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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.

The double recombination into NIH-3T3 cells of the cloned Ha-rasEJ oncogene and the cDNA gene of the human growth hormone (hGH) under the control of a heat inducible promoter (hsp70) allowed hGH production either in vitro using the mass culture of engineered cells in biogenerators, or in vivo after xeno-transplantation of the cells into an animal host. Therefore the in vivo synthesized hGH induced the production of anti-hGH polyclonal antibodies.

Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-rasEJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 micrograms albumin and 0.32 microgram transferrin per 10(6) cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminofluorene (2-AAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 micrograms albumin and 11.0 micrograms transferrin per 10(6) cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGH per 10(6) cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 micrograms albumin and 11.7 micrograms transferrin per 10(6) cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AAF per mg cell protein. Hence, Ha-rasEJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.

Lipoma cells with consistent chromosomal aberration have been transfected with plasmids carrying papilloma bovine virus subgenomic fragment (PBV 69). The successful transformation of the cells was ascerted on the changed growth pattern of the cells in liquid medium, colony formation in soft agar and modified cell appearance in electron microscopy; transfection with PBV 69 has not been, however, sufficient to immortalize lipoma cells.

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.

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.

Using "Fluorescence plus Giemsa" technique, sister chromatid exchanges (SCE) were determined on second-division metaphases in rat liver epithelial cell lines treated with 2-acetylaminofluorene (2-AAF), the procarcinogen, and N-acetoxy-2-AAF (N-OAc-2AAF), an ultimate carcinogen analog. The SCE frequency was found decreased after some conditions of 2-AAF treatment and increased with N-OAc-2-AAF. Phenobarbital (PB) decreased also the SCE frequency but cancelled the 2-AAF action when incubated after the procarcinogen. The addition of caffeine (Caf) cancelled the action of 2-AAF but not of phenobarbital. It is suggested that the mechanism of SCE may have several origins.

A new technique for the derivatization, the separation and the quantification of 2-acetylaminofluorene (2-AAF) and its metabolites biosynthetized by freshly isolated hepatocytes was developed combining gas chromatography and mass spectrometry. Analysis of the different metabolites was carried out after their derivatization into tertbutyldimethylsilyl compounds. Freshly isolated hepatocytes metabolized 2-AAF and produced five aryl-hydroxylated compounds as well as the N-hydroxy-2AAF and the 2-aminofluorene. The metabolites were found under their free and conjugated forms.

Progesterone metabolism was studied in newborn rat heart cell cultures. The following enzymatic activities have been identified: 20 alpha-hydroxysteroid dehydrogenase, 3 alpha- and 5 alpha-hydroxysteroid oxidoreductases, and 6 alpha-hydroxylate. These activities are independent of sex and are probably related to the neonatal origin of the cells.

Cardiac erucic acid metabolism has been studied with beating heart cells in culture. It has been shown that erucate is taken up by these cultured cardiac myocytes. These cells are unable to oxidize it directly but require delaying intermediary steps to shorten the erucic acid chain before its complete oxidation. This long latency time of erucate oxidation would explain its accumulation within the different cellular lipid classes. It is suggested that the occurrence of large amounts of erucate in the different phospholipids might affect cellular and mitochondrial membrane structures. Evidence is reported that erucate does not affect the oxidative capacity of the myocardial cell but because of its accumulation dilutes other fatty acids which will then be oxidized at a lower rate.

Mutagenicity of the metabolites of the expoxide-diol pathway of safrole and analogues was studied on Ames' strains with Ames' method. Safrole, eugenol, eugenolmethylether, estragol, allylbenzene and 1'-hydroxysafrole, are not mutagen on TA 1535, TA 100 (point mutation) and TA 1537, TA 1538, TA 98 (frameshift mutations) without activation system. The corresponding epoxides that we have synthetized, are mutagens and inducers of point mutation in TA 1535 and TA 100. Dose-effect curves show differences between the mutagen efficiencies of these epoxides probably in relation with their electrophilic properties. On the other hand the 2', 3'-dihydro-2',3'-dihydroxisafrole was not mutagen in Ames' test. These results confirm the promutagen character of safrole and analogues and the role of the epoxides as proximate carcinogens.

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.

The multiple effects of serum on metabolic activities and macromolecular syntheses of heart cell cultures are discussed in this chapter. This pleiotropic response, which is linearly related to the amount of serum used, allowed us to quantitatively test the growth-promoting activity of different hormones. In addition, the possible mediation of the serum effects by cyclic nucleotides is considered.

Metabolism by sexed rat liver cell lines of safrole, a natural hepatocancerogen and of metabolites and analogs were studied and compared by gas chromatography-mass spectrometry. The epoxide-diol route was evidenced for safrole, isosafrole and eugenol as well as the induction of drug metabolizing enzymes by tested compounds.

The glucuronides of safrole were studied with GC-MS. At least seven compounds were identified, among then isomers of allylcatechol and mono-hydroxysafrol glucuronides. The presence of a glucuronide of hydroxysafrole-2',3' oxide will be especially discussed.

Electrical properties of cultured newborn rat heart cells are investigated by the use of microelectrophysiological methods. Amplitudes of resting and action potentials appear close to those of in situ heart cells. Elevated spike rate of rise reveals functional fast sodium channels. An inconstant ratio of cells exhibit pacemaker-like activity but no relationship can be established between this automaticity and the tissular origin of the cultured cells. The pulsation rate appears to be linked to the action potential duration and to the pace-maker potential slope. Spontaneous arrhythmias may occur; they are mainly caused by anomalous conduction and (or) erratic pacemaker driving. Thus heart cell cultures may be considered as a precious tool in the field of the cardiac electrophysiologal and physiopathological studies.

The transport of alpha-aminoisobutyric acid has been studied as a function of age in heart cell cultures derived from new-born Rats. The specific rate of alpha-aminoisobutyric acid transport decreased slightly once confluency had been reached and then, from day 8 on, increased abruptly. This last phase may be related to the expression of specialized functions encountered in older cultures.

Rat liver epithelial cells explanted in a serum-free medium (SFM) composed of Ham's F10 basal medium plus free fatty acids adsorbed on bovine albumin gave successful rise to primary cultures and then to long-term cell lines that expressed liver functions; induction of L-tyrosine aminotransferase by glucocorticoids, hepatic pattern of progesterone metabolism, and biosynthesis of murine primary bile acids; chenodeoxycholic and cholic acid common to higher vertebrates and alpha-muricholic acid specific of the rat bile.

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 gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography-mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.

The dispersion of the chromosome number was studied in a rat liver cell line which was grown up to the 65th passage in our standard culture medium (Ham F10 containing 10% fetal calf and 10% adult human sera) and thereafter cultured in the same chemical medium supplemented with decreasing serum concentrations. Until the serum concentration changes, the parental cell line exhibited for several passages a very wide range of chromosome numbers, from 35 up to 83. This range narrowed down to 42 when the cells were passed in a 5% fetal calf serum medium. Then, after several passages in this 5% serum medium, when subculturing again in 20% serum, a second cell population, hypotetraploid, progressively appeared until a compound population, hypotetraploid, progressively appeared until a compound population reached a 45-80 bimodal state. Another sub-line grown in 1% fetal calf serum exhibited similar chromosome evolution but returned to a slight aneuploid state (43 chromosomes) in media supplemented with 20% serum of different types.

Quantification of immunoglobulins IgG, IgA, IgM, C3 complement, siderophilin, alpha 2-macroglobulin and haptoglobin in serum by a Behring laser nephelometer coupled with a date processing apparatus was compared with values obtained by the radial immunodiffusion method of Mancini. The precision was not improved by the use of the laser nephelometer as compared to Mancini radial immunodiffusion, but within run precision was better than that among runs for both methods. The analytical recovery studies for laser nephelometry showed a close concordance between the theoretical and measured values for all proteins. The two techniques were also compared by means of correlation studies.

A rat liver epithelial cell line has been propagated on microcarriers in 11 or 21 laboratory culture vessels for cell culture in suspension on microcarriers (biogenerators) with Ham F10 or DME as basal synthetic culture medium either serum-supplemented (SSM), or serum-free (SFM), or serum- and protein-free (SPFM). Without serum, the use of DME allows a cellular growth in the biogenerator at least equivalent to that obtained in culture dishes. For the cultivation on microcarriers in SFM in a biogenerator the use during the first day of culture of spent serum-free medium previously incubated (SFMI) in confluent culture dishes avoids the substratum treatment with serum. Results concerning the Vero cell line cultured in SPFM are shown.

Extensive studies of parameters conditioning selection and high plating efficiency of epithelial liver cells at primary seeding allowed us to set up a technique for the routine culture of liver cells from rats of various ages (18 day-old pc to 7 month-old) in Ham F10 medium supplemented with 10 p. cent fetal calf serum and 10 p. cent human serum. Cultures, after several passages, or sometimes at primary seeding were free of fibroblasts. The quality of water for culture medium preparation was found to be a very important parameter. G-banding caryotype showed that cells in culture were diploid until 15-20 passages. Various metabolic pathways have been studied in primary culture and in cell lines: enzymes of the anaerobic metabolism of hexoses and metabolism of steroid hormones and xenobiotics. Activity of glucose-6-phosphatase was nearly lost in all cultures. Activity of glucose-6-phosphatase was nearly lost in all cultures. Aldolase showed a specific liver activity with a cleavage ratio of phosphofructoses (F-1,6-diP/F-1-P) equal to 1 or about 1 in several primary cultures and cell lines. Many metabolites arising from incubation of cell lines with 14C-labelled corticosterone, corticosterone-21-sulfate, testosterone and progesterone have been isolated and quantitated by gas liquid chromatography (GC) and mass fragmentography coupled to GC, using 14C/12C isotope ratio measurements. These metabolites indicate the presence in cultured cells of 3 alpha/beta-steroid-reductases, 4-ene steroid reductases and hydroxylases at various positions: 2 alpha, 2 beta, 6 alpha, 6 beta, 7 alpha, 17 beta and 16 alpha. These cell lines were able to activate carcinogens through the epoxide-diol pathway and are suitable for drug metabolism study.

Intracellular investigation of bioelectrical properties is performed on cultivated rat myocardial cells. Electrophysiological parameters of cells dissociated from rat myocardium are very similar to those of in vivo cells. Fast sodium channels are found to be fonctional. Spontaneous arrhythmia are recorded from some impaled cells.

In order to analyse and quantitate the urinary 16-oxysteroids known or thought to be associated with hypertension, we have established for six 16-oxy-C19 reference steroids the following parameters: elution volume on lipophilic gel columns, gas chromatographic retention data expressed as methylene unit values of trimethylsilyl ether and O-methoxime trimethylsilyl ether derivatives on OV-1 and OV-17 packed columns and on SE-30 capillary column, and mass spectra of these compounds. These reference steroids were: 3 alpha, 16 alpha-dihydroxy-5 alpha-androstan-17-one, 3 alpha, 16 alpha-dihydroxy-5 beta-androstan-17-one, 3 beta, 16 alpha-dihydroxy-5 alpha-androstan-17-one, 3 beta, 16 alpha-dihydroxy-5-androsten-17-one, 3 beta, 16 beta-dihydroxy-5-androsten-17-one, 3 beta, 17 beta-dihydroxy-5-androsten-16-one and 3 alpha, 15 alpha-dihydroxy-5 beta-androstan-17-one. The proposed method was shown to be applicable to the specific analysis of 16-oxy-C19-steroids in biological samples since it achieved the selective isolation of these compounds from other steroids and their quantitative elution in a single fraction. The analysis of the urinary steroids of two patients with arterial hypertension demonstrated an elevated rate of 3 beta, 16 alpha-dihydroxy-5-androsten-17-one.

A noticeable increase has been observed in the production of the C 3 component of the complement by Rat liver epithelial cells cultured in vitro in the presence of small quantities of supernatant of human blood mononuclear cells cultured for 72 hrs. with phytohemagglutinin. The physicochemical properties suggest that the stimulating factor(s) is(are) protein(s).

Mechanisms and sequences of reduction and hydroxylation of progesterone into 6-hydroxypregnanolones were studied in proliferating rat liver epithelial cell cultures. These cell lines had an intense metabolic activity and all the metabolites were unconjugated. The formation of 3 alpha,6 alpha- and 3 beta,6 alpha-dihydroxy-5 alpha-pregnan-20-one was observed when the cells were incubated with progesterone, 5 alpha-pregnane-3,20-dione, 3 alpha- or 3 beta-hydroxy-5 alpha-pregnan-20-one and 6 alpha-hydroxy-5 alpha-pregnane-3,20-dione but not with 6 alpha- or 6 beta-hydroxyprogesterone, 5 beta-pregnane-3,20-dione, 3 alpha- or 3 beta-hydroxy-5 beta-pregnan-20-one. These findings indicate that the potential precursors of the 6 alpha-hydroxypregnanolones have a 5 alpha-configuration. The reduction of 5 alpha-pregnane-3,20-dione at C-3 followed by a 6 alpha-hydroxylation can be postulated as the major, if not the only, metabolic pathway. However, the possibility that 6 alpha-hydroxylation may occur prior to reduction of the C-3 oxo group cannot be entirely ruled out. The stereospecificity of reduction at C-5 and hydroxylation at C-6 are discussed and compared with 6-hydroxylated progesterone metabolites found in man and some other mammals during pregnancy and the neonatal period.

The development of a method for the serum-free culture of the Y-1 mouse adrenocortical tumor cell line has permitted a detailed search for factors regulating cellular growth and steroidogenesis. The serum-free medium (SFM) was made of Ham's F10 basal medium supplemented with free fatty acids adsorbed on albumin. The SFM complemented with calcium, arachidonic acid and cholesterol, i.e. SFM-S, induced cell proliferation to a density at confluency higher than that obtained with 1% serum-supplemented medium (1%-SSM) and allowed to sustain cell growth for more than six passages. When albumin was replaced by a dextran polymer (Mr = 2 x 10(6)) used as a carrier of lipids instead of albumin (which resulted in a serum-free and protein-free medium, SPFM), the cell number was 75% of that observed with the SFM-S. The addition to SPFM of beta-globulin alone or combined with insulin caused a 2- or 3-fold increase in the final cell density, respectively. The ability of the Y-1 cell line to produce steroids in response to ACTH was found to be higher in SFM-S or SPFM than in 1%-SSM. Furthermore, the addition of beta-globulin to SPFM stimulated steroid hormone biosynthesis with a marked increase in 11 beta-hydroxylated steroid production. These studies demonstrate that the use of a defined mixture of nutriments and of few growth factors permits to sustain not only the cellular proliferation of the Y-1 cell line but also its differentiated function of ACTH-induced steroidogenesis.

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.

The steroids in urine from normal pregnant women have been studied. After extraction of conjugate steroids, solvolysis and enzymatic hydrolysis, the liberated steroids were separated by chromatography on Sephadex LH-20, and were analysed by gas-liquid chromatography and gas chromatography mass spectrometry. The following steroids were isolated and completely identified in the LH-20 fraction 7: 5beta-pregnane-3alpha,20alpha-diol, 5beta-pregnane-3alpha,17,20alpha-triol, 5beta-pregnane-3alpha,20alpha,21-triol and 5alpha-pregnane-3beta,16alpha,20alpha-triol. In addition, two metabolites tentatively identified as 5xi-pregnane-2xi,3xi,20xi-triol and 2xi,3xi,16xi-trihydroxy-5xi-pregnan-20-one, have not been reported as occcurring in urine from pregnant women. The 5beta-pregnane-3alpha,20alpha,21-triol was detected only in the third trimester of pregnancy and the urinary excretion values are between 320 and 650 microgram per 24 h. With the present data, it is not possible to establish the precursor(s) of this steroid. However, these results tentatively suggest that 5beta-pregnane-3alpha,20alpha,21-triol arises from foeto-placental unit.

Cytotoxic effects of Bleomycin A2 on adult rat liver epithelial cell lines were evaluated by three methods: the incorporation rate of (3H) thymidine for DNA biosynthesis, the incorporation rate of L-(3H) leucine for protein biosynthesis and Giemsa dye staining of surviving cells. Chromosome investigations at successive passages of cell lines have shown that spontaneous chromosome abnormalities in distribution and structure after 15-20 passages, i.e. 50 to 60 cell generations, were the earliest morphological sign of spontaneous transformation. In this study a highly spontaneously transformed cell line was very sensitive to the drug. Another cell line at the beginning of spontaneous transformation appeared to be insensitive although on further passage it became more sensitive. The use of microtitration plates made it easier for us to undertake a comparative study of the different parameters. Following Bleomycin A2 exposure, Giemsa staining gave the best evaluation of cell killing whereas thymidine incorporation allowed the estimation of cell recovery. The antineoplastic effect of Bleomycin A2 can probably be used to evaluate the malignant potential of different rat liver epithelial cell lines.

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

A gas-liquid chromatographic (GLC) method and an isotopic dilution-mass fragmentographic (ID-MF) procedure using the same capillary chromatographic separation are described for serum cholesterol assay. GLC included silylation and separation on a highly efficient glass capillary column which allowed the separation of cholesterol from cholestanol and the use of epicoprostanol as internal standard. The concentrations were calculated from the areas of the signals and digitalized by a reporting integrator. The reproducibility was 0.5% and the correlation with the ID-MF technique was 0.997. The ID-MF technique was characterized by the use of [3,4-13C] cholesterol as the labelled standard and a chemical ionization mode. The reproducibility was 0.8%.

The derivatization of bile acids into trimethylsilyl ether isobutyl ester (IBTMS) and of neutral sterols into trimethylsilyl ether (TMS) allowed the separation on an OV-1 capillary gas chromatography column of 15 bile steroids as follows: cholesterol, 7 alpha-hydroxycholesterol, 6 beta-hydroxycholesterol, 6 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, lithocholate, deoxycholate, 25-hydroxycholesterol, chenodeoxycholate, cholate, murocholate, hyodeoxycholate, ursodeoxycholate, hyocholate, and beta-muricholate. Fragmentation data of the coupled gas chromatographic-mass spectrometric (GC-MS) analysis of these nine bile acids as IBTMS derivatives under electron impact and chemical ionizations (methane, isobutane, and ammonia) are given. The ammonia chemical ionization appears to be the best mode for compound identification and quantitation due to fragmentations into high mass ions. The comparison of methylene units of the five sterols as TMS derivatives and of each type of methyl, TMS, or isobutyl ester of the nine bile acids as TMS ethers showed that isobutyl esterification increased dramatically the retention time of the bile acids, allowing their separation after the neutral sterols. Different methods of GC-MS analysis were applied to the study of bile steroid secretion in long-term rat liver epithelial cell lines, either serum-supplemented cell lines or serum-free cell lines, growing in serum-free medium since the primary explanation or after adaptation of serum-supplemented lines to this medium. It is demonstrated for the first time that liver epithelial cell lines maintain the metabolic pathway leading from synthesized cholesterol to dioxygenated sterols and the two normal main primary bile acids of the liver, chenodeoxycholic acid and cholic acid, up to 32-47% of the in vivo daily rate, and in addition the production of alpha-muricholic acid, the bile acid marker of murine liver.

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.

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.

Palmitic acid oxidation has been studied in cultures of beating heart cells. The data reported suggest: 1. The main source of fatty acids for oxidation in cell cultures is provided by lypolysis of preexisting intracellular fatty acids. Most fatty acids that have just entered the cell are not oxidized immediately. Thus, aged heart cells in culture seem to retain their ability to utilize fatty acids as their main energy source. 2. In cultured heart cells, the amount of CO2 obtained from the carboxyl group was much higher than that obtained from the methyl group. On the other hand, beta-hydroxybutrate has been shown to be formed mainly from the methyl group. This may indicate that beta-oxidation terminates at the beta-hydroxybutyrate stage because of premature dissociation of this last intermediate from the enzyme complex.

The metabolism of glucose and palmitate by new born rat heart cell in tissue culture was investigated to ascertain the usefulness of this model system for metabolic studies. Heart cells in culture oxidize palmitate at a rate which is close to the rate reported in vivo. The fate of glucose is shown to differ from in vivo. High rates of lactate release are observed although there are many evidences that cells are not hypoxic. This impaired regulation is likely due to an impaired uptake of glucose.