscopy and demonstrated that ACE molecule obviously contains two zinc atoms.

Cell damage within the sinusoidal lining of human liver grafts during transplantation is an early event that is critical in ischemia-reperfusion injury and probably plays a key role in primary liver dysfunction after transplantation. No simple biochemical marker for sinusoidal injury is currently available. Because creatine kinase activity has been described in heart endothelial cells, we hypothesized that release of this enzyme might serve as an index of sinusoidal injury. To test this hypothesis, we used several in vivo and in vitro experimental models. Occlusion of the rat hepatic pedicle in situ for 60 min (normothermic ischemia) induced a significant increase in serum creatine kinase levels relative to those in laparotomized controls (2,530 ± 530 vs. 389 ± 64 IU/L, mean ± SEM; p < 0.005). In the isolated perfused rat liver, 60-min ischemia induced early (≤ 3 min) creatine kinase and AST release (0.87 ± 0.14 vs. 0.08 ± 0.01 IU/min/gm liver, respectively). A similar phenomenon was observed after 24-hr or 48-hr hypothermic conservation in University of Wisconsin solution. Electrophoretic analysis and immunoinhibition studies showed that creatine kinase activity comprised creatine kinase-BB (≈ 50%) and mitochondrial creatine kinase. Trypan blue infusion showed a loss of viability in sinusoidal cells, whereas hepatocytes were relatively spared. Finally, murine sinusoidal cells were isolated, cultured and then lysed by a freeze-thaw cycle and sonication. Creatine kinase activity was found in endothelial cells (creatine kinase-BB), Kupffer cells (creatine kinase-BB) and Ito cells (creatine kinase-MM). Creatine kinase-BB was not found in hepatocytes, but mitochondrial creatine kinase was detected. These results indicate that liver ischemia-reperfusion injury induces a release of creatine kinase-BB from sinusoidal cells and of mitochondrial creatine kinase from hepatocytes, suggesting that creatine kinase-BB activity could provide a simple index of cell damage within the sinusoidal lining. (HEPATOLOGY 1993;17:423–428.)

Cell-mediated immunity and macrophage activity, especially that of Kupffer cells, are impaired during cholestasis. Some evidence exists that bile acids play a role in these immune defects. The purpose of this study was to evaluate the effects of individual bile acids on immunity and to determine whether monocytes could be a target. We assessed the effects of chenodeoxycholic acid, an endogenous bile acid, ursodeoxycholic acid, which has been shown to partially correct the immunological abnormalities observed in primary biliary cirrhosis, and their tauroconjugates on the production of interleukin-1, interleukin-6 and tumor necrosis factor-alpha. Chenodeoxycholic acid had a dose-dependent inhibitory effect on interleukin-1 (inhibitory concentration 50% = 60 mumol/L), interleukin-6 (inhibitory concentration 50% = 80 mumol/L) and tumor necrosis factor-alpha (inhibitory concentration 50% = 80 mumol/L) production; inhibition was almost complete at 250 mumol/L. In contrast, ursodeoxycholic acid had lesser or minimal inhibitory effects (inhibitory concentration 50% = 100 mumol/L for interleukin-1 and above 200 mumol/L for interleukin-6 and tumor necrosis factor-alpha). The inhibitory effects of taurochenodeoxy-cholic acid and tauroursodeoxycholic acid were similar to those of chenodeoxycholic acid and ursodeoxycholic acid, respectively. Ursodeoxycholic acid did not reverse the chenodeoxycholic acid-induced inhibition of interleukin-6 or tumor necrosis factor-alpha production. In conclusion, chenodeoxycholic acid exerts strong inhibitory effects on monocyte activity in vitro, whereas the effects of ursodeoxycholic acid are minor.

We examined the possible contribution of the liver to the alterations in branched-chain amino acid (BCAA) metabolism in cirrhosis. The livers of male Sprague-Dawley rats with CCl4-induced cirrhosis were removed and placed in a recirculating perfusion system. Net amino acid uptake and release were determined over 55 min. Results were compared with those obtained with control animals, which were either pair-fed or fed ad libitum. Intrahepatic amino acid concentrations were determined at the end of the perfusion. The release of isoleucine and leucine was significantly lower in the cirrhotic livers than in the controls fed ad libitum. There was no difference between the cirrhotic and pair-fed groups with regard to the fluxes of the three BCAA. Intrahepatic concentrations of BCAA were reduced only in pair-fed controls. These results suggest that both cirrhosis and a low protein/calorie diet alter hepatic BCAA flux, but via different mechanisms. In cirrhosis, alterations could be due both to low food intake and to BCAA metabolism in non-parenchymal cells.

Primary biliary cirrhosis (PBC) is a chronic cholestatic disease in which there is a crucial need for quantitative liver-function tests. We have developed a mixed sulfobromophthalein (BSP)-indocyanine green (ICG) test and have applied it to 15 healthy subjects and 50 patients with PBC to determine its relevance to the histological severity of the disease. The two dyes were administered intravenously and sequentially as boluses. Plasma concentrations were measured over 60 min. Pharmacokinetic analysis of the plasma elimination curve permitted the calculation of clearance, constants k1 and k2, and the retention percentage at 45 min. In PBC patients, ICG kinetics were within the normal range except for those with stage IV disease (cirrhosis). BSP clearance and the k2 constant were reduced in all the patients, whereas the k1 constant was reduced only in stage III and IV disease. The BSP retention percentage at 45 min was highly correlated with histological stage (r = 0.89, P less than 0.001). The BSP-ICG mixed test may thus prove useful in the diagnosis and follow-up of patients with PBC.

We present a radiometric assay for the determination of urinary angiotensin-converting enzyme activity, using benzoyl-[1-14C]glycyl-L-histidyl-L-leucine as the substrate. An optimal pH of 8.3, an optimal chloride concentration of 0.375 mol/l and complete inhibition by EDTA-Na2, captopril and enalaprilat confirm the specificity of the assay. Comparison of dialysis and ultrafiltration for concentration of urine showed the existence of angiotensin-converting enzyme inhibitors in human urine. Dialysis against water was the more effective method for avoiding enzyme inhibition. After dialysis of urine, the assay was linear with time and with enzyme concentration; it was highly sensitive (60 mU/l) and showed good reproducibility. Under our technical conditions, we found angiotensin-converting enzyme activity in urine samples with quantitatively abnormal protein contents, but not in normal urine. Urinary angiotensin-converting enzyme did not correlate with proteinuria nor with water-salt parameters or creatinine. We confirm the kidney tubular epithelial origin of the enzyme, and propose the use of our assay to study urinary angiotensin-converting enzyme as a marker of renal tubular damage.

Conflicting evidence concerning hepatic amino acid (AA) metabolism in the isolated perfused rat liver (IPRL) led us to investigate the response of IPRL using perfusates with various AA contents. Perfusion ( n = 4) with whole rat blood diluted in Krebs buffer (1:3, v/v) led to acute proteolysis on account of AA deprivation, as shown by the large release of AA (≈ 1400 μmole in 120 min), especially branched-chain AA (BCAA) (e.g., Leu, 35.4 ± 10.4 nmole·min −1 ·g −1 the first hour, 34.3 ± 5.5 nmole·min −1 ·g −1 the second hour). In a first attempt to prevent proteolysis, livers ( n = 4) were perfused with the previous medium supplemented with AA known for their antiproteolytic activity, at twice their physiological concentrations. Results during the first hour showed uptake of several AA (mainly alanine, glutamine, and proline), reduced release of BCAA (leucine, 12.5 ± 6.3 nmole·min −1 ·g −1 ), and an increase in glucose and urea production. However, during the second hour, because of the use of a recirculating system, progressive AA depletion induced a reappearance of proteolysis. A two-step AA loading technique, i.e., the addition of antiproteolytic AA at the beginning of the perfusion and the addition of a balanced AA mixture at 60 min caused a further decrease in proteolysis during the 2 hr of perfusion ( n = 6). Under these conditions, most AA were taken up by the liver with uptake values comparable to those observed in vivo .

Angiotensin-converting enzyme (ACE) was measured in serum of 187 healthy children between the ages six months and 18 years. Results were pooled for five-year age intervals and compared with the reference values for adults that we previously determined [Clin Chem 1986;32:884-6). Results for each age group were also studied as a function of sex. Children had higher ACE activities in serum than did adults (P less than 0.001), but these activities were age-related only from age four to 18 years. Adolescents showed sex-related differences, with higher serum ACE activities in boys than in girls (P less than 0.05). Both sex- and age-related differences may be related to a steroid hormonal regulation of ACE biosynthesis. We also verified that children with sarcoidosis (n = 20) had significantly increased serum ACE activity. Such physiological variations in serum ACE activity must be taken into account for diagnosing sarcoidosis in children, for following the course of the disease, and for evaluating the accuracy of therapy.

Ornithine alpha-ketoglutarate (OKG) has been useful as an adjuvant of enteral and parenteral nutrition. However, its metabolism and mechanism of action remain unclear although it is known that alpha-ketoglutarate (alpha KG) and ornithine (ORN) follow, in part, common metabolic pathways. Six fasting healthy male subjects underwent three separate oral load tests: (i) they received 10 g of OKG (i.e., 3.6 g of alpha KG and 6.4 g of ORN); (ii) 6.4 g of ORN as ornithine hydrochloride, and (iii) 3.6 g of alpha KG as calcium alpha-ketoglutarate. Blood was drawn 15 times over a five-hour period for measurements of plasma amino acids, alpha KG, insulin, and glucagon. After OKG and ORN administration, plasma ORN peaked at 60-75 min (494 +/- 91 and 541 +/- 85 mumol/L). The increase in plasma alpha KG was very small. OKG, alpha KG, and ORN all increased glutamate concentrations at 60 min (mean: +43%, +68%, +68%, respectively, p less than 0.05 compared to basal values). However, only OKG increased proline and arginine levels at 60 min (mean: +35%, p less than 0.01 and mean: +41%, p less than 0.05). Furthermore, glutamate, proline, and arginine concentrations correlated linearly with ornithine levels at 60 min. Finally, OKG increased insulinemia and glucagonemia (mean: +24% at 15 min, p less than 0.05 and +30% at 60 min, p less than 0.01, respectively). These data provide evidence that the combination of ORN and alpha KG modifies amino acid metabolism in a way which is not observed when they are administered separately. In addition, the OKG-mediated increase in insulin levels probably does not appear to result from a direct action of ORN on pancreatic secretion.