Time course and dynamics of adipose tissue development in obese and lean Zucker rat pups.

OBJECTIVE: To evaluate the ontogeny of adipose tissue dynamics in obese and lean Zucker rat pups, from suckling to puberty. METHODS: The trial had a two-group parallel design. Sixty-two male Zucker rat pups shared within 15 litters received deuterated water for 5 days, prior killing at different age. Adipose tissues were collected for (2)H-enrichment analyses using mass spectrometry to determine fat cell proliferation and lipid synthesis rates. Rats were assigned to obese and lean rat groups by genotyping. RESULTS: The time course (from days 13 to 55) of all adipose tissue growth showed that the highest fractional rates of fat cell proliferation, triacylglycerol (TG) synthesis and de novo lipogenesis (DNL) took place during early suckling in all rat pups. The appearance of excessive fat mass growth in the obese rats, as compared with lean rats, was first shown through a significant increase in DNL at the end of suckling (P<0.05). The TG synthesis rate was enhanced (P<0.05) from the end of suckling and early postweaning until day 55 (from 122+/-10 to 498+/-78 in obese pups and from 25+/-6 to 75+/-26 mg new TG per day in lean pups (median+/-s.e.m., P<0.01)). In contrast, only by day 55 did the fractional proliferation rate of fat cells in retroperitoneal and epididymal depots in the obese rats supersede that of the lean rats (P<0.05). CONCLUSION: The early suckling period constitutes the most active period for adipose tissue development in normal rats. In the obese Zucker rat model, adipose hypertrophy primarily contributes to the early onset of obesity, while hyperplasia increases after puberty. Early onset of adipose tissue growth may play a determinant role in the development of obesity later in life.

High-throughput simultaneous determination of plasma water deuterium and 18-oxygen enrichment using a high-temperature conversion elemental analyzer with isotope ratio mass spectrometry.

This paper presents a high-throughput method for the simultaneous determination of deuterium and oxygen-18 (18O) enrichment of water samples isolated from blood. This analytical method enables rapid and simple determination of these enrichments of microgram quantities of water. Water is converted into hydrogen and carbon monoxide gases by the use of a high-temperature conversion elemental analyzer (TC-EA), that are then transferred on-line into the isotope ratio mass spectrometer. Accuracy determined with the standard light Antartic precipitation (SLAP) and Greenland ice sheet precipitation (GISP) is reliable for deuterium and 18O enrichments. The range of linearity is from 0 up to 0.09 atom percent excess (APE, i.e. -78 up to 5725 delta per mil (dpm)) for deuterium enrichment and from 0 up to 0.17 APE (-11 up to 890 dpm) for 18O enrichment. Memory effects do exist but can be avoided by analyzing the biological samples in quintuplet. This method allows the determination of 1440 samples per week, i.e. 288 biological samples per week.

Determination of cholesterol absorption in humans: from radiolabel to stable isotope studies.

Hypercholesterolemia is a major health risk. Dietary cholesterol absorption is one important factor affecting levels of plasma and tissue cholesterol. Considerable effort has thus been devoted to develop reliable in vivo clinical methodologies to determine dietary cholesterol absorption in humans. The present paper summarises radiolabelled experiments and major advances in stable isotope technologies to determine cholesterol absorption. Initially, direct methods employing gastro-intestinal intubation were developed. Later, indirect methods using oral-faecal cholesterol balance permitted calculation of cholesterol mass absorption. Once the use of radiolabelled [3H, 14C]cholesterol balance was developed in healthy humans, it was finally possible to distinguish exogenous and endogenous cholesterol. Non-invasive and safer stable isotope (2H, 13C, 18O) labelled cholesterol tracers then replaced radioisotopes for use in infants and adults. Stable isotopes and radioisotopes showed identical cholesterol kinetics. The most promising contemporary stable isotope assessment of cholesterol absorption is a dual stable isotope dual tracer approach based on simultaneous administration of oral and intravenous differentially labelled cholesterol tracers, followed by plasma sampling for 3-4 d. Online GC/Combustion/IRMS and GC/Pyrolysis/IRMS allow minimal amounts of dual stable isotope cholesterol tracers to be detected. Using the dual stable isotope dual tracer approach, the percent cholesterol absorption in adult volunteers has been determined to be 50-70%.

Effects of non-esterified stanols in a liquid emulsion on cholesterol absorption and synthesis in hypercholesterolemic men.

UNLABELLED: BACKGROUND Numerous studies have shown that dietary plant sterols (phytosterols and phytostanols) and their esters can decrease cholesterol absorption. However, few researchers have examined the effects of plant sterols on cholesterol absorption and synthesis using stable isotope tracers, instead of relying on endogenous pathway precursors. Further, we have worked with non-esterified lecithin-solubilized stanols as opposed to the more frequently studied esterified sterols and stanols. The vehicle was an oil-in-water liquid emulsion rather than the more common spread vehicle typically employed. AIM OF THE STUDY: To determine the effects of relatively low doses of lecithin-solubilized non-esterified stanols in liquid emulsions on cholesterol absorption and synthesis in mildly hypercholesterolemic subjects. METHODS: In a randomized, double blind crossover design, 12 mildly hypercholesterolemic men received either a free phytostanol supplement (3 g/d in 3 servings) or a control treatment for 3 days. Cholesterol endogenous synthesis rate was determined using the rate of incorporation of deuterium from body water into newly formed cholesterol molecules. Cholesterol absorption at the intestinal level was determined using the dual isotope method using 13C cholesterol injected intravenously and 180 cholesterol given orally. RESULTS: Cholesterol absorption was 55.7 +/- 6.5 % for the control and 33.5 +/- 5.3% for the phytostanol treatment. This massive reduction of the cholesterol absorption did not induce, on average, a difference in cholesterol endogenous synthesis which was measured at 0.074 +/- 0.0015 pool/d for plant sterols and 0.0736 +/- 0.0015 pool/d for controls (p > 0.05). CONCLUSIONS: The results demonstrated that lecithin-solubilized stanols administrated during a short period of time (3 days) in an oil-in-water emulsion can dramatically decrease cholesterol absorption, without a consistent, concomitant increase in synthesis, which is highly suggestive of effective LDL cholesterol lowering. The effects of synthesis should be verified in a longer study with more subjects.

Tandem mass spectrometric accurate mass performance of time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry: a case study with pyridine derivatives.

The interpretation of mass spectra is a key process during compound identification, and the combination of tandem mass spectrometry (MS/MS) with high-accuracy mass measurements may deliver crucial information on the identity of a compound. Obtaining accurate mass data of fragment ions in MS/MS reveals the particular problem of mass calibration when a lockmass, which is frequently used to obtain accurate masses in MS, is absent. An alternative technique is to recalibrate the MS/MS spectrum using a reference MS/MS spectrum acquired under the same conditions. We have tested and validated this approach using a hybrid quadrupole/orthogonal acceleration reflectron-type time-of-flight (TOF) mass spectrometer. The results were compared with those obtained under similar conditions on a Fourier transform ion cyclotron resonance (FT-ICR) instrument. We found that the mass accuracy observed with such an "external" recalibration on the TOF instrument in MS/MS is identical to what can be obtained on a similar instrument operating in one-dimensional MS mode using the lockmass technique. However, mass accuracy in both cases is one order of magnitude inferior to that obtained using FTMS, and also inferior to that observed using sector field MS when operated at comparable resolution. Nevertheless, for small (<200 Da) molecules, this mass accuracy was still sufficient to have the "true" elemental composition identified as the first hit in about 70% of all cases. It was possible to elucidate the fragmentation mechanism of eight azaheterocycles containing a pyridine moiety, where the accurate mass data from the TOF instrument allowed distinction between two alternative fragmentation pathways.

Chromatographic method for diaminopimelic acid detection in calcareous rocks. Presence of a bacterial biomarker in stromatolites.

The presence in the environment of diaminopimelic acid (DAP), a specific eubacterial marker, can be attributed to that of bacteria. We report a reliable and highly sensitive method for the quantification of DAP in calcareous rocks. It consists of acid hydrolysis of rock powder, purification of DAP by chromatography on Dowex 50W and Spherogel AA-NA+ columns, and quantitative analysis by high-performance liquid chromatography. Addition of tritiated DAP, the internal standard, allows one to follow the relevant fractions throughout the purification procedure and to determine their yield. The analytical step consists in pre-column derivatization with ortho-phthaldialdehyde of purified samples, and separation through a reversed-phase C18 column. Chemical controls, i.e., oxidation of samples to rule out the presence of co-eluting lanthionine and cystathionine, as well as mass spectrometry, confirm the presence of DAP in analyzed samples. Our method allows the separation of meso- from L- and/or D-stereoisomers of DAP, and reveals their presence in the examined rocks, two stromatolites of different age and geographic origin.

Practical approach to archival and retrieval of analytical data in the laboratory.

Today's analytical laboratory uses a large number of different instruments that are connected in networks. Together with increasing automation, data are produced at a rate that can easily reach gigabytes per month, which generates the problem of systematic archival. In addition, working under Good Laboratory Practice requires that archival of raw data be performed in such a way that they can be readily retrieved upon request, even years later. While systematic archival of data is already performed in most laboratories, it is the retrieval of saved information that is often far from straightforward. This paper describes a simple but systematic approach for both archival and retrieval of data files and related electronic documents. It consists of an unambiguous scheme for the naming of electronic files, an efficient backup strategy, a simple database holding information about any data acquired, and a convenient interface to this database that can be accessed from any workplace while assuring restricted access. The system is capable of handling several databases concurrently and is used in our facility to archive data from several workgroups. The use of freely available software such as the Linux operating system made it possible to implement a fast and stable solution at exceptionally low cost.

Quantification of key odorants formed by autoxidation of arachidonic acid using isotope dilution assay.

Six odor-active compounds generated by autoxidation of arachidonic acid (AA) were quantified by isotope dilution assay (IDA), i.e., hexanal (1), 1-octen-3-one (2), (E,Z)-2,4-decadienal (3), (E,E)-2,4-decadienal (4), trans-4,5-epoxy-(E)-2-decenal (5), and (E,Z,Z)-2,4,7-tridecatrienal (6). Compound 1 was the most abundant odorant with about 700 mg/100 g autoxidized AA, which corresponds to 2.2 mol% yield. Based on the odor activity values (ratio of concentration to odor threshold), odorants 3 (fatty) and 5 (metallic) showed the highest sensory contribution followed by 1 (green), 2 (mushroom-like), 6 (egg white-like), and 4 (fatty). For the first time, reliable quantitative results are reported for odorants 1-6 in autoxidized AA, in particular odorant 6, which is a characteristic compound found in autoxidized AA. Synthesis of deuterated 6, required for IDA, is described in detail. The formation of odorants 1-6 by autoxidation of AA is discussed with respect to the quantitative data.

Simultaneous assessment of cholesterol absorption and synthesis in humans using on-line gas chromatography/ combustion and gas chromatography/pyrolysis/isotope-ratio mass spectrometry.

A number of dietary components and drugs are known to inhibit the absorption of dietary and biliary cholesterol, but at the same time can compensate by increasing cholesterol synthesis. It is, therefore, necessary to have a convenient and accurate method to assess both parameters simultaneously. Hence, we validated such a method in humans using on-line gas chromatography(GC)/combustion and GC/pyrolysis/isotope-ratio mass spectrometry (IRMS). Cholesterol absorption was measured using the ratio of [(13)C]cholesterol (injected intravenously) to [(18)O]cholesterol (administered orally). Simultaneously, cholesterol synthesis was measured using the deuterium incorporation method. Our methodology was applied to 12 mildly hypercholesterolemic men that were given a diet providing 2685 +/- 178 Kcal/day (mean +/- SD) and 255 +/- 8 mg cholesterol per day. Cholesterol fractional synthesis rates ranged from 5.0 to 10.5% pool/day and averaged 7.36% +/- 1.78% pool/day (668 +/- 133 mg/day). Cholesterol absorption ranged from 36.5-79.9% with an average value of 50.8 +/- 15.4%. These values are in agreement with already known data obtained with mildly hypercholesterolemic Caucasian males placed on a diet similar to the one used for this study. However, our combined IRMS method has the advantage over existing methods that it enables simultaneous measurement of cholesterol absorption and synthesis in humans, and is therefore an important research tool for studying the impact of dietary treatments on cholesterol parameters.

Acetate, propionate and butyrate in plasma: determination of the concentration and isotopic enrichment by gas chromatography/mass spectrometry with positive chemical ionization.

This study describes a rapid and simple method to determine short-chain fatty acid (SCFA) concentrations and their isotopic enrichments (M(0) + 1 and M(0) + 2) in human plasma. Sample preparation involves SCFA extraction and derivatization with 1-(tert-butyldimethylsilyl)imidazole. Gas chromatography/mass spectrometry was performed using chemical ionization with ammonia as the reagent gas. Outstanding resolution, excellent linearity and good detection limits were obtained. Inter-assay and intra-assay repeatability was below 10% and 3% respectively for SCFA concentration. Inter-assay repeatability was below 5%, 4%, 6%, and 14% for isotopic enrichment determination of [1-(13)C]acetate and [1,2-(13)C(2)]acetate, [1-(13)C]propionate and [1-(13)C]butyrate respectively, with intra-assay being below 6%. Such SCFA concentrations and isotopic enrichments were determined in the plasma of rats infused with a (13)C-labeled SCFA. The turnovers of acetate, propionate and butyrate in rats were 19 micromol kg(-1) min(-1), 2.6 micromol kg(-1) min(-1), 0.3 micromol kg(-1) min(-1) respectively.

Analysis of steryl esters in cocoa butter by on-line liquid chromatography-gas chromatography.

On-line liquid chromatography-gas chromatography (LC-GC) has been applied to the analysis of steryl esters in cocoa butter. Separation of the steryl esters was achieved after on-line transfer to capillary GC. HPLC removes the large amount of triglycerides and pre-separates the components of interest, thus avoiding time-consuming sample preparation prior to GC analysis. The identities of the compounds were confirmed by GC-MS investigation of the collected HPLC fraction and by comparison of the mass spectra (chemical ionization using ammonia as ionization gas) to those of synthesized reference compounds. Using cholesteryl laurate as internal standard, steryl esters were quantified in commercial cocoa butter samples, the detection limit being 3 mg/kg and the quantification limit 10 mg/kg, respectively. Only slight differences in percentage distributions of steryl esters depending on the geographical origin of the material were observed. The patterns were shown to remain unchanged after deodorization. The method described might be a valuable tool for authenticity assessment of cocoa butter.

Identification of potent odorants formed by autoxidation of arachidonic acid: structure elucidation and synthesis of (E,Z,Z)-2,4,7-tridecatrienal.

The aroma composition of autoxidized arachidonic acid was characterized by aroma extract dilution analysis. The most potent odorant was trans-4,5-epoxy-(E)-2-decenal followed by 1-octen-3-one, (E,Z)-2,4-decadienal, (E,Z,Z)-2,4,7-tridecatrienal, (E,E)-2,4-decadienal, and hexanal. (E,Z,Z)-2,4,7-Tridecatrienal was unequivocally identified by mass spectrometry and nuclear magnetic resonance (NMR) data. The stereochemistry of its extended double-bond system was elucidated on the basis of NMR measurements. The target compound was synthesized in four steps starting with bromination of 2-octyn-1-ol, followed by copper-catalyzed coupling of the bromide with ethylmagnesium bromide and (E)-2-penten-4-yn-1-ol. Partial hydrogenation of the resulting C(13)-compound with triple bonds in the positions C-4 and C-7 gave rise to (E,Z,Z)-2,4,7-tridecatrien-1-ol, which was finally oxidized to the target compound. It exhibits a typical egg-white-like, marine-like odor at low concentrations, and an intense orange-citrus, animal-like odor at higher concentrations. Its odor threshold was estimated by gas chromatography-olfactometry to be 0.07 ng/L air, which is of the same order of magnitude as that reported for 1-octen-3-one and (E,E)-2,4-decadienal.

Improved method to track chlorophyll degradation.

An analytical method capable of identifying >30 chlorophyll-related compounds in plant extracts has been developed. The method employs liquid chromatography coupled to UV-vis, MS, and MS/MS detection. It can be applied without modification to analyze natural chlorophyll degradation products and other metalloporphyrines. It was successfully applied to identify chlorophyll derivatives found in rehydrated spinach powder and conventionally canned and Veri-Green-processed beans. In the Veri-Green-processed beans several degradation products were identified that are zinc-containing analogues to the chlorophyll derivatives found in vegetables after conventional canning. They have been characterized by liquid chromatography and mass spectrometry.

Selenium absorption and retention from a selenite- or selenate-fortified milk-based formula in men measured by a stable-isotope technique.

The present study was designed to determine the apparent absorption and retention of the inorganic Se compounds SeO3(2-) and SeO4(2-), which are commonly used for Se fortification of clinical nutrition products and infant formulas. Ten healthy men were fed a milk-based formula labelled with 40 microg Se as 74SeO3(2-) or 76SeO4(2-) on two consecutive days using a randomised crossover design. Se stable-isotope analysis of 9 d complete collections of urine and faeces was used to calculate apparent Se absorption and retention. Se retention from 74SeO3(2-) (41.0 (SD 8.4) %) and from 76SeO4(2-) (46.0 (SD 7.9) %) was not significantly different (P > 0.05). However, Se absorption was significantly higher from SeO4(2-) than from SeO3(2-) (91.3 (SD 1.4) % v. 50.2 (SD 7.8) %, P < 0.05). Urinary excretion of the administered dose was 9.2 (SD 1.8) % for 74SeO3(2-) and 45.3 (SD 8.2) % for 76SeO4(2-) (P < 0.05). Urinary Se excretion kinetics differed significantly for the two Se compounds; 90 % of the total urinary Se was excreted after 121 h for 74SeO32- and after 40 h for 76SeO42- These results suggest that although Se absorption and urinary excretion differ for SeO3(2-) and SeO4(2-), both Se compounds are equally well retained when administered at a relatively low dose (40 microg Se). The nutritional impact of Se fortification of foods would thus be expected to be similar when SeO4(2-) or SeO3(2-) are used.

Metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in human hepatocytes: 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid is a major detoxification pathway catalyzed by cytochrome P450 1A2.

Metabolic pathways of the mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) remain incompletely characterized in humans. In this study, the metabolism of MeIQx was investigated in primary human hepatocytes. Six metabolites were characterized by UV and mass spectroscopy. Novel metabolites were additionally characterized by 1H NMR spectroscopy. The carcinogenic metabolite, 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline, which is formed by cytochrome P450 1A2 (P450 1A2), was found to be transformed into the N(2)-glucuronide conjugate, N(2)-(beta-1-glucosiduronyl)-2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline. The phase II conjugates N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid and N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, as well as the 7-oxo derivatives of MeIQx and N-desmethyl-MeIQx, 2-amino-3,8-dimethyl-6-hydro-7H-imidazo[4,5-f]quinoxalin-7-one (7-oxo-MeIQx), and 2-amino-6-hydro-8-methyl-7H-imidazo[4,5-f]quinoxalin-7-one (N-desmethyl-7-oxo-MeIQx), thought to be formed exclusively by the intestinal flora, were also identified. A novel metabolite was characterized as 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), and it was the predominant metabolite formed in hepatocytes exposed to MeIQx at levels approaching human exposure. IQx-8-COOH formation is catalyzed by P450 1A2. This metabolite is a detoxication product and does not induce umuC gene expression in Salmonella typhimurium strain NM2009. IQx-8-COOH is also the principal oxidation product of MeIQx excreted in human urine [Turesky, R., et al. (1998) Chem. Res. Toxicol. 11, 217-225]. Thus, P450 1A2 is involved in both the metabolic activation and detoxication of this procarcinogen in humans. Analogous metabolism experiments were conducted with hepatocytes of untreated rats and rats pretreated with the P450 inducer 3-methylcholanthrene. Unlike human hepatocytes, the rat cell preparations did not produce IQx-8-COOH but catalyzed the formation of 2-amino-3,8-dimethyl-5-hydroxyimidazo[4,5-f]quinoxaline as a major P450-mediated detoxication product. In conclusion, our results provide evidence of a novel MeIQx metabolism pathway in humans through P450 1A2-mediated C(8)-oxidation of MeIQx to form IQx-8-COOH. This biotransformation pathway has not been detected in experimental animal species. Considerable interspecies differences exist in the metabolism of MeIQx by P450s, which may affect the biological activity of this mutagen and must be considered when assessing human health risk.

A high-performance liquid chromatography method for the detection of diaminopimelic acid in urine.

We describe a quantitative assay for diaminopimelic acid (DAP) in urine. It involves (i) hydrolysis of urine samples, (ii) purification by several different liquid chromatography steps, and (iii) analysis by high-performance liquid chromatography on a reversed-phase C18 column. Tritiated-DAP, the internal standard, allows one to precisely follow the DAP-containing fractions and to determine the yield during purification. Sensitive and relatively accurate quantification of DAP, with a threshold of 50 fmol, is based on ion-pairing properties of eluants and ortho-phthaldialdehyde derivatization. The presence of DAP in relevant fractions was confirmed by combined gas chromatography and mass spectrometry. The DAP concentration in adult human urine pooled over 24 h ranges from 0.69 to 2.01 microM, a result in fair agreement with previously published values obtained by ninhydrin derivatization or gas chromatography.

Synthesis of deuterated volatile lipid degradation products to be used as internal standards in isotope dilution assays. 1. Aldehydes.

The isotopically labeled compounds [5,6-(2)H(2)]hexanal (d-I), [2, 3-(2)H(2)]-(E)-2-nonenal (d-II), [3,4-(2)H(2)]-(E,E)-2,4-nonadienal (d-III), and [3,4-(2)H(2)]-(E,E)-2,4-decadienal (d-IV) were prepared in good yields using new or improved synthesis procedures. Labeling position, chemical purity, and isotopic distribution of the compounds were characterized by various MS and NMR techniques. These molecules are used as internal standards in quantification experiments based on isotope dilution assay. Synthesis of d-I, d-III, and d-IV has not yet been reported in the literature.

Synthesis of deuterated volatile lipid degradation products to be used as internal standards in isotope dilution assays. 2. Vinyl ketones.

The isotopically labeled compounds [5,6-(2)H(2)]-(Z)-1, 5-octadien-3-one (d-I) and [1-(2)H(1;2),2-(2)H(1;1)]-1-octen-3-one (d-II), as well as the unlabeled reference compound (Z)-1, 5-octadien-3-one (I) were prepared by improved synthesis procedures. Labeling position, chemical purity, and isotopic distribution of the compounds were characterized by various MS and NMR techniques. These molecules are used as internal standards in quantification experiments based on isotope dilution assay. The newly prepared compound d-II was synthesized in a simple two-step procedure, and formation of the main isotopomers was studied in model systems.