Plasma lipid peroxides in cholestatic children.

Plasma lipid peroxide levels were studied in 40 children with chronic cholestasis comprising 21 with syndromatic paucity of interlobular bile ducts (PILBD) and 19 with biliary atresia. Compared to the controls, mean lipid peroxide values were twice as high in children with biliary atresia (4.56 +/- 2.28 nmol/ml) and four times as high in those with PILBD (9.62 +/- 3.3 nmol/ml). These differences are highly significant. In patients with biliary atresia, the increase in lipid peroxide levels was clearly related to the bilirubin, cholesterol and phospholipid concentrations. In the PILBD group, however, there was little evidence of such a relationship. Vitamin E treatment seemed to have no effect on these increased lipid peroxide levels during the evolution of chronic cholestasis, and further investigations are necessary to clarify the pathological mechanisms involved.

Evidence that cycloleucine affects the high-affinity systems of amino acid uptake in cultured human fibroblasts.

The influence of cycloleucine on kinetic parameters of uptake of L-alanine, L-proline and L-leucine into cultured human fibroblasts was examined under initial-rate conditions with substrate concentrations of 0.05-10 mM and 5 mM-cycloleucine. Kinetic data obtained by computer analysis showed that, in the absence of cycloleucine, cell uptake was heterogeneous for each amino acid. L-Alanine and L-leucine entered by two transport systems with different affinities; L-proline was taken up by one saturable transport system plus a diffusion-like process. This heterogeneity disappeared in the presence of cycloleucine, since the high-affinity systems were no longer detectable. The remaining process had the same kinetic constants as the low-affinity system for alanine and leucine and a KD similar to the diffusion constant for proline. The influence of cycloleucine on the amino acid uptake was not specific either to the amino acid concerned or to a particular transport system, since the three neutral amino acid-transport systems, A, ASC and L, were involved in these experiments. This influence was shown to be unaffected by the absence of Na+ (for leucine uptake). ATP content of the cells was identical in the presence or in the absence of cycloleucine.

Plasma amino acid patterns in respiratory encephalopathy.

Serum amino acid determinations were made in 23 patients with chronic bronchitis presenting with acute respiratory insufficiency both during and after regression of signs of encephalopathy. The ratio of branched chain amino acids to aromatic amino acids was initially significantly lower than normal but returned to normal range when the signs of encephalopathy had disappeared. The modifications observed in amino acid concentrations were similar to those seen in hepatic encephalopathy. The role of the liver in the observed modifications of amino acid levels in respiratory failure is discussed.

Transport of L-alanine in cultured human fibroblasts: evidence for two kinetically distinguishable systems.

The transport of L-alanine in human diploid fibroblasts was investigated. Transport measurements were performed on subcultures between the third and eighth passages with subconfluent cells growing on glass coverslips. Kinetic analysis of approximate initial rates of transport at substrate concentrations from 0.05 to 10 mmole/liter indicate the presence of two distinguishable systems. The high affinity system has a Km of 0.24 mmole/liter and a Vmax of 6.4 nmole/100 micrograms protein/2 min. For the low affinity system, the contribution of the high affinity system to the uptake must absolutely be taken into account. The Km and Vmax values, obtained by using a computer program, are a Km of 15.0 mmole/liter and a Vmax of 14.7 nmole/100 micrograms protein/2 min. For alanine concentrations below 1 mmole/liter, the contribution of the Na+-independent uptake is less than 10%, and the kinetic constants of the high affinity system are in the same range if this contribution is taken into account. On the contrary the influence of a diffusion-like process is more significant on the low affinity system whose Km is about 49 mmole/liter after subtraction of the Na+-independent uptake from the experimental velocities. Inhibition studies were performed with NCH3-alanine. They permitted us first to confirm the existence of system A in cultured human fibroblasts in agreement with two recent works and second to show how this system contributes to L-alanine uptake. This contribution seems very small in low concentrations but it rises as the concentrations increase.

D-glucose uptake in human liver cell cultures.

The kinetics parameters for D-glucose uptake were studied in human liver cell cultures under strictly defined experimental conditions. Using a wide concentration range (0.005 to 30 mmol/l), the kinetic data obtained suggested strongly that D-glucose in human liver cell cultures can be transported by two separate systems. For the high-affinity system, the apparent Km was 0.645 +/- 0.21 mmol/l and the Vmax, 12.49 +/- 3.74 nmol/mg protein per min. For the low-affinity system, the apparent Km was 6.91 +/- 0.58 mmol/l and the Vmax, 79.90 +/- 5.27 nmol/mg protein per min. At a concentration of 2.1 x 10(-7) mol/l, cytochalasin B preferentially inhibited the high-affinity D-glucose site or transport system. The time course of D-glucose uptake, studied in two cell lines from patients with hereditary fructose intolerance, was significantly higher than for the control lines.

Glycine transport by cultured skin fibroblasts from a patient with isolated hyperglycinuria.

Glycine transport is studied in cultured skin fibroblasts from a patient with isolated hyperglycinuria and from five normal subjects. Fibroblasts from the patient take up glycine less well than do cell lines from controls. Kinetic studies are consistent with a single transport system in the patient's and controls' cell lines. Vmax value in the hyperglycinuric lines is normal, but the apparent affinity is always reduced as opposed to those of four different control lines separately tested. Statistical analysis shows significant difference between Km values.

Effect of cycloleucine on amino acid accumulation by human diploid fibroblasts.

Cycloleucine is a synthetic amino acid which produces, in vivo, biochemical abnoramlities comparable to those seen in human cystinuria-lysinuria. The effect of cycloleucine on intracellular accumulation of amino acids overlapping separate transport systems was studied using human diploid fibroblasts subcultures on glass coverslips. The data indicated that alpha-alanine, serine and proline accumulation was inhibited significantly by cycloleucine. The percentage of inhibition was approximately the same. Lysine was less affected by cycloleucine, but this amino acid accumulation proceeded at a rate slower than for neutral amino acids. In vitro, this inhibitory effect seems to be a generalized phenomenon affecting substrates. These results confirm in human fibroblasts data reported for human and rat kidney slices.