Maternal and fetal tyrosinemia type I.

A 22 year-old woman with tyrosinemia type I (HT1) married her first cousin who is heterozygous for the same FAH mutation for which the patient is homozygous. During her pregnancy she was treated with diet (prescribed tyrosine intake 300 mg/day), and nitisinone (60 mg/day). Median plasma tyrosine levels were 560 µmol/L (range: 375-838, n = 21) and nitisinone 51 µmol/L (range: 41-57, n = 3) during pregnancy. She gave birth to a clinically healthy girl affected with tyrosinemia type 1. Birth was normal (birth weight 2615 g) and the baby had normal liver function, normal plasma alpha-fetoprotein concentrations, low urinary excretion of phenolic acids and no detectable succinylacetone. At birth, the baby had hypertyrosinemia (860 µmol/L in blood cord) and nitisinone levels of 14 µmol/L. Following molecular confirmation of the diagnosis of HT1 specific treatment began on day 15 by which time she had detectable urinary succinylacetone.

Regulatory effects of galactose on galactose-1-phosphate uridyltransferase activity on human hepatoblastoma HepG2 cells.

Galactose-1-phosphate uridyltransferase (GALT) deficiency results in galactosemia in man. We have studied the regulation of the GALT gene expression on the HepG2 cell line by growing the cells in glucose or galactose medium. No difference of Km values was observed in glucose or galactose media but the Vmax value with galactose was 50% higher than that with glucose. Also in galactose medium, an increased GALT specific activity was detected suggesting the production of more enzyme proteins. Yet, slot dot quantification of GALT mRNA revealed a decreased amount of these transcripts in cells cultured with galactose or inosine while Northern blot analysis revealed the normal 1.4 kb transcript in all culture media used. Finally, IEF gel analysis displayed different isozymic patterns for the GALT enzyme in cells grown in glucose, galactose or inosine media. With glucose-free media, the major band of GALT corresponds to that found in human liver. Altogether, these results suggest that the control of GALT gene expression in HepG2 cells is located at the post-transcriptional level and correlated to the growth rate of the cell.

Inhibition of lecithin cholesterol acyltransferase by phosphatidylcholine hydroperoxides.

To gain insight into the nature of the lecithin-cholesterol acyltransferase inhibitory factor(s), we separated and collected the oxidation products from oxidized lipoproteins after lipoxygenase treatment. Isolated fractions identified by chemiluminescence, as hydroperoxides of phosphatidylcholine, were found to produce a significant reduction of lecithin-cholesterol acyltransferase activity. The reaction kinetics of lecithin-cholesterol acyltransferase with reconstitued high density lipoproteins were studied in the presence of 0.6 and 1.2 microM hydroperoxides of phosphatidylcholine. No significant changes in the apparent Vmax were observed but a concentration-dependent increase in slope of the reciprocal plots and in the apparent Km values was observed with increasing hydroperoxide concentrations. These results show that the active site of lecithin-cholesterol acyltransferase is not affected by the presence of phosphatidylcholine hydroperoxides. Nevertheless, hydroperoxides of phosphatidylcholine altered the reactivity of lecithin-cholesterol acyltransferase for reconstitued high density lipoproteins suggesting either an alteration of the binding of lecithin-cholesterol acyltransferase to the reconstitued high density lipoproteins or a competitive inhibition mechanism.

Metabolic effects of galactose on human HepG2 hepatoblastoma cells.

HepG2 cells were used as a model system to study the effects of galactose overload on the liver, a target organ of galactose toxicity in patients suffering from transferase-deficient galactosemia. In the presence of galactose, HepG2 cell growth was slow and the pattern of gene expression remained characteristic of liver cells (secretion of alpha-fetoprotein [AFP] albumin, and transferrin). Galactose-1-phosphate (Gal-1-P) accumulated, as it does in galactosemic cells, but did not affect the energetic status of the cells (no adenosine triphosphate [ATP] depletion). However, the substitution of galactose for glucose as the sole hexose in the medium affected the specific activities of the galactose-metabolizing enzymes. Galactokinase (GALK) activity was decreased, and those of galactose-1-phosphate uridyltransferase (GALT), phosphoglucomutase, and glucose-6-phosphate dehydrogenase (G6PDH) were increased. The conversion of radiolabeled galactose to glucose (CO2 production and glycogen level) was greater in galactose medium than in glucose medium after a 7-day culture. Therefore, the culture of HepG2 cells in galactose medium indicates that the enhanced utilization of this hexose is due to the increased enzyme activities regulating its own metabolism. Hence, HepG2 cells constitute a good model for the study of modulation of galactose-metabolizing enzymes by galactose.

Biomarkers of oxidative stress: an analytical approach.

Oxidative stress is implicated in many pathological processes and results from a disruption of the prooxidant/antioxidant balance. This review will focus on noninvasive biomarkers of radical-induced damage in biological fluids and particularly in blood. Special attention will be addressed to new analytical methods for the measurement of radical-mediated alterations in the integrity of lipids, proteins and DNA.

Cholesterol efflux from Fu5AH cells to the serum of patients with Alagille syndrome. Importance of the hdl-phospholipids/free cholesterol ratio and of the hdl size distribution.

We have previously described the lipoprotein abnormalities in cholestatic children with paucity of interlobular bile ducts (PILBD), and we have shown that two different profiles emerged among these patients, depending on the level of lecithin:cholesterol acyltransferase (LCAT) activity. Reduced LCAT activity was associated with hypo-alpha-lipoproteinemia (group I) whereas normal LCAT activity was associated with hyper-alpha-lipoproteinemia (group II). In both groups, high density lipoproteins (HDL) were enriched with phospholipids and LpA-I particles were predominant. Here, we have investigated the ability of serum and of isolated HDL, obtained from PILBD and control subjects, to promote cellular cholesterol efflux, from Fu5AH rat hepatoma cells. The mean fractional efflux to 5% serum in each group was, on average, following the differences in HDL concentrations (control: 30.1 +/- 4.2%; group I: 23.7 +/- 7.9%, ns; group II: 44.2 +/- 6.5%, P < 0.001). The variations in efflux values in group II were positively correlated to the variations in HDL-PL concentrations (P < 0.0001) and in HDL-PL to serum apo-AI ratio (P < 0.003). By contrast, the variation in efflux in group I was only positively related to the large range of HDL-PL to free cholesterol (FC) ratio values (P < 0.0004). Fractional efflux to isolated HDL, measured at a constant HDL-PL amount, confirmed this relationship (P < 0.0001). Two-dimensional gel electrophoresis of the HDL size and apo A-I distribution in serum, revealed that small size HDL(3) and pre-beta HDL were predominant in the serum of patients from group I, especially those exhibiting low HDL-PL to FC ratio, whereas in the serum of patients from group II, both small HDL(3) and large HDL2 were present. These results suggest that a combination of an imbalance between phospholipids and free cholesterol in the HDL particles and a deficit in large acceptors of cholesterol will be responsible for an impairment of cellular cholesterol efflux in PILBD patients with reduced lecithin:cholesterol acyltransferase activity.-Davit-Spraul, A., V. Atger, M. L. Pourci, M. Hadchouel, A. Legrand, and N. Moatti. Cholesterol efflux from Fu5AH cells in the serum of patients with Alagille syndrome: importance of the HDL-phospholipids/free cholesterol ratio and of the HDL size distribution.

Abnormal lipoprotein pattern in patients with Alagille syndrome depends on Icterus severity.

BACKGROUND & AIMS: Children with Alagille syndrome have lipid abnormalities that differ according to the severity of icteric periods. The lipoprotein profiles of 22 patients with Alagille syndrome were determined and the findings were compared with the severity of jaundice. METHODS: Plasma lipids and apolipoproteins (apos), isolated lipoprotein composition, and lecithin/ cholesterol acyltransferase (LCAT) activity were analyzed in patients. Patients were classified into two groups according to their bilirubin levels; patients in group I had total bilirubin levels of > 100 mumol/L, and patients in group II had total bilirubin levels of < 100 mumol/L. RESULTS: In patients from group II, hypercholesterolemia was associated with increased levels of high-density lipoprotein and high concentrations of apoAI and apoAII; in a few cases, an abnormal lipoprotein with a slow alpha migration was observed. In contrast, in patients from group I, the levels of high-density lipoprotein cholesterol and apoAI and apoAII were very low, and the abnormal lipoprotein X was in many cases responsible for hypercholesterolemia. In group I, the decreased LCAT activity was consistent with the very high level of unesterified cholesterol and the emergence of lipoprotein X. In both groups of patients, the levels of apoE, apoCII, and apoCIII were high, and all the lipoprotein fractions were enriched in phospholipids. CONCLUSIONS: The variations of LCAT activity caused by the degree of jaundice in patients with Alagille syndrome are implicated in the abnormal lipid profiles.

Standard treatment of alpha-tocopherol in Alagille patients with severe cholestasis is insufficient.

Alpha-tocopherol (alpha-T) is the most effective lipid-soluble antioxidant present in cells. We investigated the efficacy of alpha-T supplements for preventing lipid peroxidation in patients with Alagille syndrome, according to the severity of cholestasis. Patients were assigned to two groups on the basis of plasma bilirubin concentration (group I, bilirubin <100 microM; group II, bilirubin >100 microM). alpha-T concentrations were determined in plasma, in isolated lipoproteins, and in red blood cell membranes. In both groups of patients, alpha-T concentrations in plasma were similar to those in control subjects, but the distribution of alpha-T in lipoproteins was affected by the abnormal lipoprotein pattern in these patients. The efficacy of alpha-T was estimated by determining the amount of hydroperoxide produced from phosphatidylcholine and phosphatidylethanolamine (PE) molecular species owing to oxidative stress induced by lipoxygenase treatment. The concentrations of phosphatidylcholine molecular species and its corresponding hydroperoxides were significantly higher in both groups of patients. In group I, alpha-T and PE molecular species concentrations were similar to those in control subjects, but PE hydroperoxide concentrations were higher than those in the control subjects. In group II, alpha-T concentration was significantly lower and the concentrations of some PE molecular species and all PE hydroperoxides were lower than those in the control subjects. In conclusion, erythrocyte membrane alpha-T concentration was significantly lower only in patients with severe jaundice, despite alpha-T supplementation, raising the question as to whether the usual treatment was appropriate in this group.