Metabolism pathway of vinorelbine (Navelbine) in human: characterisation of the metabolites by HPLC-MS/MS.

The biotransformation of vinorelbine (VRL), an anti-neoplastic vinca-alkaloid derivate already marketed for nonsmall cell lung cancer and advanced breast cancer as an i.v. form and currently registered in several countries as an oral form, was investigated in human. Biological specimen from several human sources constituted the material for the metabolic identification in human. An isocratic liquid chromatographic system composed of 40 mM ammonium acetate (pH 3) and acetonitrile was used for separation of the potential metabolites of VRL. Tandem mass spectrometry with positive electrospray ionisation was used to enable the structural identification of the metabolites. A total of 17 metabolites (12 directly obtained from VRL and 5 involving sequential step pathways) were characterised with proposed structures for most of the metabolites. All metabolites went through phase I reactions by the way of deacetylation, dealkylation, oxidation and hydroxylation. No conjugates were observed. Despite the high number of metabolites quantified, VRL was the major compound observed whatever the matrix. Most of the metabolites rapidly disappeared from blood, except 4-O-deacetyl vinorelbine which was slowly cleared. Most of the enzymatic pathways involved in the metabolites strongly suggested the major role of cytochrome P450 in the biotransformation of vinorelbine.

Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography-electrospray ionization tandem mass spectrometry.

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation and programmed cell death. Qualitative and quantitative analysis of these second messengers requires sensitive and specific analytical methods to detect endogenous levels of individual ceramide species and to differentiate between them. Nine synthetic ceramides were separated by liquid chromatography coupled to tandem mass spectrometry on a C18 bonded silica column. The lipids were eluted in gradient elution mode using a mixture of water, acetonitrile and 2-propanol as mobile phase. They were detected by reaction monitoring performed on positive ion electrospray generated ions. Collision-induced fragmentations conducted on ceramides produced a well characteristic product ion at m/z 264, making multiple reaction monitoring (MRM) well suited for various ceramides quantitative measurements. After optimization of the extraction step, the proposed methodology was able to identify and quantify different ceramide species issued from human cancer cells. The method could be validated for C16, C18 and C20 ceramides, quantified at the nanogram level. The validation exhibits good results with respect to linearity, accuracy and precision.

New sensitive liquid chromatography method coupled with tandem mass spectrometric detection for the clinical analysis of vinorelbine and its metabolites in blood, plasma, urine and faeces.

A new sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and validated for the simultaneous quantitation of vinorelbine, its main metabolite, 4-O-deacetylvinorelbine and two other minor metabolites, 20'-hydroxyvinorelbine and vinorelbine 6'-oxide. All these compounds, including vinblastine (used as internal standard) were deproteinised from blood, plasma and faeces (only diluted in urine), analysed on a cyano column and detected on a Micromass Quattro II system in the positive ion mode after ionisation, using an electrospray ion source. Under tandem mass spectrometry conditions, the specific product ions led one to accurately quantify vinorelbine and its metabolites in all biological fluids. In whole blood, linearity was assessed up to 200 ng/ml for vinorelbine and up to 50 ng/ml for the metabolites. The limit of quantitation was validated at 250 pg/ml for both vinorelbine and 4-O-deacetylvinorelbine. In the other biological media, the linearity was assessed within a same range and the limit of quantitation was adjusted according to the expected concentrations of each compound. This method was initially developed in order to identify the metabolite structures and to elucidate the metabolic pathway of vinorelbine. Thanks to its high sensitivity, this method has enabled the quantitation of vinorelbine and all its metabolites in whole blood over 168 h (i.e., 4-5 elimination half lives) whilst the previous liquid chromatographic methods allowed their measurement for a maximum of 48-72 h. Therefore, using this method has improved the reliability of the pharmacokinetic data analysis of vinorelbine.

[Quality specifications and allowable standards for validation of methods used in clinical biochemistry]. / Analyses de biologie médicale: spécifications et normes d'acceptabilité à l'usage de la validation de techniques.

The purpose of this work is to provide, for a large number of analysis in the field of clinical biochemistry, appropriate criteria for the evaluation of the performance of in vitro diagnostic methods. Based on a first set of data established in 1986 and the experience cumulated by organisers in charge of internal and external quality assessment surveys, an expert group has proposed acceptable limits for a large list of analysis (n = 116). Data are reported and presented in tables divided into 7 chapters including: general biochemistry, enzymes, proteins, tumour markers, hormones, drugs (and toxic), and urinary analysis. For each analysis are given: analytical and reference ranges, three concentration levels for control specimens to be used during evaluations and the range of values within which they can be chosen, reproducibility and repeatability limits expressed as CV%. Maximal tolerable systematic error and inaccuracy are given for control and biological specimens and compared to those obtained using a reference or validated method. These data are essential for evaluations using the protocol designed by the SFBC and can serve as quality criteria for the choice and validation of in vitro diagnostic systems.

Quantitative determination of several synthetic corticosteroids by gas chromatography-mass spectrometry after purification by immunoaffinity chromatography.

A study was conducted to test a multiresidue analytical procedure for detecting and quantifying several corticosteroids on which the European Union imposes maximum residue limits (MRLs). Primary extracts from different matrices (liver, milk, urine, faeces) were first purified on C18 cartridges. A new immunoaffinity clean-up step was included. The immunoaffinity gel was used to purify several corticosteroids simultaneously with enrichment of the corresponding fractions. The extracts were treated with an aqueous solution of pyridinium chlorochromate to fully oxidise all corticosteroids and to facilitate their extraction with dichloromethane. After evaporation, the final extract was reconstituted with toluene before injection into the GC-MS apparatus. The analysis was performed in the CI-negative ionisation mode using ammonia as the reactant gas. The estimated detection and quantification limits were, respectively, 0.25 and 0.5 ppb or lower. Overall, the method is reproducible to within 20%. Recovery is between 50 and 80% according to the corticosteroid.

An atypical aortic atherosclerotic lesion in cynomolgus monkeys during hypercholesterolemia: a protection by smooth muscle cells against advanced lesions?

This study focuses on the fortuitous discovery of an atypical atherosclerotic lesion in four of 49 male adult cynomolgus monkeys (macacus fascicularis) which were maintained for a long time at a high level of hypercholesterolemia, and in seven of 19 female cynomolgus monkeys examined from the second to the 24th week of hypercholesterolemic diet: this lesion was in formation or already mature during this period of diet. This atypical lesion was formed by a collagen and elastic network surrounding synthetic smooth muscle cells without fibrofatty or fibrous plaques. Lipids were occasionally seen in the inner intima. The lesion appeared early (from the third week of diet). Once established, its morphology did not change. It became more extensive, but was not complicated by lipid overload in spite of prolonged, permanent hypercholesterolemia. This response to hypercholesterolemia is interesting because the activity of the smooth muscle cells differs from that observed in the classic lesion: they intervene earlier, their replication is very marked and rapid, their elastin secretion is greater and remains constant over time, and their phagocytic properties are reduced. This experimental study examines the installation and the maintenance of this lesion and raises the problem of its origin.

[Cutaneous apoprotein B and coronary atherosclerosis]. / Apoprotéine B cutanée et athérosclérose coronarienne.

Cutaneous and plasma lipids (cholesterol and Apoprotein B) were studied in 2 populations (average age 57.5 years), one with pathological and the other with normal coronary angiography. Skin biopsy was performed during the incision of thoracotomy. The concentrations of Apo B and cholesterol in the skin were compared to those of plasma lipids, lipoproteins and apoprotein B for the diagnosis of atherosclerosis. This study showed that skin Apo B was the best marker of coronary atherosclerosis in patients with plasma Apo B concentrations of less than 1.3 g/l. The skin Apo B concentration was closely correlated to the presence but not to the severity of this arterial pathology. The cardiovascular risk factors of this population, studied separately and in a cumulative manner, confirmed the results of previously published reports.

15- and 16-hydroxylations of androgens and estrogens in the human fetal liver: a critical step in estetrol biosynthesis.

To elucidate the main metabolic pathways which lead to the foeto-placental biosynthesis of estetrol (I), we investigated the 15 alpha- and 16 alpha-hydroxylations of potential precursors of this estrogen in the human fetal liver. We determined the 15 alpha- and 16 alpha-hydroxylation capacity of the fetal liver for each precursor by GC-MS. The results suggest that estetrol is derived only from estradiol sulfate (II) and DHEA sulfate (III). 15 alpha-Hydroxy-androstenedione (IV) can no longer be regarded as a good precursor of estetrol. The phenolic pathway appears to be a more likely route than the neutral pathway, even when derived from DHEA sulfate.

[Cutaneous cholesterol in the young and aged coronary patient]. / Le cholestérol cutané du coronarien jeune et âgé.

Serum cholesterol (ch), its lipoprotein fractions and triglycerides were measured in three populations of proven coronary patients (less than 50 years, n = 56; between 50 and 65 years, n = 56; greater than 65 years, n = 23); the risk factor total ch/HDL ch was calculated. The level of skin cholesterol was also estimated by skin biopsy in each patient and compared to that of three control populations of the same age. The results indicated that 1) there was no significant difference in skin cholesterol of patients with myocardial infarction whatever their age, 2) there was a significant difference (p less than 0,001) with control subjects of the same age except in the over 65 population, 3) the total cholesterol was normal in all three groups, 4) the HDL cholesterol of coronary patients over 50 year old was normal and slightly reduced in younger coronary patients, 5) the ratio total ch/HDL ch was increased in coronary patients under 50, but normal after this age, 6) the triglyceride level was higher in the young coronary patients than in those over 50 years old. Four conclusions are drawn: 1) the total Ch/HDL ch ratio is a good indicator of coronary risk in patients under 50 years old but shows less sensitive variations than the level of skin cholesterol, 2) the ch/HDL ch in coronary patients between 50 and 65 years old is normal; the only laboratory finding which correlates with the coronary event is skin cholesterol; after 65 years of age the skin cholesterol stabilises to the same levels as found in control subjects; 3) from the outset, at whatever age infarction occurs, skin cholesterol is increased (about 0,45 mumol/100 ngr of fresh skin), whilst the risk factor is higher in the younger population; 4) skin cholesterol shows less variation in the three coronary groups than the other blood parameters measured. It would therefore appear to be a very discriminating index of coronary atherosclerosis.

Aromatization of 15 alpha and 16 alpha hydroxylated androgens in the human placental using [1,2-3H]-substrates.

This in vitro study reports data on the aromatization of [1,2-3H]-C19 steroids in the human term placenta [androstenedione (III), testosterone (IV), 15 alpha-hydroxy-androstenedione (V), 15 alpha-hydroxy-testosterone (VI), 16 alpha-hydroxy-androstenedione (VII)]. The hydroxylated androgens were microbiologically synthesized from commercially radiolabelled [1,2-3H]-androstenedione and testosterone. Androstenedione and testosterone were good substrates for the human placental aromatase (low Km values, high Vmax); they strongly inhibited the 15 and 16 hydroxylated androgens aromatizations. On the other hand, these hydroxylated compounds acted as poor substrates and were only non-competitive inhibitors of the androstenedione and testosterone aromatizations. However, 15 alpha-hydroxy-androstenedione could not be disregarded as a potential precursor of 15 alpha-hydroxylated estrogens in the human placenta.

Skin cholesterol and skin apoprotein B in atherosclerosis.

In order to quantify the accumulation of apoprotein B in skin we determined the amount of apo B in the skin of atherosclerotic patients using a modified Hoff's method with extraction and electroimmunoassay. Skin biopsies were taken from the lower limbs of nineteen male patients with arteriosclerosis obliterans during revascularization and from the thoracic region of thirty male patients with ischemic heart disease during coronary bypass graft surgery and thirty one male patients with abnormalities of cardiac valves during valvuloplasty. A significant positive correlation between skin apo B and skin cholesterol was found in three groups. Our data support the hypothesis that cholesterol deposit in the skin of patients with atherosclerosis is derived from plasma lipoprotein B and that the content of skin lipids parallels with the development of atherosclerosis in man.

Synthesis of 16 alpha-3H androgen and estrogen substrates for 16 alpha-hydroxylase.

The synthesis of 16 alpha-3H androgens and estrogens is described. 1-(3H)-Acetic acid in the presence of zinc dust reacts with 16 alpha-bromo-17-ketosteroids to produce 16 alpha-3H-17-ketosteroids. This chemical reaction was used to prepare 16 alpha-3H-dehydroepiandrosterone (I) and 16 alpha-3H-estrone acetate (XI) from 16 alpha-bromo-dehydroepiandrosterone (X) and from 16 alpha-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16 alpha-3H-androstenedione (II) and 16 alpha-3H-estradiol-17 beta (VII). 16 alpha-3H-Estrone (VI) was obtained by the chemical hydrolysis of 16 alpha-3H-estrone acetate. The label distribution as determined by microbiological 16 alpha-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16 alpha position. These substrates can be used for measuring the 16 alpha hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).

Cutaneous cholesterol assay using a micro-method and punch-biopsy in the detection of early arteriosclerosis.

Two methods of skin biopsy and cholesterol assay are compared. Skin cholesterol assay after punch-biopsy (micro-method) is better than the macro-method: skin cholesterol assay after surgical removal of a sample. Skin biopsy which was shown to give information on the arterial wall thus becomes an easily accessible routine technique.

Measurement of progesterone and pregnenolone 16alpha-hydroxylase activities by a tritium-exchange method.

A new isotropic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group, has been developed for the measurement of progesterone and pregnenolone-16alpha-hydroxylase activity. The incubation medium consists of a phosphate buffer (pH 7; 150 mM), NADPH (1 mM), nicotinamide (10 mM) and magnesium chloride (4 mM). Tween-80 (1 mg/ml) is used to solubilize saturating concentrations of [16-3H]progesterone (200 micronM) or [16-3H]-pregnenolone (50 micronM). The microsomal fraction isolated from a male rat liver is used as the enzymic source. The enzymatically released tritium is recovered in the incubation medium as molecules of tritiated water which are distilled under reduced pressure. The amount of radioactivity present in the water exactly reflects the 16alpha-hydroxylase activity. The method is easy to perform and is completely independent of any further metabolism of the 16alpha-hydroxylated products.

Determination of papaverine in blood samples by gas-liquid chromatography and mass fragmentography.

The measurement of papaverine in blood samples by using either a glass capillary column with a flame-ionization detector or a packed column with mass fragmentographic detection is described. The two methods permit the determination of the normal range of concentrations of papaverine in blood (2-500 ng/ml). Owing to its high specificity, mass fragmentography is greatly superior to capillary chromatography, which is sometimes subject to interferences by solvent impurities.