Protein profile of exosomes from trabecular meshwork cells.

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.

Effect of sirolimus on the metabolism of apoB100- containing lipoproteins in renal transplant patients.

BACKGROUND: Sirolimus (Rapamune, rapamycin, RAPA) is a potent immunosuppressive drug that has reduced the rate of acute rejection episodes by more than 40% in phase III trials when added to an immunosuppression regimen of cyclosporine (CsA) and prednisone. However, RAPA treatment tends to increase lipid levels, particularly among patients with pre-existing hyperlipidemia. METHODS: To identify the metabolic pathway(s) leading to RAPA-mediated hyperlipidemia, five patients with renal transplants maintained on CsA+/-prednisone+/- azathioprine (AZA) were studied before and after 6 weeks of treatment with RAPA (off RAPA and on RAPA, respectively). Each study patient was infused with a single bolus of [2H4]-lysine to derive metabolic parameters for apoB100-containing lipoproteins by using kinetic analysis based upon quantitation of isotopic enrichment by gas chromatography-mass spectrometry. RESULTS: Serial lipid measurements revealed that four patients displayed increased plasma triglyceride levels after RAPA treatment, which coincided with significantly higher plasma VLDL-apoB100 concentrations (21.7+/-12.1 mg/dl off RAPA vs. 38.7+/-14.8 mg/dl on RAPA, mean+/-SD, P<0.05). Kinetic analysis showed that the RAPA-induced increase in VLDL-apoB100 concentrations was due to a significant reduction in the fractional catabolic rate (FCR) of very low-density lipoprotein (VLDL) apoB100 (0.83+/-0.65 off RAPA vs. 0.24+/-0.10 on RAPA, mean+/-SD, P<0.05), rather than an enhanced VLDL-apoB100 synthesis. In one patient, RAPA treatment induced hypercholesterolemia but not hypertriglyceridemia. This hypercholesterolemia was due to elevated low-density lipoprotein (LDL) cholesterol levels, which coincided with a decreased FCR of LDL-apoB100. Heparin-induced lipoprotein lipase activity was significantly lower in the immunosuppressed hyperlipidemic patients than in normolipidemic controls. However, RAPA treatment did not significantly alter basal lipoprotein lipase activity in renal transplant patients in this study. CONCLUSIONS: This study indicates that for renal transplant patients in whom RAPA treatment induces hyperlipidemia, this effect is the result of reduced catabolism of apoB100-containing lipoproteins.

Analysis of diacyl peroxides by Ag+ coordination ionspray tandem mass spectrometry: free radical pathways of complex decomposition.

Organic peroxides have significance in organic synthesis and biological processes. Characterization of these compounds with weak O-O bonds is sometimes difficult due to their thermal instability and sensitivity to acid or base. Coordination of diacyl peroxides with AgBF4 provides a means for analysis of these compounds by coordination ionspray tandem mass spectrometry (CIS-MS/MS). Precursor ion (Q1) scans of acetyl benzoyl peroxide give two Ag+ adducts, [M + Ag + solvent]+ and [M + Ag + M]+. These silver ion adducts can be selectively dissociated (CID) to give unique structural information about the analyte. Decomposition of the [M + Ag + solvent]+ adduct generates fragmentation products due to apparent homolytic cleavage of the O-O bond followed by decarboxylation of the resultant radicals. The bis-diacylperoxide complex, [M + Ag + M]+ gives CID pathways that involve homolysis of the (O-O bond and free radical cross-coupling of the two diacyl peroxides coordinated to the silver ion, i.e. formation of dibenzoyl peroxide, phenyl benzoate, and biphenyl from acetyl benzoyl peroxide. The observation of free radical CID modes is uncommon in mass spectrometry but these pathways are consistent with well-known solution and gas phase processes for peroxide compounds. The proposed fragmentation pathways have been supported by experiments with (18)O and deuterated substrates. This technique can be applied to analyze diacyl peroxides with different substituents as well.

Quantification of BK1-5, the stable bradykinin plasma metabolite in humans, by a highly accurate liquid-chromatographic tandem mass spectrometric assay.

Bradykinin is a vasoactive nonapeptide involved in cardiorenal physiology and inflammatory states. It has been linked to the pathophysiology of hypertension and diabetes. Correlating levels of bradykinin with disease states has been hampered by its rapid degradation, artifactual production during blood sampling, and nonspecific radioimmunoassay techniques. We previously identified BK1-5 as the stable in vivo plasma metabolite of systemic bradykinin in humans. We now report a sensitive and specific assay method for BK1-5 in human blood utilizing liquid chromatography-tandem mass spectrometry(MS) with electrospray ionization. [(13)C(2),(15)N]Glycine was incorporated into chemically synthesized BK1-5 for use as an internal standard. Blood samples (5 ml) were collected into 15-ml chilled ethanol to prevent artifactual kinin production and degradation. BK1-5 in ethanolic plasma supernatant was purified on a polymeric solid phase extraction cartridge. MS analysis was in the selective reaction monitoring mode. Precision of the assay is +/-7.5% and accuracy is 99%. Recovery of BK1-5 through sample preparation was 43% and the lower limit of detection is 4 fmol/ml blood. Concentrations of BK1-5 in 12 normal volunteers were 44.2 +/- 7.1 fmol/ml blood (mean +/- SE). During blood sampling, no artifactual production of BK1-5 was detected for up to 60 s prior to denaturing the sample. This assay provides the first accurate and precise method using MS to quantify BK1-5 in human blood as a marker for the production of systemic bradykinin in humans.

Neocytolysis on descent from altitude: a newly recognized mechanism for the control of red cell mass.

BACKGROUND: Studies of space-flight anemia have uncovered a physiologic process, neocytolysis, by which young red blood cells are selectively hemolyzed, allowing rapid adaptation when red cell mass is excessive for a new environment. OBJECTIVES: 1) To confirm that neocytolysis occurs in another situation of acute plethora-when high-altitude dwellers with polycythemia descend to sea level; and 2) to clarify the role of erythropoietin suppression. DESIGN: Prospective observational and interventional study. SETTING: Cerro de Pasco (4380 m) and Lima (sea level), Peru. PARTICIPANTS: Nine volunteers with polycythemia. INTERVENTIONS: Volunteers were transported to sea level; three received low-dose erythropoietin. MEASUREMENTS: Changes in red cell mass, hematocrit, hemoglobin concentration, reticulocyte count, ferritin level, serum erythropoietin, and enrichment of administered(13)C in heme. RESULTS: In six participants, red cell mass decreased by 7% to 10% within a few days of descent; this decrease was mirrored by a rapid increase in serum ferritin level. Reticulocyte production did not decrease, a finding that establishes a hemolytic mechanism.(13)C changes in circulating heme were consistent with hemolysis of young cells. Erythropoietin was suppressed, and administration of exogenous erythropoietin prevented the changes in red cell mass, serum ferritin level, and(13)C-heme. CONCLUSIONS: Neocytolysis and the role of erythropoietin are confirmed in persons with polycythemia who descend from high altitude. This may have implications that extend beyond space and altitude medicine to renal disease and other situations of erythropoietin suppression, hemolysis, and polycythemia.

Organization, management and operation of contemporary academic mass spectrometry service facilities.

The rapid evolution of mass spectrometry in the past 15 years has moved mass spectrometry facilities from the traditional model in which instruments were located in and used for a single department's samples to a distributed model servicing entire universities. In this paper we describe two such shared instrument facilities that have evolved from a base in a single department to facilities that service a broad clientele. The Purdue University Campus-wide Mass Spectrometry Center (CWMSC) is a decentralized facility with multiple sites on campus. The CWMSC is a limited-access facility in which samples are run by service facility personnel in close cooperation with investigators. The Vanderbilt University Mass Spectrometry Research Center (VU-MSRC) is a centralized facility in the medical school that provides services to the university at large. The VU-MSRC is an open-access facility in which users are expected to prepare and analyze their own samples under the guidance of a trained operator. Perhaps the most significant benefit achieved by these models has been the minimization of academic barriers and the resultant intellectual cross-fertilization that has greatly enriched research at institutions where this approach has been adopted. The advantages and limitations of both models are discussed in terms of the traditional academic paradigm of service, research and education.

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography-mass spectrometry.

We report here the development and validation of an LC-MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C18 reversed-phase column using an acetonitrile-water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS-MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477-266), DMLOP (m/z 463-->252) and A-LOP (m/z 519-->266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3+/-1.50% (range: 70.7-73.7%) and 79.4+/-12.8% (64.9-88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.

Structural analysis of diacyl peroxides by electrospray tandem mass spectrometry with ammonium acetate: bond homolysis of peroxide-ammonium and peroxide-proton adducts.

Organic peroxides have significant implications in organic chemistry and biological processes. The weak O-O bond makes them extremely difficult to characterize by conventional analytical methods. Diacyl peroxides are one of the major radical sources in polymerization and organic synthesis. It is well known that diacyl peroxides are thermal labile and thus are not amenable to study by gas chromatography/mass spectrometry (GC/MS). Electrospray tandem mass spectrometry (ESI-MS/MS) has been applied to the structural analysis of diacyl peroxides by formation of ammonium adducts. Collision induced dissociation (CID) studies of the ammonium adducts of the peroxide [M + NH(4)](+) give collision energy dependent fragments. For most diacyl peroxides, homolysis of the peroxy bond predominates the fragmentation pathways of the peroxide-ammonium adducts. Deuterated substrates have been employed to provide evidence for typical fragmentation pathways. The CID studies were also used to locate the O-18 in some O-18 specifically labeled diacyl peroxides. For branched alkyl or alkoxy substrates, McLafferty rearrangement and decarboxylation become a major pathway. By comparison with some anhydride analogues, ESI-MS/MS can also be used to study this class of compounds.

Metabolism of bradykinin In vivo in humans: identification of BK1-5 as a stable plasma peptide metabolite.

Studies investigating the role of bradykinin in disease states such as hypertension, sepsis, and asthma have been confounded by difficulties in measuring the concentration of this short-lived peptide. The purpose of this study was to determine a stable metabolite of bradykinin in the systemic circulation of humans. Bradykinin (containing trace concentrations of [(3)H]bradykinin) was administered i.v. into three human volunteers in increasing amounts up to a maintenance rate of 200 ng/kg/min until a total dose of 1 mg was given. Metabolic products were purified and identified by HPLC and by electrospray ionization mass spectrometry. Infused bradykinin was rapidly degraded, such that no exogenous bradykinin was detected in venous plasma sampled during infusion. BK1-5 (Arg-Pro-Pro-Gly-Phe), the 1-to-5 amino acid fragment of bradykinin, was identified as a major stable plasma metabolite of bradykinin. Plasma concentrations of BK1-5 correlated with dose of bradykinin infused and concentrations at the end of bradykinin infusion were 1510 to 4600 fmol/ml of blood. BK1-5 was cleared from blood with a terminal half-life of 86 to 101 min. Thus, in humans, bradykinin is rapidly degraded in vivo to BK1-5, a stable metabolite. Measurement of this metabolite could provide a tool to assess pathophysiologic and pharmacologic alterations in systemic bradykinin generation associated with human disease.

Formation of novel D-ring and E-ring isoprostane-like compounds (D4/E4-neuroprostanes) in vivo from docosahexaenoic acid.

Free radical-mediated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. We have reported that bioactive prostaglandin D2/E2-like compounds, termed D2/E2-isoprostanes, are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid. Docosahexaenoic acid, in contrast to arachidonic acid, is the most abundant unsaturated fatty acid in brain. We therefore questioned whether D/E-isoprostane-like compounds (D4/E4-neuroprostanes) are formed from the oxidation of docosahexaenoic acid. Levels of putative D4/E4-neuroprostanes increased 380-fold after oxidation of docosahexaenoic acid in vitro from 15.2 +/- 6.3 to 5773 +/- 1024 ng/mg of docosahexaenoic acid. Subsequently, chemical approaches and liquid chromatography electrospray ionization tandem mass spectrometry definitively identified these compounds as D4/E4-neuroprostanes. We then explored the formation of D4/E4-neuroprostanes from a biological source, rat brain synaptosomes. Basal levels of D4/E4-neuroprostanes were 3.8 +/- 0.6 ng/mg of protein and increased 54-fold after oxidation (n = 4). We also detected these compounds in fresh brain tissue from rats at levels of 12.1 +/- 2.4 ng/g of brain tissue (n = 3) and in human brain tissue at levels of 9.2 +/- 4.1 ng/g of brain tissue (n = 4). Thus, these studies have identified novel D/E-ring isoprostane-like compounds that are derived from docosahexaenoic acid and that are formed in brain in vivo. The fact that they are readily detectable suggests that ongoing oxidative stress is present in the central nervous system of humans and animals. Further, identification of these compounds provides a rationale for examining their role in neurological disorders associated with oxidant stress.

Hepatic microsomal and peroxisomal docosahexaenoate biosynthesis during piglet development.

The roles of peroxisomes and microsomes on the biosynthetic pathway for docosahexaenoic acid (DHA) from alpha-linolenic acid (ALA) were investigated. Microsomes and peroxisomes were prepared from livers of fetal and neonatal piglets by a combination of differential and gradient layer centrifugation. Microsomes, peroxisomes, and combined cell fractions were incubated with [13C-U]18:3n-3. The [M] and [M + 18] isotopomers of the fatty acids in the long-chain polyunsaturated fatty acid (LCPUFA) n-3 pathway were detected by gas chromatography-mass spectrometry. The quantity of each fatty acid was determined by gas chromatography, and synthesis of each fatty acid was calculated for a 30-min period. Synthesis of DHA was not detected in combined fetal liver fractions. The data suggest that DHA in the fetus is probably supplied from maternal sources through the placenta. In either singly incubated microsomal or peroxisomal preparations from neonatal livers, no DHA synthesis was detected. After combination of the microsomal and peroxisomal fractions, DHA synthesis was evident and increased rapidly between birth and 2 wk of age. This is the first demonstration of the entire biosynthetic LCPUFA n-3 pathway in subcellular organelles starting from isotopically labeled ALA to the final product, DHA, with all the intermediates present and isotopically labeled. The primary importance of the data is that it unequivocally demonstrates that peroxisomes are required for biosynthesis of DHA from ALA.

Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment.

Assessment of free fatty acid (FFA) concentration and isotopic enrichment is useful for studies of FFA kinetics in vivo. A new procedure to recover the major FFA from plasma for concentration and isotopic enrichment measurements is described and validated. The procedure involves extraction of plasma lipids with hexane, methylation with iodomethane (CH(3)I) to form fatty acid methyl esters (FAME), and subsequent purification of FAME by solid phase extraction (SPE) chromatography. The new method was compared with a traditional method using thin-layer chromatography (TLC) to recover plasma FFA, with subsequent methylation by BF(3)/methanol. The TLC method was found to be less reliable than the new CH(3)I method because of contamination with extraneous fatty acids, chemical fractionation of FFA species, and incomplete recovery of FFA associated with TLC. In contrast, the CH(3)I/SPE method was free of contamination, did not exhibit chemical fractionation, and had higher recovery. The iodomethane reaction was specific for free fatty acids; no FAME were formed when esterified fatty acids (triglycerides, cholesteryl esters, phospholipids) were subjected to the methylation reaction. We conclude that the CH(3)I/SPE method provides rapid and convenient recovery of plasma fatty acids for quantification or GC/MS analysis as methyl esters, and is not subject to the problems of contamination, reduced recovery, and chemical fractionation associated with recovery of FFA by TLC.

Quantitation of monosaccharide isotopic enrichment in physiologic fluids by electron ionization or negative chemical ionization GC/MS using di-O-isopropylidene derivatives.

The aldonitrile pentaacetate and other derivatives lack ions in the electron ionization (EI) spectra possessing an intact hexose structure and thus must be analyzed by chemical ionization GC/MS in order to study multiple isotopomers. We report methods for quantitation of hexose di-O-isopropylidene acetate (IPAc) or pentafluorobenzoyl (PFBz) esters. These were prepared in a two-step procedure using inexpensive reagents that do not adversely impact the isotopomer structure of the sugar. The acetate derivative possesses an abundant [M - CH3] ion in the EI spectrum which is suitable for quantitative analysis of isotopomers. The negative chemical ionization (NCI) spectrum of the corresponding pentafluorobenzoyl derivative has a dominant molecular anion. Moreover, the PFBz derivative is about 100-fold more sensitive than the acetate, which offers some advantages for analysis of minor hexoses found in plasma. Isotopic calibration curves of [U-13C]glucose are linear over the 0.1-60% tracer/tracee range tested. The useful range for isotopic tracer studies is 25-2500 pmol for EI analysis of the acetate derivative and 0.1-55 pmol for NCI analysis of PFBz derivative (sample amount injected). For most studies where sample size is not limited, EI-GC/MS analysis of the IPAc derivative is preferred. NCI-GC/MS analysis is reserved when sample size is limiting or when studies involve hexoses other than glucose that are normally present at low concentration.

Neocytolysis contributes to the anemia of renal disease.

Neocytolysis is a recently described physiological process affecting the selective hemolysis of young red blood cells in circumstances of plethora. Erythropoietin (EPO) depression appears to initiate the process, providing the rationale to investigate its contributions to the anemia of renal disease. When EPO therapy was withheld, four of five stable hemodialysis patients showed chromium 51 (51Cr)-red cell survival patterns indicative of neocytolysis; red cell survival was short in the first 9 days, then normalized. Two of these four patients received oral 13C-glycine and 15N-glycine, and there was a suggestion of pathological isotope enrichment of stool porphyrins when EPO therapy was held, again supporting selective hemolysis of newly released red cells that take up the isotope (one patient had chronic hemolysis indicated by isotope studies of blood and stool). Thus, neocytolysis can contribute to the anemia of renal disease and explain some unresolved issues about such anemia. One implication is the prediction that intravenous bolus EPO therapy is metabolically and economically inefficient compared with lower doses administered more frequently subcutaneously.

Cholesterol-lowering effect of soy protein in normocholesterolemic and hypercholesterolemic men.

Cardiovascular heart disease is a major health problem in the United States. Elevated blood cholesterol has been shown to significantly increase the risk of cardiovascular heart disease. The National Cholesterol Educational Program (NCEP) Step I diet, which restricts fat and cholesterol intakes, is usually recommended as the initial treatment to lower blood cholesterol. Soy protein has been shown to be hypocholesterolemic, particularly in hypercholesterolemic subjects. However, the hypocholesterolemic effect of soy protein in subjects with a blood total cholesterol concentration <5.17 mmol/L is not clear. To determine whether soy protein could enhance the hypocholesterolemic effect of the NCEP Step I diet, 13 normocholesterolemic and 13 hypercholesterolemic men aged 20-50 y were enrolled in a randomized, 2-part, crossover study. Subjects were fed either an NCEP Step I soy-protein diet or an NCEP Step I animal protein diet for 5 wk. After a washout period of 10-15 wk, the subjects were fed the alternate diet for 5 wk. The hypocholesterolemic effect of soy protein was found to be independent of age, body weight, pretreatment plasma lipid concentrations, and sequence of dietary treatment. Regardless of plasma lipid status, the soy-protein diet was associated with a statistically significant decrease in the plasma concentrations of LDL cholesterol (P = 0.029) as well as the in the ratio of plasma LDL cholesterol to HDL cholesterol (P = 0.005). Our results indicate that soy protein enhances the hypocholesterolemic effect of the NCEP Step I diet in both normocholesterolemic and hypercholesterolemic men.

The interaction between nutritional status and growth hormone in young cattle: differential responsiveness of fat and protein metabolism.

The effect of dietary intake level on in vivo plasma leucine and plasma palmitate flux rates and on the response to a bolus injection of bovine growth hormone (GH) was investigated in six young steers. Animals were fed on a pelleted diet of dried grass-barley (0.7:0.3, w/w) in quantities sufficient to supply 0.8, 1.2, 1.6, 2.0, 2.4 or 2.65 x maintenance energy requirement, offered in hourly portions. Continuous intravenous infusions of [1-13C]leucine or [1-13C]palmitate were used to determine the flux of amino acid and fatty acid through the plasma pool before, immediately (1-3 h) after and 22-24 h after a subcutaneous injection of bovine GH (0.55 mg/kg body weight). Hourly blood samples were taken for 27 h to monitor the temporal responses of circulating hormones and metabolites following GH administration. The animal on the lowest plane of nutrition had elevated plasma GH and reduced insulin-like growth factor-1 concentrations compared with those fed on higher intake levels. Plasma leucine flux and leucine concentration increased with intake while palmitate flux and plasma non-esterified fatty acid (NEFA) concentrations were inversely related to intake. Leucine flux rate decreased in the animals fed on the two highest intake levels in response to GH 22-24 h after administration, but plasma leucine concentrations were reduced in all animals at this time. Only the animal fed on the lowest intake level showed an immediate response to GH (within 3 h of administration) with increased palmitate flux and plasma NEFA concentrations but a lipolytic response was apparent in other animals 22-24 h post-administration although the magnitude of the response was markedly reduced at high intakes. We conclude that lipid and protein metabolism are differentially responsive to GH and nutritional status.

Torpedo maculopathy.

BACKGROUND: Congenital nevi of the retinal pigment epithelium (RPE) may manifest variable degrees of pigmentation. These nevi, which are almost always asymptomatic, can be either solitary or grouped. Torpedo maculopathy is a recently described congenital RPE nevus. METHODS: A review of congenital nevi of the RPE is presented to include torpedo maculopathy. RESULTS: Torpedo maculopathy is a solitary congenital RPE nevus; it is oval, variably pigmented, and located in the temporal macula. Diagnosis of this lesion is made on the basis of its characteristic shape and location. The etiology may be related to alterations in the choroidal vasculature in the macular area during the embryologic development of the eye. Because of the benign nature of the nevus, yearly evaluations are recommended. CONCLUSIONS: Classification of congenital nevi of the RPE is still evolving. As more is learned, a better system of organizing these lesions will be developed.

New insights into the metabolism of long chain polyunsaturated fatty acids during infancy.

Data obtained with stable isotope methodology have demonstrated that preterm and term infants can convert LA and ALA, respectively, to AA and DHA. In addition, they have clarified the pathways by which infants convert LA and ALA to LCPUFA and have demonstrated the importance of factors such as the dietary LA/ALA ratio and postnatal age on biosynthesis of AA and DHA. Further work is needed to clarify the role of other influential factors on endogenous synthesis of LCPUFA and to determine the absolute amounts of endogenous LCPUFA synthesis. Such data are necessary to define more precisely the LCPUFA requirements of growing infants.

Intermediates in endogenous synthesis of C22:6 omega 3 and C20:4 omega 6 by term and preterm infants.

An alternative pathway of omega 3 and omega 6 fatty acid metabolism has been described in isolated rate hepatocytes and human fibroblasts. This alternative pathway, which is independent of delta 4 desaturation, involves elongation of C22 5 omega 3 and C22:4 omega 6 to C24 fatty acids, delta 6 desaturation of the C24 fatty acids and subsequent beta oxidation of the desaturated products to C22:6 omega 3 and C22:5 omega 6. To determine whether this alternative pathway is operative in the human infant and also to obtain additional information concerning endogenous conversion of C18:3 omega 3 and C18:2 omega 6 to longer chain more unsaturated fatty acids, presence of [M + 18] isotopomers of omega 3 and omega 6 fatty acids in the plasma phospholipid fraction of term and preterm infants after administration of [U-13C]18:3 omega 3 and [U-13C]18:2 omega 6 was determined by negative chemical ionization gas chromatography/mass spectrometry. [M + 18] isotopomers of the following omega 3 fatty acids were detected: C18:3, C18:4, C20:3, C20:4, C20:5, C22:4, C22:5, C22:6, C24:4 (two infants only), C24:5, and C24:6. [M + 18] isotopomers of omega 6 fatty acids detected included only C18:2, C18:3, C20:2, C20:3, and C20:4, but sensitivity was insufficient to detect [M + 18] isotopomers of C22 and C24 omega 6 fatty acids. Presence of [M + 18] isotopomers of C24:5 omega 3 and C24:6 omega 3 indicates that these fatty acids were synthesized endogenously from C18:3 omega 3. This plus the in vitro data strongly suggests that infants use the recently described alternative pathway in endogenous synthesis of C22:6 omega 3. However, involvement also of delta 4 desaturation cannot be excluded. Detection of [M + 18] isotopomers of C20:3 omega 3, C20:2 omega 6, and C22:4 omega 3 suggests that C18:3 omega 3, C18:2 omega 6, and C20:4 omega 3 are elongated as well as desaturated. The specific fate of these elongation products and their importance in endogenous synthesis of omega 3 and omega 6 long chain polyunsaturated fatty acids remain to be determined.

Quantification of cholesterol tracers by gas chromatography--negative ion chemical ionization mass spectrometry.

Because of its high sensitivity, gas chromatography negative ion chemical ionization mass spectrometry (GC-NCI-MS) is a potentially valuable analytical tool for the study of cholesterol metabolism. Of several derivatives prepared for potential use in tracer studies pentafluorobenzoyl cholesterol was selected because it formed rapidly at ambient temperature and was stable for long periods, could be detected at a level of 1 fmol, and yielded a mass spectrum in which the molecular ion was the principal component. Hexadeuterated cholesterol tracer ([26,26,26,27,27,27-2H6]cholesterol) could be detected in dilutions up to 2700 in unlabeled cholesterol by selected ion monitoring with a coefficient of variation averaging 3.2%. In seven normal subjects tracer cholesterol was infused intravenously and plasma cholesterol enrichment was determined after 4 h. The measured rapidly miscible cholesterol pool was 391.0 +/- 38.6 mg cholesterol/kg. Negative ion mass spectrometry of pentafluorobenzyol cholesterol will facilitate analysis of both small amounts of natural cholesterol and labeled cholesterol in applications where sensitivity is critical.