Alterations of HDL cholesterol distribution induced by incubation of human serum.

The study of high density lipoprotein (HDL) alterations induced by serum incubation was undertaken by a new approach. Subfractions of HDL were separated by gradient gel electrophoresis, without preliminary ultracentrifugation, and were characterized by their size range. After dissolution of the polyacrylamide gel, each subfraction was analyzed for its total- and unesterified-cholesterol content by gas chromatography. We have observed that a general displacement of HDL cholesterol towards the subspecies of high size range occurred during serum incubation at 37 degrees C, contemporaneously with cholesterol esterification. This displacement could not be identified with a HDL3 to HDL2 conversion since it occurred within HDL3 and HDL2. It is probably the indication of a complex HDL conversion leading to particles of increased sizes. HDL alterations occurring upon serum incubation appear to be the consequence of the activity of an HDL conversion factor, which is thermolabile, non-dialysable, present in the d greater than 1.25 serum fraction and differs from lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein. They could be considered as preliminary enzymatic transformations, necessary for the action of lecithin:cholesterol acyltransferase and as the first step of a metabolic sequence including, successively, HDL conversion, cholesterol esterification by lecithin:cholesterol acyltransferase and cholesteryl ester transfer.

Biosynthesis of sterols and bile acids in rat liver epithelial cell lines.

A rat liver epithelial cell line growing in a serum-supplemented medium expressed biosynthetic pathways of bile sterols and of free and conjugated chenodeoxycholic and cholic acids, the main primary bile acids of the liver. They were identified and measured by gas chromatography-mass spectrometry. The bile steroid secretion in the serum-supplemented cell line was established upon incubation in a serum-free medium which was demonstrated to sustain cell growth, allowing elimination of the interference of exogenous bile steroids and effectors. The free bile acid secretion was also expressed in a subline adapted to proliferate in this serum-free medium, i.e., a basal medium supplemented with 4 g/l albumin carrying 7.6 muequiv./l of a mixture of six long-chain free fatty acids but without any addition of hormones and growth factors. In addition, the rat liver epithelial cell line growing in the serum-supplemented medium maintained, with time, a steady-state of bile acid secretion over a lifespan of 500 days. In the two types of liver epithelial cell lines, dexamethasone and chenodeoxycholic acid supplementation exerted, individually, either a stimulating or an inhibiting effect on the bile acid secretion concurrently with the hydroxylation of chenodeoxycholic acid into alpha-muricholic acid.

Pleiotypic response of rat heart cells in culture to serum stimulation.

The pleiotypic effects of medium replacement were studied in rat heart cell cultures. After each medium change alpha-aminoisobutyric acid and glucose transport are increased, RNA and protein syntheses are activated. DNA synthesis did not begin before 12 hours and was followed by a wave of mitoses. This sequence of events suggests that the stimulated cells were in early G 1 phase. DNA synthesis, following the shift to a fresh medium, is linearly related to the amount of serum used as is protein synthesis. However when serum concentrations higher than 20 percent were used no increased protein synthesis could be observed suggesting the existence of another limiting factor, which was probably the isoleucine content of the medium. The serum stimulating factor is heat stable, dialysable and was found in both human and fetal calf sera.

Metformin induces an agonist-specific increase in albumin production by primary cultured rat hepatocytes.

Metformin (MET) is known to increase several biological effects of insulin (INS), but there is no information concerning its direct effects on protein synthesis. We studied the action of MET on albumin production by primary cultures of freshly isolated rat hepatocytes, alone or in combination with various agonists: INS, IGF-1, EGF, thyroxin, and dexamethasone. While having no effect alone, MET in vitro potentiates the effects of INS, IGF-1, and EGF. When this increasing effect toward INS was studied over a broad concentration range, MET appeared to improve low-acting INS levels and to intensify the maximal INS effects. In contrast, MET did not change the production of albumin stimulated by thyroxin or dexamethasone. Animals chronically pretreated with MET in vivo showed a higher yield of isolated hepatocytes, better attachment, and especially higher viability after liver perfusion and during cell culture. This may largely explain why basal albumin rates were higher than in in vitro-treated cells. The effect of MET in the presence of the agonists exhibited the same agonist-specificity as in vitro. Our data provide new insights into the pharmacology of MET by showing that hepatic protein synthesis is increased by MET and INS. From the specificity of action of MET towards INS, IGF-1, and EGF (but not thyroxin or dexamethasone), we hypothesize that this biguanide may act on intracellular pathways located between membrane receptors and sites of branching in the signaling cascades shared by these agonists.

Glyceride content of amniotic fluid cells as an index of fetal maturity.

A simple biochemical method has been used to determine the content of neutral lipid of washed cell pellets prepared from 114 samples of amniotic fluid. This was done to improve the large standard deviation of the conventional orange fat cell measurements. By relating the content of neutral lipids to the total protein content of the pellet, the error of cell counting is minimized. The resultant glyceride/protein ratio increases 20 times within the last 8 weeks of pregnancy. The results are not altered by associated diabetes, hydramnios or Rh isoimmunization.

Cholesterol gas-liquid chromatographic microassay in serum lipoproteins separated by polyacrylamide gel electrophoresis.

A new method of cholesterol assay in serum lipoprotein fractions separated by electrophoresis on polyacrylamide plates is described: each lipoprotein fraction was collected by cutting the gel and, after gel dissolution, cholesterol was extracted and assayed by gas-liquid chromatography. This method associates the specificity and sensitivity of gas-liquid chromatography with the resolution of polyacrylamide gel electrophoresis, a highly reliable technique for hyperlipoproteinemia phenotyping. It is precise (variation coefficient below 2%), fast and needs only 2.5 microlitres of serum. Direct assay of cholesterol is feasible not only on each of the three main fractions, very low density lipoproteins, low density lipoproteins and high density lipoproteins, but also on intermediary fractions such as Lp(a) lipoprotein. This method should allow a better evaluation of the relationship between the serum cholesterol fractions and development of atherosclerosis.

Cholesterol content of serum lipoprotein fractions in children maintained on chronic hemodialysis.

Lipoprotein analyses were performed on serum samples from nine hemodialyzed children, 8 to 18 years old, and from nine pair-matched control children. Cholesterol was measured by gas-liquid chromatography in lipoprotein fractions separated by polyacrylamide gel electrophoresis. Total serum triglycerides and cholesterol levels were higher in the hemodialyzed group. Very low density lipoprotein cholesterol concentration was higher. The increase of low density lipoprotein cholesterol concentration and the decrease of high density lipoprotein cholesterol concentration were not significant, but the ratio of the low density lipoprotein cholesterol to the high density lipoprotein cholesterol was increased. These results outline the potential risk of premature atherosclerosis in uremic children on maintenance hemodialysis. In addition, lipoprotein cholesterol reference values are presented from a group of 113 healthy children.

Synthesis and gas chromatographic-mass spectrometric analysis of reduced compounds derived from 6 alpha- and 6 beta-hydroxy-11-deoxycorticosterone.

A series of thirty two 6-hydroxylated steroids were synthesized by selective reduction of the 4-5 double bond, the 3-oxo group, and/or the 20-oxo group of 6 alpha- and 6 beta-hydroxyDOC. The different reactions leading to the production of specific isomers are discussed. The gas chromatographic and spectrometric characteristics of the methoxime-trimethylsilyl (MO-TMS) or trimethylsilyl (TMS) derivatives of the isomers obtained are given. The gas chromatographic separation of the syn- and anti-isomers of the methoxime in position 3 was found to be characteristic of the configuration of the hydroxyl in position 6. The difference between methylene unit values of syn- and anti- isomers is much larger for the 6 alpha-series than for the 6 beta-series. The mass spectral analysis showed that many ions are specific of the MO-TMS derivatives of steroids with 3,6-dihydroxy-4-ene or 3-oxo-6-hydroxy-4-ene structure. In the case of steroids with a saturated ring A no significant ions characteristic of the presence of a 6-trimethylsilyloxy substituent were found. This work provides previously unavailable reference data on 6-hydroxylated steroids which should facilitate the study of corticosteroid metabolism.

[Enzymatic activity assays in the hepatic cell by mass fragmentography associated with gas-liquid chromatography]. / Mesure d'activités enzymatiques dans la cellule hépatique par la fragmentographie de masse couplée à la chromatographie gaz-liquide

The most generalized methods in enzymology are based on the quantitative assay of compounds, substrate or coenzyme, by spectrophotometry without any separation. Such a method is ruled out if the colorimetric reaction is not specific of the compound. In liver enzymology, aside the classical metabolic pathways, such assays are difficult to apply, especially when several metabolic steps are investigated. It is therefore necessary to use separative methods to isolate the metabolized substrate(s). For instance, the reductive catabolism of corticosterone leads to fourteen isomers (two dihydrocompounds, four tetrahydrocompounds and eight hexahydrocompounds) in which their respective productions are sex and age-linked. A position isomer of corticosterone, the 18-hydroxy-11-deoxy-corticosterone, follows the same reductive route. In adrenals some reduced metabolites arise from these two steroid hormones and are age dependent. When such metabolites are amenable to volatilization for gas chromatography, the interfacing of the gas chromatograph to the mass spectrometer allows to identify each compound introduced in the spectrometer. Among the ions produced by fragmentation of a compound or of a family of compounds, several specific fragments can be selected to be monitored along the chromatographic run leading to mass peaks which are quantitatively proportional to the amount of compounds, as far as other foreign molecules do not contribute fo fragment productions. These methods called mass fragmentography or multiple ion detection, or selected ion monitoring, allow with the help of all the resources of gas chromatography such the derivatization of studied molecules with heavy isotope labeled reagents to use the same unlabeled derivatized molecules as carriers and internal standards at once. This method allows to quantitate at the level of the picomolecule or less. Examples will be given with the study of the metabolism hormone steroids and xenobiotic compounds by the liver and adrenals in the animal and by isolated liver and adrenal cell cultures.

[A case of virilizing adrenal cortical adenoma whose steroid profile evokes a partial 21-hydroxylase deficiency (author's transl)]. / Un cas d'adénome corticosurrénalien virilisant présentant le profil hormonal d'un déficit partiel en 21-hydroxylase.

The C19 and C21 urinary steroids from a virilizing adrenal tumour with high levels of plasma 17 alpha-hydroxyprogesterone and its urinary metabolites have been identified and quantitated gas chromatography and mass spectrometry of sephadex fractions of the total urinary extract. A of the fifty-five identified steroids thirteen were compounds not found before in such a case. The actiology of the apparent 21-steroid hydroxtlase deficiency is discussed at the light of these analytical results and of the hormonogenesis enzymatic induction of the tumour biopsy.