Direct measurement of apolipoprotein B synthesis in human very low density lipoprotein using stable isotopes and mass spectrometry

Stable isotope methodology has been adapted to the study of lipoprotein turnover in human subjects. Using endogenous [15N]glycine labeling and gas-liquid chromatographic-mass spectrometric analysis, synthesis of apolipoprotein B in very low density lipoprotein (VLDL) was measured directly in five normal and two hyperlipidemic subjects. An isotopic precursor steady state was achieved during the studies by utilizing a priming dose and constant infusion containing [15N]glycine. Measurement of the plateau in 15N enrichment in the urinary hippurate produced during each study was used to estimate the 15N enrichment of the hepatic glycine precursor pool. The range of values for the fractional synthetic rate of VLDL apoB in the normal subjects obtained by this method was 5.9 to 11.5 day-1, with a mean of 9.2 +/- 2.4 (SD). This value agrees with the results of previous investigations which have utilized other methods. The method was also tested in two hypertriglyceridemic subjects and gave fractional synthetic rates of VLDL apoB that were significantly lower than in normals (1.5 and 2.8 day-1). This stable isotope method allows calculation of the fractional synthetic rate of VLDL apoB by maintaining an isotopic steady state throughout the study. It makes possible repeated studies in the same individual since no risk of exposure to radioisotopes is involved.     

Omega-cyclohexyl fatty acids in acidophilic thermophilic bacteria. Studies on their presence, structure, and biosynthesis using precursors labeled with stable isotopes and radioisotopes.

Omega-Cyclohexyl undecanoic acid and omega-cyclohexyl tridecanoic acid were found in 10 strains of acido-thermophilic bacteria isolated from different Japanese hot springs. These unusual fatty acids were found in the esterified form in glyceride type complex lipids and constituted 74 to 93% of the total fatty acids in the bacteria. The fatty acids other than omega-cyclohexyl fatty acids found were 14-methyl hexadecanoic acid (3 to 15%) and 15-methyl hexadecanoic acid (1 to 6%), and trace amounts of straight chain and methyl-branched tetra- and penta-decanoic acids. Biosynthesis of omega-cyclohexyl fatty acids increased with increase in the concentration of glucose in the culture medium. The metabolism of omega-cyclohexyl fatty acids was studied using deuterium-labeled precursors by mass fragmentation analysis. The deuterium of [2-D]glucose was specifically incorporated into position 2 of the cyclohexyl ring of the fatty acids, indicating that the ring was synthesized from the glucose molecule. Radioactivity was efficiently incorporated into the omega-cyclohexyl fatty acids from labeled glucose, shikimate, and cyclohexyl carboxylate. These findings indicate that omega-cyclohexyl fatty acids are synthesized with glucose through shikimic acid and probably cyclohexyl carboxylyl-CoA derivative as the intermediates.     

Determination of amino sugars and amino acids in glycoconjugates using precolumn derivatization with o-phthalaldehyde.

This report examines the RP-HPLC separation of o-phthalaldehyde derivatives of amino acids, amino sugars, and amino sugar alcohols using either 2-mercaptoethanol or 3-mercaptopropionic acid. A method with pmol sensitivity for the analysis of N-acetylamino sugars of glycoconjugates was elaborated. Upon hydrolysis, amino sugars are reduced with borohydride. Automated precolumn derivatization and chromatographic conditions for the resulting hexosaminitols are the same as those used for the analysis of amino acids. The method has been tested with as little as 2 micrograms of bovine fetuin, with a glycopeptide from bromelain and with an oligosaccharide after periodate oxidation.     

Quantitative proteomics using stable isotope labeling with amino acids in cell culture

Stable isotope labeling with amino acids in cell culture (SILAC) is a simple in vivo labeling strategy for mass spectrometry-based quantitative proteomics. It relies on the metabolic incorporation of nonradioactive heavy isotopic forms of amino acids into cellular proteins, which can be readily distinguished in a mass spectrometer. As the samples are mixed before processing in the SILAC methodology, the sample handling errors are also minimized. Here we present protocols for using SILAC in the following types of experiments: (i) studying inducible protein complexes, (ii) identification of Tyr kinase substrates, (iii) differential membrane proteomics and (iv) studying temporal dynamics using SILAC 5-plexing. Although the overall time is largely dependent on the rate of cell growth and various sample processing steps employed, a typical SILAC experiment from start to finish, including data analysis, should take anywhere between 20 and 25 d.     

Analysis of lung surfactant phosphatidylcholine metabolism in transgenic mice using stable isotopes

Stable isotope labelling of lipid precursors coupled with mass spectrometry-based lipidomic analyses and determination of isotope enrichment in substrate, intermediate and product pools provide the parameters needed to determine absolute flux rates through lipid pathways in vivo. Here, as an illustration of the power of such analyses we investigated lung phosphatidylcholine (PC) synthesis in Surfactant Protein-D (SP-D) null mice. These animals develop emphysema, foamy alveolar macrophages and an alveolar lipoproteinosis with increasing age. We used the incorporation of methyl-9-[²H] choline chloride coupled with ESI-MS/MS to quantify absolute rates of lung surfactant PC synthesis and secretion in an SP-D^−/− mouse model, together with an analysis of the molecular specificity of lung PC synthesis. PC synthetic rates were comparable in control (0.52 μmol/lung/h) and SP-D^−/− (0.69 μmol/lung/h) mice, as were rates of surfactant PC secretion (29.8 and 30.6 nmol/lung/h, respectively). Increased lung PC in the SP-D^−/− mouse was due to impaired catabolism, with a rate of accumulation of 0.057 μmol/lung/h. The relatively low rates of surfactant PC secretion compared with total lung PC synthesis were compatible with a suggested ABCA1-mediated basolateral lipid efflux from alveolar type II epithelial cells. Finally, PC molecular species analysis suggested that a proportion of newly synthesised PC is secreted rapidly into the lung air spaces in both control and SP-D^−/− mice before significant PC acyl remodelling occurs.     

Chiroptical studies of fluorescamine labeled amino acids

Absorption and circular dichroism studies of fluorescamine condensation products with α-amino acids, dipeptides and phenylethylamine in the 300–450 nm region are reported. The results make a major revision of the previously suggested rule for absolute configuration determination necessary.     

Preparative-scale isolation of isotopically labeled amino acids

Over 10 g of individual ²H, ¹⁵N-labeled amino acids was resolved and recovered on a laboratory-scale ion-exchange system from a crude bacterial protein hydrolyzate derived from 20 g of lyophilized cells. The 17 amino acids (cystine was not isolated) were recovered containing less than 1.0% of other contaminating amino acids except for proline (4.0%). The aromatic and basic amino acids were isolated on a dual-column carrier displacement system (390-ml resin bed volume) while most of the neutral and acidic amino acids were separated on a pyrazolium chloride elution system (560-ml resin bed volume). The two remaining overlapping pairs were resolved on small carrier displacement columns. In addition, the overlapping fractions from adjacent peaks of the pyrazolium chloride elution system represent only 3.5% (0.37 g) of the total sample.     

Determination of food sources of marine invertebrates from a subtidal sand community using analyses of fatty acids and stable isotopes

The fatty acid compositions and stable isotope ratios of carbon, nitrogen, and sulfur were analyzed in the bivalve mollusks Mactra chinensis, Pandora pulchella, Felaniella usta, and Megangulus zyonoensis, the polychaete Chaetopterus cautus, and the main sources of organic matter in a subtidal sand bottom community in Vostok Bay (Sea of Japan). The fatty acid composition and stable isotope ratios of some bivalves is likely to be indicative of substantial inputs from benthic microalgae and an important role of microbial food chains. Only the filter-feeding polychaete C. cautus showed similarity in these characteristics to suspended particulate organic matter synthesized by phytoplankton. It is suggested that the contribution of benthic microalgae to the diet of a consumer organism, inferred solely from the carbon stable isotope analysis, can be significantly overestimated.     

Determination of amino acids in saliva using capillary electrophoresis with fluorimetric detection

In the present study a sensitive method for the quantification of main free amino acids in saliva using capillary electrophoresis with laser induced fluorescence detection was developed. As background electrolyte 20 mM borate buffer pH 9.5 was used. Amino acids were separated after derivatization were optimized. The main amino acids occurring in saliva (Pro, Ser, Gly and Glu) were separated in less than 7 min. The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.1 to 2.4 nM. The developed method was employed for determination of amino acids in real saliva samples.     

Determination of specific radioactivity of plant amino acids using dansylation

A sensitive procedure is described for measuring the absolute specific activity of all the protein amino acids, both free and in total protein, as well as of some nonprotein amino acids. It is illustrated with developing pea cotyledons. Particular attention is paid to optimization of tlc loading and resolution, protection of cysteine, and stabilization of fluorescence of the eluted derivatives.     

Experimental concerns in tracer kinetic investigations of apolipoprotein metabolism

The complexity of the metabolism of the plasma lipoproteins makes it impossible to integrate the details of the reactions of specific apolipoproteins and their associated lipids without the use of computerized modeling methods. Because apolipoproteins impart specificity in the transport and chemical processing of plasma lipids, they have been the focus of many in vivo kinetic tracer investigations. The analysis of such kinetic data by modeling techniques has provided important advances in understanding lipoprotein metabolism. An example is the Delipidation Chain, an hypothesis explaining VLDL metabolism in terms of a sequential delipidation process. As a consequence of the advance in knowledge of apolipoprotein structure and metabolism, coupled with progress in computerized modeling of large systems, it has become important to refine the design of in vivo tracer kinetic investigations of the apolipoproteins. Considerations of particular importance include the selection of apolipoprotein tracers which can be shown to undergo the same reactions as the apolipoproteins whose metabolism they trace. If the physical and chemical processes which convert apolipoproteins from one metabolic pool to another are to be analyzed correctly, it is necessary to describe precisely and to measure accurately these pools. Current methods for delineating metabolic pools of apolipoproteins in vivo need to be refined. When accomplished, this will provide new opportunities to investigate the metabolic pathways of the apolipoproteins and their associated lipids. A very important challenge is to design experiments which will differentiate transfer processes, which result in net transport of a reactant, from exchange processes, whereby a tracer and a tracee are exchanged between pools without a net transport event occuring. Since both types of processes occur readily with apolipoproteins, it is important to develop methods to examine them separately. Computerized kinetic modeling provides a means for describing and understanding the complexities of lipoprotein metabolism. A major challenge is for the experimentalist to acquire data which accurately reflect the physiological processes involved in lipoprotein metabolism.     

A food web analysis of the juvenile blue crab, Callinectes sapidus, using stable isotopes in whole animals and individual amino acids

The stable isotope compositions (C and N) of plants and animals of a marsh dominated by Spartina alterniflora in the Delaware Estuary were determined. The study focused on the juvenile stage of the Atlantic blue crab, Callinectes sapidus, and the importance of marsh-derived diets in supporting growth during this stage. Laboratory growth experiments and field data indicated that early juvenile blue crabs living in the Delaware Bay habitat fed primarily on zooplankton, while marsh-dwelling crabs, which were enriched in ¹³C relative to bay juveniles, utilized marsh-derived carbon for growth. In laboratory experiments, the degree to which juvenile blue crabs isotopically fractionated dietary nitrogen, as well as the growth rate, depended on the protein quality of the diet. The range of δ¹³C of amino acids in laboratory-reared crabs and their diets was almost 20‰, similar to the isotopic range of amino acids of other organisms. In laboratory studies, the δ¹³C of nonessential and essential amino acids in the diet were compared to those in juvenile crabs. Isotopic fractionation at the molecular level depended on diet quality and the crabs' physiological requirements. Comparison of whole-animal isotope data with individual amino acid C isotope measurements of wild juvenile blue crabs from the bay and marsh suggested a different source of total dietary carbon, yet a shared protein component, such as zooplankton. Received: 1 July 1998 / Accepted: 15 March 1999     

Determination of stable molybdenum isotopes by thermionic mass spectrometry for tracer studies of molybdenum plant metabolism

A tracer technique based on multiple stable Mo isotopes and thermionic quadrupole mass spectrometry for isotopic analysis of plant tissue was developed and has been applied in the long-term study of foliar absorption of Mo by potato plants. As several tracers have been used the multivariate linear regression methods has been applied to calculate the portions of tracer Mo present in the potato samples from the isotopic ratios measured and to estimate their reproducibilities.In this paper solely the tracer portions transferred from the leaf into the tuber of the potato have been determined. Dependent on the time of contamination, tracer portions of 0.2 to 12% have been measured, corresponding to a maximum of 2.3% of the foliar application of the tracer molybdate. The reproducibilities of the tracer method as a whole (analytical determination and calculation of the tracer portions in the tracer plant samples) amounted to 7% at the maximum (with one exception); by contrast, the individual differences between the three plants investigated were much larger (up to 80%).     

Esterification of fatty acids using nylon-immobilized lipase in n-hexane: kinetic parameters and chain-length effects.

The esterification of long-chain fatty acids in n-hexane catalyzed by nylon-immobilized lipase from Candida rugosa has been investigated. Butyl oleate (22 carbon atoms), oleyl butyrate (22 carbon atoms) and oleyl oleate (36 carbon atoms) were produced at maximum reaction rates of approximately equal to 60 mmol h(-1) g(-1) immobilized enzyme when the substrates were present in equimolar proportions at an initial concentration of 0.6 mol l(-1). The observed kinetic behavior of all the esterification reactions is found to follow a ping-pong bi-bi mechanism with competitive inhibition by both substrates. The effect of the chain-length of the fatty acids and the alcohols could be correlated to some mechanistic models, in accordance with the calculated kinetic parameters.